A systematic review on the association of mitochondrial DNA copy number (mtDNA-CN) with Alzheimer's disease (AD) and Parkinson disease (PD) is lacking and the causal relationship remains unclear.

We aimed to conduct a systematic review of observational studies on the association of mtDNA-CN with AD and PD and perform a bidirectional 2-sample Mendelian randomization (MR) study to investigate their causal relationships.

PubMed, Embase, and Web of Science were searched for eligible studies before Jan 2025. The causal links were conducted with inverse-variance weighted (IVW) method as the main analysis.

Fourteen case-control and 2 cohort studies investigated the association between mtDNA-CN and AD, with 13 reporting decreased mtDNA-CN associated with increased risk of AD and 3 showing no significant association. All the studies (9 case-control, 1 cross-sectional, 2 cohort studies) observed the relation between mtDNA-CN and PD except for 3 studies reporting no significant association. In MR analysis, genetically predicted mtDNA-CN was not associated with AD and PD, whereas genetically predicted AD (β -0.085, 95 % CI -0.156 to -0.013; P = 0.02) but not PD was associated with mtDNA-CN. Sensitivity and replication analyses showed a stable finding.

The systematic review found limited observational studies on mtDNA-CN and AD and PD and majority were case-control study. Findings of the bidirectional MR study did not support a causal effect of mtDNA-CN in the development of AD and PD but found that AD can lead to decreased levels of mtDNA-CN, which suggest mtDNA-CN as a potential biomarker of AD.

Mitochondrial DNA (mtDNA) is vulnerable to environmental exposure and is related to various diseases. However, the relationship of green space exposure with mtDNA copy number (mtDNAcn) is not yet well clarified. In this study, we sought to explore how green space exposure influences mtDNAcn in children and adolescents. This cross-sectional study involved 1151 participants aged 6-18 years from Liuzhou, China. Green space exposure was quantified using the normalized difference vegetation index (NDVI) within several circular buffers (250-2000 m). Leukocyte mtDNAcn was measured using the qPCR method. Multiple linear regression was employed to estimate the relationship of green space exposure with mtDNAcn. Per interquartile range increment in NDVI250m, NDVI500m, NDVI1000m, and NDVI2000m were related to 3.33% (95% confidence interval [CI]: 1.00%, 5.72%), 3.37% (95% CI: 1.02%, 5.78%), 3.34% (95% CI: 0.93%, 5.81%), and 3.25% (95% CI: 0.49%, 6.08%) increase in mtDNAcn, respectively. Subgroup analyses showed that the positive relationship of green space exposure with mtDNAcn was more evident in children, females, participants with normal weight, and those not exposed to passive smoking. Our findings indicate a positive relationship of green space exposure with mtDNAcn in children and adolescents. Given the significance of mtDNAcn during childhood and adolescence, it is essential to implement strategies that enhance green environments to support the health and development of children and adolescents.

Mitochondrial dysfunction has been associated with neurodegenerative diseases (NDDs). This study aimed to explore the association between blood mitochondrial DNA copy number (mtDNA-CN) and development of NDDs. This study was based on two-sample Mendelian randomization (MR) analysis. The genome wide association study (GWAS) data of NDDs including Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), age-related macular degeneration (AMD), multiple sclerosis (MS), Parkinson's disease (PD), primary open-angle glaucoma (POAG), and vascular dementia (VD) was obtained from FinnGen consortium. Inverse-variance weighted (IVW) was applied as the primary approach for MR estimation. MR results revealed that blood mtDNA-CN exhibited a significant relationship with the incidence of AD (IVW-P = 0.011, odds ratio [OR] = 0.65) and AMD (IVW-P = 0.038, OR = 0.64). However, there was no significant association observed between blood mtDNA-CN and other NDDs (IVW-P > 0.05). Our findings supported the relationship between mitochondrial dysfunction and development of AD and AMD, and that blood mtDNA-CN may serve as a potential biomarker for the incidence of these two NDDs.

The etiology of adenomyosis remains unclear. The association between epithelial-mesenchymal transition (EMT) and mitochondrial dysfunction is involved in fibrotic diseases. Adenomyosis is defined as the existence of endometrial glands and stroma in the myometrium with EMT and ultimate fibrosis. This study was designed to investigate the involvement of mitochondrial dysfunction in fibrotic adenomyosis. Mitochondrial integrity was examined in mouse and human adenomyotic tissues. Control and tamoxifen-treated mice were treated with 3-nitropropionic acid (a mitochondrial dysfunction inducer) and NG-nitro-L-arginine methyl ester (a mitochondrial dysfunction inhibitor), respectively, at postnatal day 21, followed by an evaluation of adenomyosis, EMT, and fibrosis as well as the expression of mitophagy, oxidative stress, and transforming growth factor-β1 (TGF-β1). The gene profiles of adenomyotic uteri were examined at postnatal day 42. Adenomyotic mice exhibited increased development of EMT and fibrosis. Adenomyotic tissues showed consistent mitochondrial destruction with increased fission, mitophagosomes, and lysosomes. Besides, mitophagy, oxidative stress, and TGF-β1 levels were consistently increased. The mitochondrial dysfunction, the development of mitophagy and fibrosis, and TGF-β1 expression were induced by 3-nitropropionic acid in control uteri. In contrast, NG-nitro-L-arginine methyl ester attenuated mitochondrial dysfunction, mitophagy, fibrosis, and TGF-β1 in adenomyotic uteri. Gene profiling demonstrated increased expression of mitochondrial dysfunction-related genes in adenomyotic uteri. This indicates that mitochondrial dysfunction-induced TGF-β1 dysregulation and fibrosis are associated with the development of adenomyosis.

Insect fat body serves as a central hub for energy mobilization and protein synthesis. During larval metamorphosis, fat body undergoes programmed cell death and tissue disassembly. Following adult eclosion, fat body reconstructs with cell proliferation and becomes competent for large-scale vitellogenin (Vg) synthesis required for the maturation of dozens of eggs.

This study aims to uncover the molecular mechanisms underlying the remodeling of fat body in acquisition of competence for massive Vg production.

RNA-seq and metabolomics were used for identification of differentially expressed genes and metabolites. RNAi was applied for gene knockdown. Transmission electron microscope, MitoTracker staining, mitochondrial DNA quantification, ATP and citrate synthase assays were employed for examining mitochondrial biogenesis. Dual-luciferase reporter assay and EMSA were performed for transcriptional regulation. qRT-PCR and western blot were performed for measuring Vg synthesis.

