Amount Of Mitochondrial DNA Research Articles

Deep sequencing shows that accumulation of potentially pathogenic mtDNA mutations rather than mtDNA copy numbers may be associated with early embryonic loss

PurposeTo explore the relationship between mitochondrial DNA quantity and heteroplasmy and early embryonic loss.MethodsA total of 150 villous samples from patients with spontaneous abortion (SA, n = 75) or induced abortion (IA, n = 75) were collected. qPCR and next-generation sequencing (NGS) were used to test mitochondrial DNA quantity and heteroplasmy. Missense mutations with a CADD score > 15 and heteroplasmy ≥ 70% were defined as potentially pathogenic mutations.ResultsWith respect to mitochondrial DNA copy numbers, there was no significant difference between the SA and IA groups (median (IQR), 566 (397–791) vs. 614 (457–739); P = 0.768) or between the euploid and aneuploid groups (median (IQR), 516 (345–730) vs. 599 (423–839); P = 0.107). mtDNA copy numbers were not associated with spontaneous abortion using logistic regression analysis (P = 0.196, 95% CI 1.000–1.001). In addition, more patients harbored possibly pathogenic mtDNA mutations in their chorionic villi in the SA group (70.7%, 53/75) compared with the IA group (54.7%, 41/75; P < 0.05). However, there was no statistical difference between the euploid (80%, 24/30) and aneuploid groups (64.4%, 29/45; p = 0.147).ConclusionEarly embryonic loss and the formation of aneuploidy were not related to mtDNA copy number. Patients with spontaneous abortion were more likely to have possibly pathogenic mutations in their mtDNA, and this may assist in purifying pathogenic mtDNA. However, whether the accumulation of these potentially morbific mtDNA mutations caused early embryonic loss requires further investigation.

Value of mtDNA and ctDNA in patients with hepatocellular carcinoma

Objective The aim was to evaluate the diagnostic value of mitochondrial DNA and circulating tumor DNA in patients with hepatocellular carcinoma (HCC). Background HCC is one of the most common malignant tumors worldwide. Not all patients with HCC are diagnosed using α-fetoprotein alone. Mitochondrial DNA and circulating tumor DNA have been extensively studied over the past few decades and have a high sensitivity and specificity in the detection of HCC. Patients and methods Blood samples were obtained from 150 individuals, comprising 50 patients with HCC and 100 apparently healthy participants as a control group. The blood samples were collected in the time period from August 2013 to September 2016. All patients were subjected to routine laboratory investigations and quantification of mitochondrial DNA and circulating tumor DNA by real-time PCR. Results There was a significant increase in the levels of mitochondrial DNA and circulating tumor DNA in patients with HCC in comparison with the healthy control group. By using receiver operator characteristic curve, sensitivity and specificity of mtDNA for HCC group were 70% and 75%, respectively with a cutoff of 480 GE/ml, whereas sensitivity and specificity of ctDNA for HCC group was 81% and 88%, respectively, with a cutoff of 348 GE/ml. Conclusion Increase sensitivity and specificity of circulating tumor DNA and mitochondrial DNA in HCC make us consider them as potential tumor markers, and analysis of circulating tumor DNA and mitochondrial DNA with other tumor markers could improve the diagnostic sensitivity for HCC.

1947-P: RE1-Silencing Transcription Factor (REST) Regulates Mitochondrial Activity in White Adipocytes

