Mitochondrial epigenetic modifications and nuclear-mitochondrial communication: A new dimension towards understanding and attenuating the pathogenesis in women with PCOS

Abstract

Mitochondrial DNA (mtDNA) epigenetic modifications have recently gained attention in a plethora of complex diseases, including polycystic ovary syndrome (PCOS), a common cause of infertility in women of reproductive age. Herein we discussed mtDNA epigenetic modifications and their impact on nuclear-mitochondrial interactions in general and the latest advances indicating the role of mtDNA methylation in the pathophysiology of PCOS. We highlighted epigenetic changes in nuclear-related mitochondrial genes, including nuclear transcription factors that regulate mitochondrial function and may be involved in the development of PCOS or its related traits. Additionally, therapies targeting mitochondrial epigenetics, including time-restricted eating (TRE), which has been shown to have beneficial effects by improving mitochondrial function and may be mediated by epigenetic modifications, have also been discussed. As PCOS has become a major metabolic disorder and a risk factor for obesity, cardiometabolic disorders, and diabetes, lifestyle/behavior intervention using TRE that reinforces feeding-fasting rhythms without reducing caloric intake may be a promising therapeutic strategy for attenuating the pathogenesis. Furthermore, future perspectives in the area of mitochondrial epigenetics are described.

1. Mitochondria and its genome

Mitochondria play key roles in ATP production, cellular homeostasis, metabolism, and Reactive Oxygen Species (ROS) production [1]. They are solely maternally inherited and contain a 16 kbps circular genome [2]. Human mitochondrial DNA encodes 37 genes, that include 13 protein-coding genes encoding part of the OXPHOS complexes, 22 tRNA genes, and 2 rRNA genes [3]. The displacement loop (D-loop), a non-coding region of mtDNA, contains a promoter for mtDNA replication and various elements necessary for mtDNA transcription and replication. Unlike nuclear DNA (nDNA), mtDNA is not protected by histones, although it is organized into nucleoids with mitochondrial transcription factor A (TFAM) as a primary component [4, 5]. However, a large number of mitochondrial proteins are encoded by the nuclear DNA and imported via mitochondrial localization signal. These proteins play a vital role in mitochondrial function and nuclear-mitochondrial crosstalk [6]. Multiple copies of the mitochondrial genome express the mtDNA copy number, an indirect measurement of mitochondrial function [3].

2. Mitochondrial epigenetic modifications

Mitochondrial epigenetic modifications, including DNA methylation, the most common one, are less studied. Unlike nuclear DNA methylation, mitochondrial methylation changes occur at non-CpG sites [7]. Apart from aberrant methylation, other epigenetic changes are also observed in the mitochondrial genome. The genome-wide human mitochondrial methylome was first studied in 2014 by Ghosh et al., who confirmed the occurrence of a conserved methylation pattern throughout the genome [8]. mtDNA methylation could modulate the mitochondrial gene expression via the mitochondrial transcriptional machinery [9]. DNA methyltransferases (DNMTs) are localized in the mitochondria, suggesting that methylation occurs within the organelle. Evidence suggests that there are molecular isoforms of DNMTs that methylate cytosines in the mitochondrial genome and function mechanistically similar to nuclear DNMTs [10]. In particular, the enzyme nuclear-encoded DNA methyltransferase 1 (DNMT1), involved in the nuclear methylation process, has a mitochondrial targeting sequence (MTS) that promotes localization in mitochondria and is thought to be involved in the mitochondrial methylation mechanism [10]. DNA methyltransferase 3 (DNMT3) also modulates mtDNA methylation de-novo [10]. The mtDNA methylation/demethylation conversion process in response to environmental stimuli and diseases is possible via mechanisms of demethylation within mitochondria by the ten-eleven translocation (TET) group of genes [11]. It has been reported that the enzymes TET1 and TET2, which contribute to the demethylation of mitochondrial-methylated cytosines to 5-hydroxymethylcytosine (5-hmC), act in mitochondria [10, 12, 13]. However, unlike 5-methylcytosine (5-mC) marks, 5-hmC marks are characterized by the fact that they are not conserved in samples and are enriched in the upstream regions of gene start sites and the gene body [14]. It is also known that mitochondrial protein acetylation occurs in response to nutrient availability or energy status in the cell and is regulated by mitochondrial sirtuins [15].