Transcriptomic and metabolomic analyses revealed significant upregulation of genes and metabolites involved in mitochondrial biogenesis in the fat body of adult locusts. PGC-1α was highly expressed in adult fat body. Knockdown of PGC-1α reduced mitochondrial biogenesis, fat body cell number, Vg synthesis and ovarian development. CREBB bound to PGC-1α promoter and activated its transcription. CREBB depletion impaired mitochondrial biogenesis and fat body remodeling. Moreover, loss of TORC1 function suppressed CREBB function and PGC-1α expression, subsequently disrupting mitochondrial biogenesis and fat body remodeling. Juvenile hormone (JH) deprivation also decreased CREBB function and PGC-1α expression, which was reversible with JH treatment. Our results suggest that TORC1 and JH coordinate CREBB-upregulated PGC-1α expression, which promotes mitochondrial biogenesis and fat body remodeling for Vg synthesis and egg production.

The findings provide new insights into the molecular mechanisms of post-metamorphic fat body development, and highlight the role of JH/TORC1/CREBB/PGC-1α/mitochondrial biogenesis axis in insect reproduction. The data also offer potential targets for insect pest control.

ATAD3A, a nuclear gene encoding the ATAD3A protein, has diverse roles in mitochondrial processes, encompassing mitochondrial dynamics, mitochondrial DNA maintenance, metabolic pathways and inter-organellar interactions. Pathogenic variants in this gene cause neurological diseases in humans with recognizable genotype-phenotype correlations. Yet, gaps in knowledge remain regarding the underlying pathogenesis.

To further investigate the gene function and its implication in health and disease, we utilized CRISPR/Cas9 genome editing to generate a knockout model of the zebrafish ortholog gene, atad3. We characterized the phenotype of the null model, performed mitochondrial and functional tests, and compared the transcriptome of null embryos to their healthy siblings.

Analysis of atad3-null zebrafish embryos revealed microcephaly, small eyes, pericardial edema and musculature thinning, closely mirroring the human rare disease phenotype. Larvae exhibited delayed hatching and embryonic lethality by 13 days post-fertilization (dpf). Locomotor activity, ATP content, mitochondrial content, and mitochondrial activity were all reduced in the mutant embryos. Transcriptome analysis at 3 dpf via RNA-sequencing indicated decline in most mitochondrial pathways, accompanied by a global upregulation of cytosolic tRNA synthetases, presumably secondary to mitochondrial stress and possibly endoplasmic reticulum (ER)-stress. Differential expression of select genes was corroborated in fibroblasts from an affected individual.

The atad3-null zebrafish model emerges as a reliable representation of human ATAD3A-associated disorders, with similarities in differentially expressed pathways and processes. Furthermore, our study underscores mitochondrial dysfunction as the primary underlying pathogenic mechanism in ATAD3A-associated disorders and identifies potential readouts for therapeutic studies.

Venous thromboembolism (VTE), including pulmonary embolism (PE) and deep vein thrombosis (DVT), is the third most common cardiovascular disease. A low amount of mitochondrial DNA copy number (mtDNA-CN) reflects mitochondrial dysfunctions and has been associations with arterial cardiovascular diseases. However, the role of mtDNA-CN in venous cardiovascular disease was unclear. We aimed to implement a 2-sample Mendelian randomization analysis to approximate the causal nature of these relationships.

Genetic instruments for VTE, PE, and DVT were derived from the largest available genome-wide association study datasets. The inverse variance weighted method was used as the primary analytical approach, with sensitivity analyses performed to assess horizontal pleiotropy and heterogeneity. The reverse Mendelian randomization analysis was conducted using genetic instruments for mtDNA-CN.

The genetically instrumented mtDNA-CN levels did not exhibit a causal effect on VTE (P = 0.224), DVT (P = 0.190), and PE (P = 0.571). However, genetically predicted VTE (odds ratio (OR) = 0.569, 95% confidence interval (CI) = 0.341-0.952; P = 0.032), PE (OR = 0.991, 95% CI = 0.983-0.999; P = 0.037), and DVT (OR = 0.429, 95% CI = 0.207-0.890; P = 0.023) were associated with decreased mtDNA-CN levels in the inverse variance weighted analysis. Sensitivity and replication analyses confirmed the robustness of these findings.

Our findings did not support a causal effect of mtDNA-CN in the development of VTE, but provide direct evidence that VTE may lead to reduced mtDNA-CN levels. These results suggest that mtDNA-CN as a biomarker of VTE in clinical practice.

Previous studies have reported that mitochondrial DNA copy number (mtDNA-CN) of blood was associated with a series of aging-related diseases. However, it remains unknown whether mtDNA-CN can be a potential biomarker of acute aortic syndromes (AASs). The mtDNA-CN in blood of 190 male patients with AAS and 207 healthy controls were detected by standardized real-time quantitative PCR-based assay. The mtDNA sequencing data of blood and myocardial muscle in 134 individuals were used to analyze mtDNA somatic mutations in blood. mtDNA-CN in peripheral blood was negatively correlated with age of individuals. Further analysis based on next-generation sequencing data demonstrated numbers and heteroplasmy of mtDNA mutations were positively correlated with age. Remarkably, mtDNA-CN of patients with AAS was lower than that of healthy controls. Logistic regression also showed that mtDNA-CN was independently associated with risk of AAS. During follow-up, patients with the lowest mtDNA-CN quartile had a hazard ratio of 2.543 for all-cause-mortality and 1.964 for composite end points compared with the other patients. Moreover, multivariate Cox regression indicated that lowest mtDNA-CN quartile was independently associated with all-cause mortality in patients with AAS. Our study demonstrated a negative correlation between mtDNA-CN and age. Moreover, lower mtDNA-CN in peripheral blood was significantly associated with higher risk and worse prognosis of AAS. It provided crucial evidence supporting the potential of mtDNA-CN as a novel biomarker of AAS.