RE1-silencing transcription factor (REST) is a transcriptional factor which negatively regulates the expression of numerous neuronal genes. In this study, model mice lacking REST in adipocytes were generated to investigate the role of REST in adipocytes. This was achieved by mating Rest2lox/2lox mice with Adipoq-Cre mice, and the resultant Adipoq-Cre/Rest2lox/2lox mice were compared with Adipoq-Cre mice. Adipoq-Cre/Rest2lox/2lox mice exceeding 10 weeks of age showed increased body weight, fat mass, and adipocyte size; however no difference was observed in food intake. Glucose tolerance deteriorated under a high fat diet, but not under normal diets in Adipoq-Cre/Rest2lox/2lox mice. Oxygen consumption decreased in Adipoq-Cre/Rest2lox/2lox mice. The characteristics of gene expression in epididymal fat, i.e., white adipose tissue (WAT) and brown adipose tissue (BAT), isolated from Adipoq-Cre/Rest2lox/2lox mice, were compared by microarray with those from Adipoq-Cre mice. Although expression of numerous neuronal genes increased in Adipoq-Cre/Rest2lox/2lox mice, there were no differences in adipocyte-specific genes. Gene analyses utilizing a cluster one program detected a significant decrease in the mitochondrial translation gene group in WAT, but not BAT. There was no difference in the amount of mitochondrial DNA, whereas the expression of cytochrome C protein was reduced in WAT but not BAT, which was isolated from Adipoq-Cre/Rest2lox/2lox mice. These results indicate that obesity in Adipoq-Cre/Rest2lox/2lox mice is due to a mitochondrial dysfunction in WAT. It is also suggested that REST might regulate the mitochondrial function in WAT. Disclosure M. Fuwa: None. K. Kajita: None. K. Taguchi: None. T. Ikeda: None. T. Ishizuka: None. H. Morita: None.

Mangiferin induces the expression of a thermogenic signature via AMPK signaling during brown-adipocyte differentiation

Mangiferin (MF) from Mangifera indica has been serendipitously found to ameliorate obesity and is used as an antioxidant, anti-inflammatory, antimicrobial, and anticancer agent. Nonetheless, the mechanism of MF-induced brown-adipose-tissue activation has not been studied. Therefore, we investigated the effect of MF on thermogenic features during brown-adipocyte differentiation. Treatment with MF improved the expression of a brown-fat signature and of mitochondrial-mass–related genes, thus resulting in UCP1 induction. MF also raised the expression of other thermogenic regulators, including peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1α), PR domain–containing protein 16 (PRDM16), and peroxisome proliferator-activated receptors alpha and gamma (PPAR-α and -γ). MF promoted mitochondrial biogenesis, judging by increased expression of cell death–inducing DNA fragmentation factor α–like effector A (CIDEA), mitochondrial transcription factor A (TFAM), iodothyronine deiodinase 2 (DIO2), cytochrome c oxidase subunit 7A (COX7A), cyclooxygenase 2 (COX2), sirtuin 1 (SIRT1), and nuclear respiratory factor 1 (NRF1). MF treatment increased the mitochondrial DNA amount and improved mitochondrial respiratory function by increasing the oxygen consumption rate during brown-adipocyte differentiation. A gene knockdown assay involving small interfering RNA and competitive inhibition with dorsomorphin revealed that MF may promote thermogenesis in brown preadipocytes via activation of AMPK signaling. Collectively, our findings suggest that MF may be a novel pharmaceutical agent that can ameliorate obesity via activation of brown adipose tissue.

Quantification of Neurospora crassa mitochondrial DNA using quantitative real-time PCR.

The filamentous fungus Neurospora crassa is a popular model organism used in a wide range of biochemical and genetic studies and vastly used in mitochondrial research. Despite the relevance of mitochondria in N. crassa biology, no method for quantification of mitochondrial DNA (mtDNA) is currently available. Quantitative real-time PCR (qPCR) is a powerful tool, with a wide range of applications, and has been used for the quantification of nucleic acids in humans and a few other species. Here we present a new qPCR assay for relative quantification of N. crassa mtDNA. Three sets of qPCR primers targeting different regions of the mitochondrial genome were tested for mtDNA quantification. The qPCR was successfully validated in N. crassa strains from different geographical locations, representing the vast genetic diversity of this species, and knockout mutant strains. Moreover the assay proved to be efficient in templates with varied amounts of mitochondria, obtained through different DNA extraction methods. The qPCR performed well in all tested samples revealing a higher amount of mtDNA than nuclear DNA in all cases. This technique will facilitate the characterization of mtDNA of N. crassa in future studies and can be used as a tool to validate methods of mitochondria isolation. SIGNIFICANCE AND IMPACT OF THE STUDY: The standardization of quantitative real-time PCR (qPCR) techniques is essential to enable and facilitate future comparisons. Neurospora crassa is a model organism with a lot of potential in different fields of study. Here we use N. crassa to develop and establish an assay to quantify mitochondrial DNA using qPCR. We tested strains with different geographical background and our data demonstrated the usefulness of this assay to quantify mitochondrial DNA in N. crassa. This technique can be useful in a wide variety of applications and in different types of studies.