3. The primary role of mitochondria in regulating the nuclear epigenome

The role of the mitochondria in modulating the nuclear epigenome is well known. S-adenosylmethionine (SAM), a universal methyl group donor, is synthesized in the mitochondria via a one-carbon metabolic cycle. In the cytosol, methionine is formed by biosynthesis of SAM in an enzymatic reaction using adenosine 5’-triphosphate (ATP) as part of the methionine cycle. The methionine cycle in the cytosol is coupled to the folate cycle [16] (Fig. 1). 5-methyl tetrahydrofolate (5-MTHF) donates a methyl group to homocysteine (Hcy), forms tetrahydrofolate (THF) and L-methionine in a reaction catalyzed by the vitamin B12-dependent enzyme methionine synthase (MS), and remethylates Hcy [16]. Thus, the production of SAM is regulated by ATP synthesis and folate. The reactions of the folate cycle occur in both the mitochondria and cytosol and are linked by the exchange of the serine and glycine amino acid pool via methylenetetrahydrofolate (methionine-THF) formed from THF [17]. The mitochondrion plays a pivotal role in regulating the switch between nucleotide synthesis and SAM via the mitochondrial bifunctional enzyme (MBE), which is found to be active in embryonic and cancer cells to felicitate nucleotide synthesis and is deactivated at the adult stage for the synthesis of SAM [11]. SAM which is synthesized in the cytosol, is transported to the mitochondria by a specific mitochondrial carrier, SAM carrier (SAMC), where it modulates the mitochondrial methylation pattern [18]. Thus, a defect in one-carbon metabolism would alter DNA methylation patterns in mtDNA and affect nuclear-mitochondrial communication, which occurs via the transport of metabolites from mitochondria to the nucleus via the cytosol and vice versa. Non-coding small RNAs, such as microRNAs (miRNAs), appear to be localized in the mitochondria [19]. Nuclear-encoded miRNAs are responsible for the epigenetic regulation of both nuclear and mtDNA-encoded genes and participate in nuclear-mitochondrial coordination. Furthermore, intermediates of mitochondrial tricarboxylic acid (TCA) metabolites, such as α-ketoglutarate (αKG), succinate, and fumarate, influence the activity of TET proteins [20], indicating a possible role of mitochondria in the demethylation mechanism (Fig. 1). Non-coding small RNAs have also been found to be encoded by both murine and human mitochondrial genomes, but they have been less studied [21]. The TCA cycle or one-carbon cycle metabolites generated in the mitochondria control epigenetic modifications through DNA methylation, histone methylation, and acetylation by acting as cofactors via nuclear-mitochondrial communication [22]. αKG and succinate, metabolites of the TCA cycle, are important regulators of DNA and histone methylation and TET demethylase activity, whereas acetyl-CoA regulates histone acetylation [22] (Fig. 1).

Fig. 1.

Diagram representing the role of mitochondrial epigenetics and nuclear mitochondrial communication via tricarboxylic acid (TCA) metabolites and one-carbon (1-C) metabolic pathway and its possible contribution resulting in polycystic ovaries, mitochondrial dysfunction, and metabolic risk in women with PCOS.