Venous thromboembolism (VTE) is a common and serious complication among cancer patients. Mitochondrial DNA (mtDNA) copy number is known to influence various cellular pathways involved in cancer development. While an association between reduced mtDNA and VTE risk in non-cancer patients was previously reported, its relationship with VTE in cancer patients remains unclear. Therefore, we aimed to investigate the association between mtDNA copy number and VTE risk in a nested-case control study of 48 patients from the Vienna Cancer and Thrombosis Study (CATS), a prospective observational cohort study. The mtDNA copy number was measured in equally distributed age, sex, cancer type, and stage matched patients with and without VTE using a qPCR-based method. Of the 48 patients, 24 were diagnosed with VTE (median age [IQR] 62 [57-60] years, 54.2 % female) and 24 had no VTE event (median age [IQR] 63 [58-71] years, 54.2 % female). We found that patients who developed VTE had lower mtDNA copy numbers compared to those without VTE (216.73 [167.99-401.39] vs 301.47 [210.66-526.84]). Multivariable analysis adjusting for chronological age, D-dimer, sex, cancer stage and BMI revealed that each 10-unit increase in mtDNA copy number decreased the odds of VTE occurrence by 5.9 % (p = 0.021). Patients with distant metastatic cancer (M1) had lower mtDNA copy numbers than those without distant metastasis at study inclusion (220.34 [172.67-323.70] vs 328.48 [213.89-556.68; p = 0.052). Overall, our findings suggest a potential link between reduced mtDNA copy number and increased VTE risk in cancer patients.

Human genetic diversity in today's world has been shaped by evolutionary history, demographic shifts and environmental exposures, influencing complex traits, disease susceptibility and drug responses. Capturing this diversity is essential for advancing precision medicine and promoting equitable healthcare. Despite the great progress achieved with initiatives such as the human Pangenome and large biobanks that aim for a better representation of human diversity, important challenges remain. In this Perspective, we discuss the importance of diversity in clinical genomics through an evolutionary lens. We highlight progress and challenges and outline key clinical applications of diverse genetic data. We argue that diversifying both datasets and methodologies-integrating ancestral and environmental factors-is crucial for fully understanding the genetic basis of human health and disease.

Ageing is a degenerative process characterised by a decline in physiological functioning of the organism. One of the core regulators of cellular ageing are telomeres, repetitive DNA sequences of TTAGGG that cap the ends of chromosomes and are maintained by the ribonucleoprotein complex, telomerase. Age-dependent progressive loss of the telomere ends eventually induces cell cycle arrest for the induction of either replicative senescence or apoptosis. It was previously established that overexpression of the 37 kDa/ 67 kDa laminin receptor (LRP/LR) increased telomerase activity and telomere length while concomitantly reducing senescence markers in aged normal cells in vitro. Therefore, it was hypothesized that elevating LRP/LR in vivo may increase telomerase activity and hinder the ageing process on an organism scale. To this end, aged C57BL/6J mice were treated/transfected to induce an overexpression of LRP::FLAG. Various physiological tests and histological analyses were performed to assess overall organism fitness as well as to discern the treatments' ability at reducing tissue degeneration and atrophy. It was found that mice overexpressing LRP::FLAG displayed improved physiological characteristics and markedly less tissue degeneration and atrophy when compared to control and non-treated mice. Alongside these improvements, certain organs displayed increased telomerase activity with a corresponding elongation in average telomere length. In addition the overexpression of LRP::FLAG significantly improved various proliferative and anti-ageing associated proteins while causing a concomitant decrease in senescence associated proteins. These findings are therefore indicative of a novel function of LRP/LR delaying the onset of senescence, while also promoting healthier ageing through elevating TERT and telomerase activity.

SLE is associated with significant morbidity, especially in the case of renal involvement. Mitochondrial dysfunction plays a significant role in SLE and may be assessed by measuring mitochondrial DNA (mtDNA) and cytokines reflecting mitochondrial stress (mitokines). Circulating mtDNA is a promising biomarker in SLE and appears to be reduced in severe SLE. However, measuring circulating mtDNA is challenging and reported methods are heterogenous. Our study aimed at evaluating whole blood mtDNA to nuclear DNA (nucDNA) ratio using droplet-digital PCR and circulating mitokines, growth differentiation factor 15 (GDF-15) and fibroblast growth factor 21 in SLE with and without renal involvement.

Cross-sectional study involving 195 patients with SLE and age-matched healthy volunteers (HV) as control. Biomarkers were compared in patients with and without renal involvement (defined by estimated glomerular filtration rate <60 mL/min or proteinuria >0.5 g/day) and in those with active and inactive SLE.

Compared with HV, patients with SLE displayed lower mtDNA/nucDNA ratios, especially in the case of renal involvement. Accordingly, mitokines were increased in patients with SLE with renal involvement. We found no correlation between mtDNA/nucDNA ratio and global disease activity. Mitokine levels, on the other hand, correlated with disease activity, in particular GDF-15 even after adjusting for renal involvement.

Our findings suggest that lower whole blood mtDNA/nucDNA ratio, a surrogate marker for mitochondrial dysfunction, reflects renal damage, while GDF-15 may also reflect disease activity in SLE. Further studies are needed to assess the clinical value of these markers as predictors for active lupus nephritis.

Mitochondrial DNA content (mtDNAc) at birth is a sensitive biomarker to environmental exposures that may play an important role in later life health. We investigated sensitive time windows for the association between prenatal ambient temperature exposure and newborn mtDNAc.

In the ENVIRONAGE birth cohort (Belgium), we measured cord blood and placental mtDNAc in 911 participants using a quantitative real-time polymerase chain reaction. We associated newborn mtDNAc with average weekly mean temperature during pregnancy using distributed lag nonlinear models (DLNMs). Double-threshold DLNMs were used to study the relationships between ambient temperature and mtDNAc below predefined low (5th, 10th, 15th percentile of the temperature distribution) and above predefined high temperature thresholds (95th, 90th, 85th percentile of the temperature distribution).

Prenatal temperature exposure above the used high temperature thresholds was linked to lower cord blood mtDNAc, with the strongest effect in trimester 2 (cumulative estimates ranging from -21.4% to -25.6%). Placental mtDNAc showed positive and negative associations for high temperature exposure depending on the applied high temperature threshold. Negative associations were observed during trimester 1 using the 90th and 95th percentile threshold (-26.1% and -33.2% lower mtDNAc respectively), and a positive association in trimester 3 when applying the most stringent 95th percentile threshold (127.0%). Low temperature exposure was associated with higher mtDNAc for both cord blood and placenta. Cord blood mtDNAc showed a positive association in trimester 2 when using the 10th percentile threshold (11.3%), while placental mtDNAc showed positive associations during the whole gestation and for all applied thresholds (estimates ranging from 80.8% - 320.6%).

Our study shows that in utero temperature exposure is associated with differences in newborn mtDNAc at birth, with stronger associations observed in the placenta. These findings highlight the impact of prenatal ambient temperature exposure on mtDNAc during pregnancy.

Adipose tissue (AT), is a major endocrine organ that plays a key role in health and disease. However, adipose dysfunctions, especially altered energy metabolism, have been under-investigated as white adipocytes have relatively low mitochondrial density. Nevertheless, recent studies suggest that mitochondria could play a major role in AT disorders and that AT mitochondrial activity could depend on adiposity level and location. This clinical study aimed to evaluate mitochondrial respiration and metabolism in human visceral (vAT) and subcutaneous (scAT) AT and their relationship with body mass index (BMI). This clinical study enrolled 67 patients (30 females/37 males) scheduled for digestive surgery without chemotherapy and parietal infection. BMI ranged from 15.4 to 51.9 kg·m-2 and body composition was estimated by computed tomographic images. Mitochondrial respiration was measured in situ in digitonin-permeabilized AT using high-resolution respirometry and a substrate/inhibitor titration approach. Protein levels of mitochondrial and lipid metabolism key elements were evaluated by Western blot. Maximal mitochondrial respiration correlated negatively with BMI (p < .01) and AT area (p < .001) regardless of the anatomical location. However, oxidative phosphorylation respiration was significantly higher in vAT (2.22 ± 0.15 pmol·sec-1·mg-1) than scAT (1.79 ± 0.17 pmol·sec-1·mg-1) (p < 0.001). In line with oxygraphy results, there were higher levels of mitochondrial respiratory chain complexes in low-BMI patients and vAT. Mitochondrial respiration decreased with increasing BMI in both scAT and vAT, without sex-associated difference. Mitochondrial respiration appeared to be higher in vAT than scAT. These differences were both qualitative and quantitative. Clinical Trials Registration IDNCT05417581.

This study aimed to investigate whether there are differences in cognitive impairment between medication-free patients with bipolar depression (BD) and major depressive disorder (MDD) and whether these differences are related to circulating cell-free mtDNA (ccf-mtDNA).

For this cross-sectional study, 76 outpatients with BD, 86 outpatients with MDD and 70 healthy controls (HCs) were enrolled. Sociodemographic and clinical data were collected. The Repeatable Battery for the Assessment of Neuropsychological Status (RBANS) and the Stroop Color-Word Test were used to assess cognitive function. Plasma ccf-mtDNA levels were measured via qPCR.

BD and MDD patients had similar scores for immediate memory, language, attention, delayed memory, the RBANS total score, Stroop color, Stroop word, and Stroop total score, which were significantly lower than the HCs. The visuospatial/constructive scores of the BD patients were significantly lower than those of the HCs (p < 0.001) and MDD patients (p = 0.008), but there was no difference between the HCs and MDD patients. The ccf-mtDNA levels in the BD and MDD patient groups were significantly higher than those in the HC group, and those in the MDD group were higher than those in the BD group (p = 0.016). Multiple stepwise regression analysis showed that ccf-mtDNA was negatively correlated with language in patients with depression (t = -2.11, p = 0.039).

There were differences in specific cognitive dimensions between patients with BD and MDD. Increased ccf-mtDNA levels were found in BD and MDD patients, suggesting ccf-mtDNA may be involved in the pathophysiology of MDD and BD.

Diabetic periodontitis is a common oral complication of diabetes characterized by progressive destruction of periodontal tissues. Recent evidence suggests that mitochondrial dysfunction plays a crucial role in the pathogenesis and progression of this condition. This review aims to systematically summarize the role and potential mechanisms of mitochondrial dysfunction in diabetic periodontitis. We first explore the relationship between diabetes and mitochondrial dysfunction, then analyze the specific manifestations of mitochondrial dysfunction in diabetic periodontitis, including morphological changes, energy metabolism disorders, increased oxidative stress, and enhanced apoptosis. We further delve into the connections between mitochondrial dysfunction and the pathogenic mechanisms of diabetic periodontitis, such as exacerbated inflammatory responses, decreased tissue repair capacity, and autophagy dysregulation. Finally, we discuss potential therapeutic targets based on mitochondrial function, including antioxidant strategies, mitochondria-targeted drugs, and autophagy regulators. We also propose future research directions, emphasizing the need for in-depth exploration of molecular mechanisms, development of new diagnostic markers and therapeutic strategies, and personalized treatment approaches. This review provides new insights into understanding the pathogenic mechanisms of diabetic periodontitis and offers a theoretical basis for developing targeted prevention and treatment strategies to improve oral health in diabetic patients.

The athlete's paradox states that intramyocellular triglyceride accumulation associates with insulin resistance in sedentary but not in endurance-trained humans. Underlying mechanisms and the role of muscle lipid distribution and composition on glucose metabolism remain unclear. We compared highly trained athletes (ATHL) with sedentary normal weight (LEAN) and overweight-to-obese (OVWE) male and female individuals. This observational study found that ATHL show higher insulin sensitivity, muscle mitochondrial content, and capacity, but lower activation of novel protein kinase C (nPKC) isoforms, despite higher diacylglycerol concentrations. Notably, sedentary but insulin sensitive OVWE feature lower plasma membrane-to-mitochondria sn-1,2-diacylglycerol ratios. In ATHL, calpain-2, which cleaves nPKC, negatively associates with PKCε activation and positively with insulin sensitivity along with higher GLUT4 and hexokinase II content. These findings contribute to explaining the athletes' paradox by demonstrating lower nPKC activation, increased calpain, and mitochondrial partitioning of bioactive diacylglycerols, the latter further identifying an obesity subtype with increased insulin sensitivity (NCT03314714).

Mitochondrial DNA copy number (mtDNA-CN) in human body fluids is widely used as a biomarker of mitochondrial dysfunction in common metabolic diseases. Here we describe protocols to measure cellular and/or cell free (cf)-mtDNA-CN in human peripheral blood and urine. Cellular mtDNA is located inside the mitochondria where it encodes key subunits of the respiratory complexes in mitochondria and is usually normalized with reference to the nuclear genome as the mitochondrial genome to nuclear genome ratio (Mt/N) in either whole blood, peripheral blood mononuclear cells (PBMCs), or whole urine. Cf -mtDNA is usually found outside of the mitochondria, often released following mitochondrial damage, can trigger inflammatory pathways, and is usually measured as mtDNA-CN per volume of the starting material. Here we describe how to (1) separate whole blood into PBMCs, plasma, and serum fractions and whole urine into urinary supernatant and pellet, (2) prepare DNA from each of these fractions, (3) prepare reference standards for absolute quantification, (4) carry out qPCR for either relative or absolute quantification from test samples, (5) analyze qPCR data, and (6) calculate the sample size to adequately power studies. The protocol presented here is suitable for high throughput use and can be modified to quantify mtDNA from other body fluids, human cells, and tissues.