4. Nuclear–mitochondrial communication via epigenetic mechanisms

Tightly regulated coordination between the mitochondria and the nucleus is critical for cellular homeostasis. This occurs through bidirectional communication via anterograde signaling, in which the nucleus controls mitochondrial gene expression and posttranslational modifications, and retrograde signaling, in which the mitochondria control nuclear gene expression [23, 24] (Fig. 1). There are several known epigenetic mechanisms through which communication between the nucleus and mitochondria occurs in cells. First, cellular mitochondrial DNA content or copy number determines the nuclear epigenetic landscape. Modulation of the nuclear epigenetic landscape has been observed as a consequence of the depletion of mtDNA content [25, 26]. Second, mtDNA variants also impact the global nuclear DNA methylation levels resulting in altered nuclear gene expression patterns [27]. Unique haplogroups belonging to different ethnicities are known to possess distinct nuclear epigenetic importance. For example, the increased expression of a nuclear-encoded gene, methionine adenosyltransferase 1 A (MAT1A), was hypermethylated in cybrids belonging to the J haplogroup [27]. The MAT1A gene encodes an enzyme that catalyzes a reaction involved in the transfer of the adenosyl moiety of ATP to methionine to biosynthesize SAM. Furthermore, it has also been suggested that the activity of transcription factors involved in MAT1A gene expression may be regulated by ATP. In a study carried out among different mtDNA haplogroups (H, J, U, X, T, I, K, W, and V) in the study population, J haplogroups showed increased global DNA methylation levels accompanied by lower intracellular ROS and ATP levels compared to all other haplogroups [13, 27]. In-vitro analysis using J-cybrids showed overexpression of the MAT1A gene accompanied by decreased ATP levels and ROS production [27]. Moreover, in vitro experiments have shown that different mtDNA haplogroups alter the expression of nuclear genes and DNA methylation in the nucleus, which play a crucial role in determining cell fate [28]. Diverse haplogroups express differences in mtDNA copy numbers and nuclear- and mtDNA-encoded OXPHOS complex genes [28]. Third, the expression of nuclear-encoded mtDNA genes is controlled by epigenetic mechanisms that control the expression of the nuclear genome [13]. Fourth, mtDNA itself is marked by epigenetic modifications, particularly 5-mC and 5-hmC, and hence modulate nuclear gene expression [13]. Fifth, epigenetic enzymes and RNAs that are encoded in the nucleus translocate to the mitochondria and play a role in the regulation of metabolic pathways in the mitochondria [29]. Sixth, nuclear-encoded epitranscriptomic enzymes modulate mtRNAs implicated in the regulation of mitochondrial function [29].

This nuclear–mitochondrial communication is mediated by a variety of nuclear-encoded transcription factors and non-coding RNAs (ncRNAs) in response to environmental cues and nutrient availability. Genes of the peroxisome proliferator-activated receptor (PPAR) family play important roles in regulating mtDNA and metabolism [30]. One of the most important transcription factors is PGC-1α (encoded by the PPARGC1A gene), which is a crucial cofactor for mitochondrial biogenesis and regulates transcriptional activation of nuclear genes encoding mitochondrial proteins [31, 32]. Other transcription factors that contribute to this process are nuclear respiratory factor 1 (NRF-1) and nuclear respiratory factor (NRF-2), which activate nuclear-encoded genes coding for mitochondrial respiratory chain function, activation, and assembly of OXPHOS complexes. Mitochondrial transcription factor A (TFAM) contributes to mtDNA replication and maintenance, and mitochondrial RNA polymerase (POLRMT) binds to TFAM and mtDNA and initiates mtDNA transcription. The mitochondrial transcription factor B2 (TFB2M) stabilizes open mtDNA and interacts with POLRMT to initiate transcription. Transcription of mtDNA is controlled by mitochondrial transcription termination factors (mTERFs) [31]. Moreover, the sirtuins (SIRTs) family of proteins involved in histone acetylation are nuclear-encoded. Among seven sirtuins (SIRT1-7) in mammals, three sirtuins (SIRT3-5) are located in the mitochondria and regulate mtDNA epigenetic modifications [33]. Numerous non-coding RNAs (e.g., lncRNAs, miRNAs, and circRNAs) are also involved in anterograde and retrograde signaling. For example, lncRNAs may either be encoded in the mitochondria and participate in retrograde signaling or are encoded in the nucleus and modulate mitochondrial metabolism and function after being transported to the mitochondria by anterograde signaling [34].

5. Polycystic ovary syndrome (PCOS)

Polycystic ovary syndrome (PCOS) is the most common endocrinopathy, with clinical heterogeneity affecting a large population of reproductive-aged women worldwide [35]. Clinical manifestations of PCOS may include irregular menstruation, hyperandrogenism, and polycystic ovaries. The majority of women with PCOS are obese and insulin resistant [35, 36]. In addition, metabolic disturbances in PCOS pose an increased risk of various comorbidities such as type 2 diabetes, metabolic syndrome, cardiovascular disease, and endometrial cancer later in life [36]. The etiopathophysiology of PCOS is still not known. Genetic and epigenetic factors, together with various environmental risk factors, including unhealthy diet and poor lifestyle habits, may lead to PCOS [37]. Recent evidence indicates mitochondrial dysfunction and epigenome dysregulation in PCOS pathophysiology. Communication between the mitochondria and the nucleus is crucial for regulating the metabolic needs of cells [38]. Altered methylation of both mtDNA and nuclear-related mitochondrial genes has been implicated in the pathophysiology of PCOS.

6. Altered methylation in mtDNA genes in PCOS

Mitochondria are the most abundant organelles in oocytes and play crucial roles in oocyte maturation, fertilization, and pregnancy outcomes. Good quality oocytes and fertilization rates are related to successful ART outcomes [39]. Jia and his group detected significant hypermethylation in mtDNA-encoded genes, the D-loop, 12 S rRNA, 16 S rRNA, and the ND4 gene, a member of OXPHOS complex I, in polycystic ovaries of gilts, resulting in poor oocyte quality and hyperhomocysteinemia in follicular fluid [40] (Table 1). Altered methylation in the D-loop affects mtDNA replication and transcription. The gene encoding the 12 S rRNA protein regulates insulin sensitivity, acts as an inhibitor of the folate cycle, and activates AMPK, which is involved in metabolic regulation, thereby protecting against diet-induced obesity. These epigenetic alterations result in mitochondrial dysfunction, including reduced mtDNA copy numbers. The one-carbon metabolic pathway was found to be altered in the polycystic ovary group with significant upregulation of methyltransferases, DNA methyltransferase (DNMT1), betaine homocysteine methyltransferase (BHMT), and glycine N-methyltransferase (GNMT) at both the gene and protein levels, and significantly increased Hcy in follicular fluid [40]. Moreover, Hcy concentration in follicular fluid was associated with significantly upregulated one-carbon metabolic enzymes [40]. Jia et al. (2019) demonstrated that Hcy treatment of oocytes derived from gilts exhibited poor quality and developmental competence and induced mitochondrial dysfunction (increased mitochondrial ROS and reduced mtDNA copy number). Hcy treatment reduced the mRNA expression levels of nine mtDNA-encoded genes belonging to OXPHOS complex 1 (ND1, ND2, ND3, ND4, ND4L, and ND5), OXPHOS complex IV (COX2) and ribosomal mtRNA genes (12 S rRNA and 16 S rRNA) [41]. Methylation status was examined in mtRNA genes, which showed significantly increased methylation of 16 S rRNA in the Hcy group (Table 1). Furthermore, Hcy treatment enhanced the expression of one-carbon metabolic enzymes (DNMT1, BHMT, and GNMT). They further demonstrated that the DNMT inhibitor 5’AZA reversed Hcy-induced mitochondrial dysfunction and impaired oocyte quality, developmental competence, expression of methyltransferases, and methylation status of the 16 S rRNA gene. This suggests that the one-carbon pathway leading to hypermethylated mtDNA and mitochondrial dysfunction may be a novel target for improving oocyte quality in women with PCOS [41]. These results suggest the importance of the one-carbon pathway and the underlying mitochondrial epigenetic mechanism in women with PCOS, as serum Hcy levels are elevated in women with PCOS who have poor oocyte quality, leading to poor fertilization, implantation, and pregnancy outcomes [41].

Table 1.

Epigenetic modifications in mtDNA encoded genes and nuclear related mtDNA genes in women with PCOS