Varicocele has been associated with reduced male fertility potential. Treatment modalities for varicocele improve semen parameters, yet more than 50% of cases remain infertile. Varicocele-induced heat and hypoxia stress may affect sperm mitochondrial functions, possibly leading to aberrant epigenetic modifications. This study includes 30 fertile men and 40 infertile men with clinical varicocele. The effect of varicocele treatment (antioxidant supplementation and or varicocelectomy) was evaluated after 3 months of treatment. Mitochondrial membrane potential (MMP) and intracellular reactive oxygen species (iROS) were measured by flow cytometry using JC-1 and DCFDA, respectively. mtDNA copy number and deletions were determined by PCR. DNA methylation was analysed by pyrosequencing. Present investigations suggest that infertile men with varicocele have abnormal semen parameters; significantly low MMP, high iROS, and high mtDNA copy number. Semen parameters were improved in a subset of men of both the treatment modalities; however, it was noted that varicocelectomy helped better in improving sperm parameters compared to antioxidant treatment. Both treatment modalities helped in reducing iROS and mtDNA copy number significantly; however, they were noneffective in improving MMP. Altered DNA methylation at mitochondria D loop and mitochondrial structure and function genes UQCRC2, MIC60, TOM22, and LETM1 (promoter region) were observed in varicocele group. The DNA methylation levels were restored after varicocele treatment; however, the restoration was not consistent at all CpG sites. Both the treatment modalities helped in restoring the altered DNA methylation levels of mitochondrial genes but the restoration is nonhomogeneous across the studied CpG sites.

Prolonged occupational noise exposure poses potential health risks, but its impact on mitochondrial DNA (mtDNA) damage and methylation patterns remains unclear.

We recruited 306 factory workers, using average binaural high-frequency hearing thresholds from pure-tone audiometry to assess noise exposure. MtDNA damage was evaluated through mitochondrial DNA copy number (mtDNAcn) and lesion rate, and mtDNA methylation changes were identified via pyrophosphate sequencing.

There was a reduction in MT-RNR1 methylation of 4.52% (95% CI: -7.43% to -1.62%) among workers with abnormal hearing, whereas changes in the D-loop region were not statistically significant (β = -2.06%, 95% CI: -4.44% to 0.31%). MtDNAcn showed a negative association with MT-RNR1 methylation (β = -0.95, 95% CI: -1.23 to -0.66), while no significant link was found with D-loop methylation (β = -0.05, 95% CI: -0.58 to 0.48). Mediation analysis indicated a significant increase in mtDNAcn by 10.75 units (95% CI: 3.00 to 21.26) in those with abnormal hearing, with MT-RNR1 methylation mediating 35.9% of this effect.

These findings suggest that occupational noise exposure may influence compensatory increases in mtDNA content through altered MT-RNR1 methylation.

Increasing studies have explored the correlation of mitochondrial DNA copy number (mtDNA-CN) abnormalities with cardiovascular disease (CVD) and all-cause mortality; however, their findings are contradictory. This systematic review and meta-analysis sought to quantitatively summarize current studies to elucidate the impact of mtDNA-CN on CVD outcomes and all-cause mortality. Relevant studies were searched for in PubMed, Embase and Web of Science databases, up to 23 October 2023. Summary relative risks (RRs) and 95% confidence intervals (CIs) were calculated with the random-effects model. In total, 22 articles were included in the systematic review, 13 of which were included in the meta-analysis of CVD outcomes and 8 in all-cause mortality. Compared to the highest mtDNA-CN level, the summary RR (95% CI) for the lowest mtDNA-CN level was 2.09 (95% CI 1.59-2.75) for CVD, 1.70 (95% CI 1.29-2.24) for coronary heart disease (CHD), 1.43 (95% CI 1.15-1.79) for heart failure (HF), 1.88 (95% CI 1.08-3.28) for stroke and 1.33 (95% CI 1.21-1.47) for all-cause mortality. Lower mtDNA-CN may increase the risk of CVD, including CHD, HF and stroke, as well as all-cause mortality. MtDNA-CN is a potential predictor of CVD and all-cause mortality.

Sodium butyrate can reduce inflammation, but it is not known if butyrate can improve mitochondrial dysfunction during sepsis. We tested butyrate to prevent or reverse lipopolysaccharide (LPS)-induced mitochondrial dysfunction in murine kidney and liver. C57BL/6 mice were grouped as control (n = 9), intraperitoneal (IP) LPS (n = 8), pretreatment with IP butyrate 600 (n = 3) or 1200 mg/kg (n = 8) followed 2 h later by LPS, posttreatment with IP butyrate 600 (n = 3) or 1200 mg/kg (n = 7) 1 h after LPS, or butyrate 1200 mg/kg only (n = 8). Kidney and liver tissue were collected at 24 h to measure mitochondrial respiration, electron transport system (ETS) complex activity and subunit expression, and content (citrate synthase [CS] activity and mtDNA/nDNA). Kidney mitochondrial respiration was decreased after LPS compared to controls. Pretreatment with butyrate 1200 mg/kg increased kidney OXPHOSCI+II, ETSCI+II, ETSCII, and CIV respiration compared to LPS; posttreatment did not achieve significant increases except for OXPHOSCI. Liver mitochondrial respiration exhibited a similar pattern as in kidney, but differences were not significant. ETS complex and CS activity did not differ between groups, but CI and CII subunit expression trended higher with butyrate in kidney. Changes in mtDNA/nDNA followed a similar pattern as respiration in kidney and liver with a decrease after LPS that was not present with butyrate pretreatment. These data show that butyrate can prevent-but not significantly reverse-the LPS-induced decrease in kidney mitochondrial respiration without a clear effect in liver. Mitochondrial protection was not attributable to changes in ETS complex activity but may reflect maintenance of ETS subunit expression.

This study presents a comprehensive analysis of mitochondrial DNA (mtDNA) variations in dogs diagnosed with primary and recurrent tumours, employing Oxford Nanopore Technologies (ONT) for sequencing. Our investigation focused on mtDNA extracted from blood and tumour tissues of three dogs, aiming to pinpoint polymorphisms, mutations, and heteroplasmy levels that could influence mitochondrial function in cancer pathogenesis. Notably, we observed the presence of mutations in the D-loop region, especially in the VNTR region, which may be crucial for mitochondrial replication, transcription, and genome stability, suggesting its potential role in cancer progression. The study is pioneering in its use of long-read sequencing to explore the mutational landscape of mtDNA in canine tumours, revealing that while the overall mutational load did not differ between primary and recurrent tumours, specific changes in m.16168A/G, m.16188G/A, and m.16298A/G are linked with tumour tissues. Interestingly, the heteroplasmy outside the D-loop region was not specific to tumour tissues and did not provoke any malignant damage in protein-coding sequences, which in turn may be a tolerant effect of the reactive oxygen species (ROS) cellular stress mechanism.