Encoded genome	Gene	Gene Function	Experimental model	Tissue/ sample	Epigenetic modifications	References
mtDNA encoded genes	D-loop	mtDNA non-coding region, act as a promoter and contains essential mtDNA transcription and replication elements	Animal	Oocytes of polycystic ovaries	Hypermethylation	[40]
	12 S rRNA	Involved in mtDNA transcription, regulate insulin sensitivity and act as inhibitor of folate cycle and activate AMPK	Animal	Oocytes of polycystic ovaries	Hypermethylation	[40]
	16 S rRNA	Involved in mtDNA transcription	Animal	Oocytes of polycystic ovaries	Hypermethylation	[40, 41]
	ND4	Subunit of ETC complex chain, member of OXPHOS complex I, involved in ROS and ATP production	Animal	Oocytes of polycystic ovaries	Hypermethylation	[40]
Nuclear related mitochondrial genes	PPARGC1A	Nuclear Transcription factor involved in mitochondrial biogenesis, plays a central role in lipid and lipoprotein metabolism	Women with PCOS	Leukocytes	Promoter methylation	[42]
	APP	Involved in mitochondrial activity	Women with PCOS	Cord Blood	Hypomethylation	[44]
	PARK2	Involved in mitochondrial activity	Women with PCOS	Cord Blood	Hypermethylation	[44]
	PPAR gene family (PPARG, PPARD, PPARGC1A, PPARGC1B)	Family of nuclear transcription factors involved in mitochondrial biogenesis, regulation of lipid metabolism, energy balance, metabolic function, cell differentiation, and tumorigenesis	Women with PCOS	Adipose tissues	hypermethylation	[45]
	DNMT1, DNMT3A, DNMT3B, DNMT3L	Genes encoding DNA methyltras- ferases involved in methylation process	Women with PCOS	Adipose tissues	hypermethylation	[45]

D-loop: Displacement loop, 12 S rRNA: 12 S Ribosomal ribonucleic acid, 16 S rRNA: 16 S Ribosomal ribonucleic acid, ND4: NADH dehydrogenase 4, PPAR: Peroxisome proliferator-activated receptors, PPARGC1A: Peroxisome Proliferator-activated Receptor Gamma Coactivator-1 Alpha, APP: Amyloid precursor protein, PARK2: Parkin RBR E3 ubiquitin protein ligase, PPARG: Peroxisome Proliferator Activated Receptor Gamma, PPARD: Peroxisome Proliferator Activated Receptor Delta, PPARGC1B: Peroxisome Proliferator-activated Receptor Gamma Coactivator-1 Beta, DNMT1: DNA methyltransferase 1, DNMT3A: DNA methyltransferase 3 alpha, DNMT3B: DNA methyltransferase 3 Beta, DNMT3L: DNA methyltransferase 3 Like

7. Altered methylation in nuclear-related mitochondrial genes in PCOS

In a study of leukocytes from women with PCOS, increased PGC-1α promoter methylation was found to be associated with decreased mtDNA copy number compared with that in control women [42]. It was observed that the changes in PGC-1α promoter methylation and mtDNA copy number were more pronounced in women with PCOS with unfavorable metabolic risk [42] (Table 1). Altered expression of PGC-1α results in decreased expression of nuclear-encoded genes belonging to the OXPHOS complexes (I-V) (NDUFA3, SDHD, UCRC, COX7C, and ATP5H) in the skeletal muscle of women with PCOS who are insulin resistant, indicating a link between insulin resistance and mitochondrial dysfunction at an early stage that may lead to type 2 diabetes later in life [43]. Analysis of global methylation in the cord blood of women with PCOS suggests a gene network involving genes involved in mitochondrial activity, APP, and PARK2, indicating intrauterine reprogramming and epigenetic signatures that increase the risk for PCOS. These signatures may be detected early at birth and may have diagnostic implications [44]. Epigenetic and transcriptional alterations in human adipose tissue in PCOS suggest altered signaling pathways regulating mitochondrial biogenesis and the NRF2-mediated oxidative stress response pathway [45]. In this study, multiple PPAR family genes (PPARG, PPARD, PPARGC1A, PPARGC1B) and genes encoding DNA methyltransferases, such as DNMT1, DNMT3A, DNMT3B, and DNMT3L, were found to be differentially methylated in the adipose tissue of PCOS patients compared to controls [45] (Table 1).