Sperm mitochondria are vital organelles for energy production and pre- and post-fertilization sperm functions. The potential influence of the age of the bull and season on the sperm-borne mitochondrial copy number and the transcription activity has not yet been investigated. Therefore, the expression patterns of all protein-coding mitochondrial genes were identified throughout the year along with mitochondrial copy numbers in young and old bulls' spermatozoa. For that, high-quality semen samples (n = 32) with more than 80% quality for the morphological parameters, from young (n = 4, aged 18-24 months old) and old (n = 4, aged 40-54 months old) Holstein bulls, were collected during the four seasons (n = 4 samples each animal/season). The DNA and RNA were isolated from sperm cells and subjected to the DNA copy number and expression analyses using qPCR. Furthermore, an in silico analysis using gene ontology online tools for the abundantly expressed genes was utilized. The data were statistically analyzed using Prism10 software. There was a significant reduction in the mitochondria copy number of young bulls' spermatozoa compared to their old counterparts during the summer (29 ± 3 vs. 51 ± 6, p < 0.001) and winter (27 ± 3 vs. 43 ± 7, p < 0.01) seasons. However, sperm-borne mitochondrial protein-coding genes were transcriptionally higher in young bulls throughout the year. Within the same group of bulls, unlike the old bulls, there was a significant (p < 0.05) induction in the transcription activity accompanied by a significant (p < 0.05) reduction in the mitochondrial copy numbers in the summer (29 ± 3) and winter (27 ± 3) compared to the spring (42 ± 9) and autumn (36 ± 5) seasons in young bulls. Additionally, the pathway enrichment of the top six expressed genes differed between age groups and seasons. In conclusion, under the same quality of semen, the early stages of age are associated with mitochondrial biogenesis and transcription activity dysregulation in a season-dependent manner.

Self-renewal of glioma stem cells (GSCs) is responsible for glioblastoma (GBM) therapy resistance and recurrence. Tumor necrosis factor α (TNFα) and TNF signaling pathway display an antitumor activity in preclinical models and in tumor patients. However, TNFα exhibits no significance for glioma clinical prognosis based on the Glioma Genome Atlas database. This study aimed to explore whether TNFα of tumor microenvironment maintains self-renewal of GSCs and promotes worse prognosis in glioma patients.

Spatial transcriptomics, immunoblotting, sphere formation assay, extreme limiting dilution, and gene expression analysis were used to determine the role of TNFα on GSC's self-renewal. Mass spectrometry, RNA-sequencing detection, bioinformatic analyses, qRT-RNA, immunofluorescence, immunohistochemistry, single-cell RNA sequencing, in vitro and in vivo models were used to uncover the mechanism of TNFα-induced GSC self-renewal.

A low level of TNFα displays a promoting effect on GSC self-renewal and worse glioma prognosis. Mechanistically, Vasorin (VASN) mediated TNFα-induced self-renewal by potentiating glycolysis. Lactate produced by glycolysis inhibits the TNFα secretion of tumor-associated macrophages (TAMs) and maintains TNFα at a low level.

TNFα-induced GSC self-renewal mediated by VASN provides a possible explanation for the failures of endogenous TNFα effect on GBM. A combination of targeting VASN and TNFα antitumor effect may be an effective approach for treating GBM.

This study delves into the relationship between environmental metal exposure and QT interval corrected for heart rate (QTc) prolongation, a critical marker for cardiovascular risk in the elderly. Although the interplay between metal exposure and QTc prolongation is important for predicting sudden cardiac death, it remains underexplored. Our analysis of 6478 participants from the Shenzhen aging-related disorder cohort involved measuring urinary concentrations of 22 trace metals and using mitochondrial DNA copy number (mtDNA-CN) as an indicator of mitochondrial dysfunction. Utilizing Bayesian kernel machine regression, and structural equation modeling, we assessed the effects of mixed trace metals on QTc prolongation. Our findings indicated a direct association between certain metals (Sb, Cu, Zn) and a 7 % increase in QTc prolongation risk, while Li, V, and Rb were associated with a 5 % reduction in risk. Elevated levels of V, Ti, and Cr corresponded to higher mtDNA-CN. Notably, restricted cubic splines revealed a U-shaped, nonlinear relationship between mtDNA-CN and QTc prolongation. After adjusting for metal exposure, an inverse correlation was observed between mtDNA-CN and QTc prolongation, suggesting mitochondrial dysfunction as a partial mediator.

Knee osteoarthritis (KOA) is characterized by mitochondrial damage and increased inflammation. Circulating cell-free mitochondrial DNA (ccf-mtDNA), which originates from damaged mitochondria, is an endogenous damage-associated molecular pattern (DAMPs) molecule that may trigger inflammation and is recognized as a potential biomarker for various diseases. In this study, we investigated the potential association between plasma ccf-mtDNA content and its use as a diagnostic biomarker in patients with KOA.

We collected plasma samples from patients with KOA and healthy controls (HC). Subsequently, quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect ccf-mtDNA content in the plasma samples. We used the Kellgren-Lawrence (K-L) classification criteria to classify patients with KOA into four grades: I-IV. Disease severity in patients with KOA was assessed using the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC). Next, Spearman analysis was performed to observe the correlation between ccf-mtDNA content and the K-L classification and WOMAC score. Logistic regression analysis was used to evaluate the relationship between ccf-mtDNA and KOA risk.

In total, we enrolled 60 patients with KOA and HC who were matched for age, sex, and body mass index (BMI). We found that plasma ccf-mtDNA contents were significantly higher in patients with KOA (median, 2.44; quartile range, 1.10-3.79) than in HC (median, 1.08; quartile range, 0.52-2.12) (P < 0.0001). Plasma ccf-mtDNA content sequentially increased following the KOA class I-IV group (P = 0.040) and positively correlated with the K-L classification (r = 0.369, P = 0.004) and WOMAC scores (r = 0.343, P = 0.007). The ccf-mtDNA content did not significantly differ between patients with bilateral and those with single KOA (P = 0.083). Patients with high levels of ccf-mtDNA had a significantly increased risk of KOA compared with those with low levels of ccf-mtDNA (odds ratio [OR], 4.15, 95% confidence interval [CI], 1.71-10.07; P = 0.002). Quartile analysis revealed a significant dose-dependent association (P trend < 0.001).