8. Shreds of evidence for altered TCA metabolites in PCOS

Metabolomics analysis in women with PCOS suggests a marked impairment in the TCA cycle pathway, as evidenced by significantly decreased plasma citrate levels and increased levels of amino acids such as threonine, valine, phenylalanine, and tyrosine, resulting in decreased levels of succinyl-CoA and fumarate [46]. In a review of metabolic abnormalities in women with PCOS, several reports indicated increased TCA cycle metabolites, such as succinate, malate, oxaloacetate, acetate, acetoacetate, 3-hydroxybutyrate, 4a-methylzymosterol-4-carboxylic acid, and α-keto acids in follicular fluid and decreased serum/plasma citrate levels in women with PCOS compared to controls [47]. A recent study by Liu et al. also showed impaired TCA cycle metabolism (increased citric acid and iso-citric acid) in the follicular fluid of women with PCOS belonging to the insulin-resistant group compared to women without insulin resistance [48].

9. mtDNA epigenetic modifications as therapeutic targets

Due to their dynamic nature, the epigenetic modifications of mtDNA may be reversible and, therefore, can be exploited for novel therapeutic strategies. In-vitro studies of pharmacological modulation with valproic acid resulted in significantly altered 5hmC content of mtDNA [49]. The DNMT inhibitor RG108 is well known to ameliorate altered effects associated with increased nuclear DNA and mtDNA methylation [50]. However, new molecular strategies are needed to selectively target epigenetic drugs in the mitochondria. RG108 directly binds to DNMT proteins [50]. Labeled RG108 could be used to investigate the mechanism by which it is imported into the mitochondria.

In a prenatal AMH-induced PCOS-like animal model, SAM supplementation in F3 female offspring corrected defects in a wide variety of genes associated with insulin signaling, ovarian function, inflammation, and immune response. Supplementation with a methyl donor and high-dose folate improves metabolic function in obese women with PCOS [51].

Since the majority of women with PCOS are obese, dietary intervention in women with PCOS may modulate nuclear-mitochondrial communication via epigenetic regulation. It is possible that time-restricted eating may help in reversing mitochondrial epigenetic modifications.

10. Attenuation of PCOS-induced metabolic dysregulation with time-restricted eating

In recent years, time-restricted eating (TRE) has been used as a potential therapeutic strategy for the regulation of metabolic disorders, weight loss management, and maintenance of the overall health span [52–55]. TRE is a lifestyle intervention that maintains a daily rhythm of feeding-fasting, without reducing calorie intake, simply by limiting the total feeding time to 8–12 h of the active phase [52–55]. This behavior intervention mediated via TRE is more sustainable compared to calorie restriction as TRE only limits the eating windows but not the total food consumption, potentially allowing for greater sustainability without the potential side effects. Using animal models and pilot-based human studies, TRE-mediated benefits to combat metabolic disorders and various obesogenic challenges have been shown, including improved insulin sensitivity, reduced triglycerides and cholesterol, reduced systemic inflammation, and improved endurance [53, 54, 56].

More recent works have been focused on unraveling the mechanistic basis of TRE-mediated benefits associated with cardiometabolic aging and other metabolic disorders [52, 55, 56]. For example, TRE prevents age-associated cardiac disorders by regulating mitochondrial electron transport chain (ETC) function, circadian clock, and CCT machinery [52, 55]. Moreover, using diet and genetic obesity models, it has been shown that TRE-mediated intervention can attenuate muscle performance by suppressing abnormal intramuscular fat deposits, attenuating mitochondrial aberrations, reducing insulin resistance, and increasing ATP levels in muscle [56, 57]. Furthermore, transcriptomic analyses followed by genetic validation in the muscle revealed the involvement of lipid signaling, mitochondrial homeostasis, and other energy-sensing pathways with TRE-mediated benefits in the obesity models [56, 57]. Under both diet and genetically obese conditions, implementation of the TRE paradigm resulted in upregulated genes involved in glycine production (Sardh and CG5955) and SAM regulation utilizing glycine (Gnmt), as well as downregulation of a key gene (Dgat2) involved in triglyceride synthesis. Moreover, in a diet-induced obesity model, genes and metabolites linked to the purine cycle were upregulated under TRE. However, under genetically obese conditions, genes and metabolites linked to AMPK signaling, glycogen metabolism, glycolysis, TCA, and mitochondrial ETC were found to be upregulated under TRE [56, 57]. Both obesity models led to increased levels of ATP upon implementation of TRE, this suggesting TRE’s ability to mediate muscle function attenuation under metabolic challenges [56, 57]. Both SIRT1 and SIRT3 are known to interact with circadian clock machinery [58]. Impaired circadian rhythms are observed in women with PCOS due to poor lifestyles and irregular sleep patterns. This results in impaired clock genes, abnormal secretion of gonadotropins, and reproductive hormones that affect female fertility, contributing to the etiology of PCOS. Several of the above-mentioned genes are also associated with mitochondrial epigenetic dysregulation and function via improving mitochondrial function, thus suggesting TRE’s role in mitigating PCOS. More specifically, recently, a small pilot-based study, five weeks of TRE implementation able to mitigate PCOS by reducing body fat and weight loss, improving menstruation, hyperandrogenemia, insulin resistance, and chronic inflammation in eighteen PCOS women aged between 18 and 31 [59]. This is a small study; however, the outcome of these findings suggests that there must be some common mechanism for the TRE-mediated regulation of PCOS and other metabolic disorders, including obesity.