Our study's findings showed that plasma ccf-mtDNA was highly expressed in patients with KOA compared with HC. Furthermore, ccf-mtDNA content is significantly associated with the severity and risk of KOA. Therefore, its detection may provide insight into the prevention and treatment of KOA.

Endometriosis is a chronic gynecological inflammatory disease. The mitochondrial DNA copy number (mtDNA CN) and mitochondrial transcription factor A (TFAM) are known to contribute to human pathologies and cancer. Therefore, this study aims to reveal the association of mtDNA CN and TFAM+35G/C (rs1937) polymorphism with the risk of endometriosis in Egyptian females.

This case-control study involved 160 Egyptian females divided into two groups: 80 endometriosis cases and 80 controls. The mtDNA CN was quantified using a real-time quantitative PCR (qPCR), and the TFAM +35G/C SNP (rs1937) was genotyped using the TaqMan allelic discrimination assay technique.

The mtDNA CN was markedly decreased in endometriosis cases compared to controls (P < 0. 001). TFAM rs1937 genotypes and allele distributions were all in Hardy-Weinberg equilibrium. The GC genotype and the 'C' allele frequency (P = 0.015 and P = 0.017, respectively) were substantially greater in endometriosis cases.

Decreased mtDNA CN and the GC genotype of TFAM +35G/C polymorphism were significantly associated with the risk of endometriosis in Egyptian females.

Hepatocellular carcinoma (HCC) is a leading liver cancer that significantly impacts global life expectancy and remains challenging to treat due to often late diagnoses. Despite advances in treatment, the prognosis is still poor, especially in advanced stages. Studies have pointed out that investigations into the molecular mechanisms underlying HCC, including mitochondrial dysfunction and epigenetic regulators, are potentially important targets for diagnosis and therapy. Mitoepigenetics, or the epigenetic modifications of mitochondrial DNA, have drawn wide attention for their role in HCC progression. Besides, molecular biomarkers such as mitochondrial DNA alterations and non-coding RNAs showed early diagnosis and prognosis potential. Additionally, natural compounds like alkaloids, resveratrol, curcumin, and flavonoids show promise in HCC show promise in modulating mitochondrial and epigenetic pathways involved in cancer-related processes. This review discusses how mitochondrial dysfunction and epigenetic modifications, especially mitoepigenetics, influence HCC and delves into the potential of natural products as new adjuvant treatments against HCC.

Oxidative stress (OS) is believed to be a significant factor in the decline of semen quality, with mitochondrial DNA copy number (mtDNAcn) serving as a sensitive biomarker for both semen quality and mitochondrial dysfunction resulting from oxidative stress. While glutathione S-transferases (GSTs) are commonly known as 'antioxidant' enzymes, there is ongoing debate regarding the relationship between GST genotypes and semen quality. In a study involving 568 male volunteers from the outpatient department of Puyang Reproductive Medicine Center, sperm mtDNAcn, semen quality, and GSTM1/GSTT1 genotypes were analyzed to investigate the potential link between GSTM1/GSTT1 gene variations and semen quality, as well as the impact of GSTs gene variations on the connection between sperm mtDNAcn and semen quality. Adjusting for variables such as age, BMI, smoking, and alcohol consumption, it was found that mtDNAcn was significantly correlated with decreased sperm concentration and total sperm count (b = - 0.109, - 0.128, respectively; P = 0.002, 0.001, respectively). GSTM1 was associated with progressive motility (OR 0.390, 95% CI 0.218, 0.697), Straight line velocity (VSL) (OR = 0.606, 95% CI 0.385, 0.953), and Straightness (STR) (OR 0.604, 95% CI 0.367, 0.994), while GSTT1 was linked to progressive motility (OR 0.554, 95% CI 0.324, 0.944) and Beat crossover frequency (OR 0.624, 95% CI 0.397, 0.982). The GSTT1 was found to moderate the relationship between mtDNAcn and sperm motility parameters linearity (LIN), STR, and Wobble (WOB), with additive interaction effects observed between GSTT1 and mtDNAcn on LIN, STR, and WOB (P for interaction = 0.008, 0.034, 0.010, respectively). Overall, this study suggests that GSTT1 and GSTM1 gene variations may play a role in sperm motility, with GSTT1 potentially influencing the impact of oxidative stress on sperm motility.

Cancer cells are constantly evolving to adapt to environmental changes, particularly during exposure to drug treatment. In this work, we aimed to characterize genetic and epigenetic changes in mitochondrial DNA (mtDNA) that may increase the resistance of oral squamous cell carcinoma (OSCC) to cisplatin. We first derived drug-resistant cells from two human OSCC cell lines, namely SAS and H103, by continual cisplatin treatments for about 4 months. To determine mtDNA changes induced by cisplatin, we performed nanopore sequencing and quantitative polymerase chain reaction analysis of mtDNA extracted from the cells pre- and post-treatment. We also assessed the mitochondrial functions of the cells and their capacity to generate intracellular reactive oxygen species (ROS). We found that in the cisplatin-resistant cells derived from SAS, there was a reduction in mtDNA content and significant enrichment of a m.3910G > C mutation in the MT-ND1 gene. However, such changes were not detected in cisplatin-resistant H103 cells. The cisplatin treatment also altered methylation patterns in both SAS and H103 cells and decreased their sensitivity to ROS-induced cytotoxicity. We suggest that the sequence alterations and epigenetic changes in mtDNA and the reduction in mtDNA content could be key drivers of cisplatin resistance in OSCC. These mtDNA alterations may participate in cellular adaptation that serves as a response to adverse changes in the environment, particularly exposure to cytotoxic agents. Importantly, the observed mtDNA changes may be influenced by the distinct genetic landscapes of various cancer subtypes. Overall, this study reveals significant insights into cisplatin resistance driven by complex mtDNA dynamics, particularly in OSCC. This underscores the need for targeted therapies tailored to the genetic profiles of individual OSCC patients to improve disease prognosis.