Overall, circadian clock components interact with feeding/fasting signals synergistically and ensure transcribing, processing, translating, and degrading of hundreds or thousands of gene products participating in the energy-consuming process are restricted to times of the day when nutrients are available [54, 55, 60]. Daily rhythms in the food availability drive systemic signals that interact with the circadian oscillator at the molecular level to increase the robustness of these transcriptional oscillations to maintain metabolic homeostasis [54, 55, 60]. These transcripts then mediate metabolic (anabolic and catabolic) processes that function during specific phases of the feeding/fasting cycle. In the presence of a disrupted circadian clock, feeding- and fasting-driven pathways can drive some oscillations in transcription and downstream metabolites but cannot fully compensate for the loss.

Importantly, TRE findings shift the paradigm away from this decades-long work centered on diet composition and towards diet timing. Moreover, these interventions are needed to explore PCOS, which is a major reproductive endocrine and metabolic disease. There is a pressing need to investigate new approaches that can reduce this major metabolic disorder using model organisms. Overall, similar to other metabolic disorders, nutritional quality, excess caloric intake, and obesity are well-recognized risk factors for PCOS. There is a prominent gap in the genetic model system where these risks, potential lifestyle interventions to mitigate these risks, and the underlying genetic basis can be systematically examined. Therefore, uncovering novel TRE-mediated molecular pathways will be useful for mitigating PCOS and other disorders.

11. Conclusion and future perspectives

In summary, mitochondrial epigenetics is an emerging area of research, and initial pieces of evidence are promising for developing mitochondrial epigenetic modifications as biomarkers or therapeutic targets. With the advent of high-throughput next-generation sequencing techniques, mitochondrial epigenetics and epigenetic modifications in nuclear-related mitochondrial genes have been comprehensively characterized. Designing novel in-vitro studies and studies using animal model organisms may help in modulating the mitochondrial epigenome and studying the impact. So far, nuclear mitochondrial communication has been studied in various diseases; however, studies in women with PCOS are scanty.

Mitochondrial epigenetic modifications may be a mediator between environmental factors such as diet and the metabolic condition of obesity in women with PCOS. Obesity might also perturb nuclear-mitochondrial homeostasis affecting the availability of nutrients acting as intermediates of the one-carbon cycle [61]. Obese, insulin resistant women with PCOS had aberrant skeletal muscle mitochondrial physiology compared with lean, insulin-sensitive women with PCOS [62]. Hence, it would be interesting to assess the mitochondrial epigenetic alterations that differ between obese and normal-weight infertile women with PCOS, as increased BMI is significantly associated with reduced clinical pregnancy rate, increased miscarriage rate, and poor In-vitro fertilization (IVF) outcomes [63]. Identifying these mitochondrial epigenetic signatures that differentiate obese and normal-weight women may be used to identify the risk of PCOS in obese individuals and may help in providing proper genetic counseling and developing novel therapeutic strategies for tailored treatment in obese women with PCOS.

The main cause of pregnancy failure is fertilization failure or embryo arrest during preimplantation development. Chromosome aneuploidies are the main cause of infertility and reduced fertility due to age in women. Preimplantation genetic testing for aneuploidies (PGT-A) helps reduce multiple IVF cycles to achieve pregnancy. Assessing mtDNA copy number in the embryo is a new strategy for evaluating embryo quality [64]. A high mtDNA copy number is indicative of lower embryo viability and implantation, whereas a reduced mtDNA copy number in oocytes represents poor oocyte quality and may affect pregnancy outcomes. Supplementation of an autologous population of mitochondrial isolates during fertilization in porcine oocytes with mitochondrial deficiency enhances oocyte developmental competence, embryo quality, and the development of blastocysts without compromising genetic identity [65]. These findings have clinical relevance for rescuing in vitro-matured compromised oocytes in women with PCOS. In addition, tests to assess mitochondrial epigenetic defects such as elevated Hcy levels in serum and follicular fluid and methylation defects in the above-mentioned mtDNA or nuclear-related mtDNA genes in granulosa cells surrounding the oocyte may be an added advantage in decreasing the number of IVF cycles to achieve successful pregnancy in infertile women with or without PCOS in the future.

The pleiotropic beneficial effects of TRE in animal models for mitigating multiple metabolic disorders have opened up a potential lifestyle intervention strategy to combat metabolic disorders, including PCOS. Clinical trials and multicenter studies in women with PCOS, particularly obese PCOS, are required to study the effect of TRE in attenuating the pathogenesis of PCOS and its link with the mitochondrial epigenetic mechanism.

It is speculated that with the continuous progress in mitochondrial research on the epigenetics front, mitochondrial epigenetic modifications could be potential biomarkers and promising therapeutic targets to prevent or treat complex diseases such as PCOS.

Abbreviations

5-hmC

5 Hydroxymethylcytosine

5-mC

5-methylcytosine

5-MTHF

5-methyl tetrahydrofolate

αKG

Alpha-ketoglutarate

AMP

Adenosine Monophosphate

AMPK

AMP-activated protein kinase

ATP

Adenosine Triphosphate

BHMT

Betaine homocysteine methyltransferase

D-loop

Displacement loop

DNMT

DNA methyltransferase

DNMT1

DNA methyltransferase 1

DNMT3

DNA methyltransferase 3

D NMT3A

DNA methyltransferase 3 alpha

DNMT3B

DNA methyltransferase 3 Beta

DNMT3L

DNA methyltransferase 3 Like

ETC

Electron transport chain

GNMT

Glycine N-methyltransferase

Hcy

Homocysteine

IVF

In-vitro fertilization

MBE

Mitochondrial bifunctional enzyme

Met

Methionine

miRNA

micro-RNAs

MS

Methionine synthase

mtDNA

Mitochondrial DNA

mTERFs

Mitochondrial transcription termination factors

MTHF

Methyl tetrahydrofolate

MTS

Mitochondrial targeting sequence

nDNA

Nuclear DNA

ncRNA

non-coding RNAs

NRF-1

Nuclear respiratory factor 1

NRF-2

Nuclear respiratory factor 2

OXPHOS

Oxidative Phosphorylation

PCOS

Polycystic ovary syndrome

PGC-1α

Peroxisome proliferator-activated receptor-gamma coactivator

PGT-A

Preimplantation genetic testing for aneuploidies

POLRMT

Mitochondrial RNA polymerase

PPAR

Peroxisome proliferator-activated receptors

PPARG

Peroxisome Proliferator-Activated Receptor Gamma

PPARGC1A

Peroxisome Proliferator-activated Receptor Gamma Coactivator-1 Alpha

PPARGC1B

Peroxisome Proliferator-activated Receptor Gamma Coactivator-1 Beta

PPARD

Peroxisome Proliferator-Activated Receptor Delta

ROS

Reactive Oxygen Species

rRNA

Ribosomal ribonucleic acid

SAM

S-adenosylmethionine

SAMC

SAM carrier

SIRTs

Sirtuins

TCA

Tricarboxylic acid cycle

TET

Ten-eleven translocation

TFAM

Mitochondrial transcription Factor A

TFB2M

Mitochondrial transcription Factor B2

THF

Tetrahydrofolate

TRE

Time-restricted eating

tRNA

Transfer ribonucleic acid