Background: Mitochondria are considered the powerhouse of cells, and skeletal muscle cells are no exception. However, information regarding muscle mitochondria from different species is limited. Methods: Different muscles from cattle, pigs and chickens were analyzed for mitochondrial DNA (mtDNA), protein and oxygen consumption. Results: Bovine oxidative muscle mitochondria contain greater mtDNA (p < 0.05), protein (succinate dehydrogenase, SDHA, p < 0.01; citrate synthase, CS, p < 0.01; complex I, CI, p < 0.05), and oxygen consumption (p < 0.01) than their glycolytic counterpart. Likewise, porcine oxidative muscle contains greater mtDNA (p < 0.01), mitochondrial proteins (SDHA, p < 0.05; CS, p < 0.001; CI, p < 0.01) and oxidative phosphorylation capacity (OXPHOS, p < 0.05) in comparison to glycolytic muscle. However, avian oxidative skeletal muscle showed no differences in absolute mtDNA, SDHA, CI, complex II, lactate dehydrogenase, or glyceraldehyde 3 phosphate dehydrogenase compared to their glycolytic counterpart. Even so, avian mitochondria isolated from oxidative muscles had greater OXPHOS capacity (p < 0.05) than glycolytic muscle. Conclusions: These data show avian mitochondria function is independent of absolute mtDNA content and protein abundance, and argue that multiple levels of inquiry are warranted to determine the wholistic role of mitochondria in skeletal muscle.

Diabetic ketoacidosis (DKA) is a severe and potentially fatal acute complication in diabetic patients, commonly occurring in type 1 diabetes (T1D) but also seen in type 2 diabetes (T2D). The pathogenesis of DKA involves complex physiological processes that are not fully understood, especially the role of mitochondria. Mitochondria, known as the powerhouse of cells, plays a crucial role in oxidative phosphorylation and ATP production, which is vital in various metabolic diseases, including diabetes. However, the exact causal relationship between mitochondrial dysfunction and DKA remains unclear.

This study employed Mendelian randomization (MR) analysis and protein-protein interaction (PPI) networks to systematically explore the causal relationships between mitochondrial DNA copy number (mtDNA-CN) and specific mitochondrial proteins with DKA. We used bidirectional MR analysis and genome-wide association study (GWAS) data from openGWAS database to investigate the causal effects of mtDNA-CN and 64 mitochondrial-related proteins on DKA and its subtypes (T1DKA, T2DKA, unspecified-DKA).

The study revealed that increased mtDNA-CN significantly reduces the risk of DKA, whereas the effect of DKA on mtDNA-CN was not significant. Mitochondrial-related proteins such as MRPL32, MRPL33, COX5B, DNAJC19, and NDUFB8 showed a negative causal relationship with DKA, indicating their potential protective roles. Conversely, ATP5F1B and COX4I2 have a positive causal relationship with DKA, indicating that excessive ATP production in diabetic patients may be detrimental to health and increase the risk of severe complications such as DKA.

The results emphasize the necessity of protecting mitochondrial function in order to reduce the risk of DKA. The study offers novel perspectives on the molecular pathways involved in DKA, emphasizing the critical functions of mt-DNA and distinct proteins. These evidences not only enhance our comprehension of the implications of mitochondrial dysfunction in diabetes-related complications but also identify potential therapeutic targets for individualized treatment approaches, thereby making a substantial contribution to clinical care and public health initiatives.

Mercury (Hg) pro-oxidant role on biological systems and its biogeochemical cycle represent a serious threat due to its persistence in marine environment. As the mitochondrial genome is exposed to reactive oxygen species (ROS), the aim of the present study is the validation of the variation in the number of mitochondrial DNA copies (mtDNAcn) as biomarker of oxidative stress in aquatic environment. During summer 2021, three selected fish species (Mullus barbatus, Diplodus annularis and Pagellus erythrinus) were collected in Augusta Bay, one of the most Mediterranean contaminated areas remarkable by past Hg inputs, and in a control area, both in the south-east of Sicily. The relative mtDNAcn was evaluated by qPCR on specimens of each species from both sites, characterized respectively by higher and lower Hg bioaccumulation. M. barbatus and P. erythrinus collected in Augusta showed a dramatic mtDNAcn reduction compared to their control groups while D. annularis showed an incredible mtDNAcn rising suggesting a higher resilience of this species. These results align with the mitochondrial dynamics of fission and fusion triggered by environmental toxicants. In conclusion, we suggest the implementation of the mtDNAcn variation as a valid tool for the early warning stress-related impacts in aquatic system.

Mitochondria are vital for most cells' functions. Viruses hijack mitochondria machinery for misappropriation of energy supply or to bypass defense mechanisms. Many of these mitochondrial dysfunctions persist after recovery from treated or untreated viral infections, particularly when mitochondrial DNA is permanently damaged. Quantitative defects and structural rearrangements of mitochondrial DNA accumulate in post-mitotic tissues as recently reported long after SARS-CoV-2 or HIV infection, or following antiviral therapy. These observations are consistent with the "hit-and-run" concept proposed decades ago to explain viro-induced cell transformation and it could apply to delayed post-viral onsets of symptoms and advocate for complementary supportive care. Thus, according to this concept, following exposure to viruses or antiviral agents, mitochondrial damage could evolve into an autonomous clinical condition. It also establishes a pathogenic link between communicable and non-communicable chronic diseases.

Background/Objectives: Intra-amniotic infection (IAI) is a rare but serious condition with potential complications such as preterm labor and intrauterine fetal death. Diagnosing IAI is challenging due to varied clinical signs. Oxidative stress and mitochondrial dysfunction have been hypothesized to evolve around IAI. This study focused on measuring circulating mtDNA levels, a proposed biomarker for mitochondrial dysfunction, in maternal serum and placenta of women with confirmed IAI and healthy controls. Methods: 12 women with confirmed IAI (IAI group) were enrolled following premature preterm rupture of the membranes (PPROM) and compared to 21 healthy women (control group). Maternal blood was obtained two weeks pre-partum and peripartum; furthermore, postpartum placental blood was taken. In the IAI group, maternal blood was taken once weekly until delivery as well as peripartum, as was placental blood. Circulating cell-free mtDNA was quantified by real-time quantitative PCR. Results: Upon admission, in the IAI group, mean plasma mtDNA levels were 735.8 fg/μL compared to 134.0 fg/μL in the control group (p < 0.05). After delivery, in the IAI group, mean mtDNA levels in the placenta were 3010 fg/μL versus 652.4 fg/μL (p < 0.05). Conclusions: Circulating cell-free mtDNA could serve as a valuable biomarker for IAI prediction and diagnosis. Future research should establish reference values for sensitivity in predicting IAI.