Role of Mitochondrial Unfolding Protein Response in Reproduction and Aging with Utilization of Autologous Mitochondrial Injections in Mitochondrial Diseases along with in Aging Oocytes

There is a very essential role that mitochondria play in controlling energy metabolism with respect to oocytes maturation as well as Preimplantation embryos in which both quantity as well as morphology of mitochondria along with mitochondrial DNA amount get very closely controlled. Lot of mouse experimental kinds having impaired mitochondrial function cause infertility that validates the crucial part played by mitochondria in female reproduction. On finding abnormalities of mitochondrial action both cells as well as organisms utilize methods that are aimed at recuperating or help in get the mitochondria rescued and delete mitochondria which can’t in anyway be salvaged. Of these methods Mitochondrial Unfolded Protein Response (mtUPR) has been associated with ageing avoidance recently, since mitochondrial aids in age associated collection of injured proteins, decreased oxidative phosphorylation as well as reactive oxygen species (ROS) synthesis. Importance of these lies in female reproduction since if mtUPR genes like Clpp are removed following specific targeting it causes female infertility, causing problems in maturing of oocyte along with 2 cell embryo generation and inability to develop blastocyst. Additionally if CLPP is missing it results in exaggerated follicular removal with a phenotype mimic long premature ovarian failure. More studies will give understanding of both physiological and pathological regulation of oocyte as well as initial embryonic mitochondrial action that workers are trying to use in generating therapies that are innovative in aging oocytes and in women presenting with mitochondrial inheritent abnormalities and thus treating female infertility with elderly females.

Молекулярные основы рака легких, вызванных радоном

Lung cancer is the most common form of malignant neoplasia and is one of the leading causes of cancer death. According to the World Health Organization (WHO), radon is the second leading cause of lung cancer after smoking. Exposure to radon leads to the death of cells of the bronchial epithelium of the lungs, which is accompanied by the release of a large amount of mitochondrial DNA. Free-circulating mitochondrial DNA (fc mtDNA) can serve as a kind of trigger for the transduction of pro-inflammatory signals in animal cells. The production of fc mtDNA can occur as a result of apoptosis processes. Any ionizing radiation, including alpha radiation, to which radon belongs, promotes the promotion of cell death both as a result of direct mechanical action on the cell due to reactive oxygen species (ROS), and due to the appearance of unrepairable damage to the genetic material. In addition, radon can alter the expression profile of miRNAs, which in turn can activate apoptosis and inflammation. Free-circulating mitochondrial DNA by activating the Nf-kb signaling pathway mediates the synthesis of proinflammatory cytokines, and as a result of this activation, inflammation develops, which is one of the key factors in lung cancer oncogenesis. This short review analyzes the molecular mechanisms of lung cancer associated with the carcinogenic effect of radon based on the analysis of the fc mtDNA level, the profile of the tissue-specific microRNA fraction, the level and spectrum of pro- and anti-inflammatory cytokines

Heterogeneity of the nucleic acid repertoire of plasma extracellular vesicles demonstrated using high-sensitivity fluorescence-activated sorting

ABSTRACT The aim of this study was to investigate cell source-dependent nucleic acids repertoire of diverse subpopulations of plasma extracellular vesicles (EVs). Blood plasma from nine healthy volunteers was used for the analysis. Samples of EVs were obtained by differential centrifugation of plasma. The application of high-sensitivity fluorescence-activated vesicles sorting (hs-FAVS) using fluorophore-conjugated anti-CD41-FITC (Fluorescein isothiocyanate) and anti-CD235a-PE antibodies allowed the isolation of three subpopulations of EVs, namely CD41+ CD235a-, CD41-CD235a+ and CD41-CD235a dim. The high purity (>97%) of the sorted subpopulations was verified by high-sensitivity flow cytometry. Presence of nanosized objects in sorted samples was confirmed by combination of low-voltage scanning electron microscopy and dynamic light scattering. The amount of material in sorted samples was enough to perform Quantitative polymerase chain reaction (qPCR)-based nucleic acid quantification. The most prominent differences in the nucleic acid repertoire were noted between CD41+ CD235- vs. CD41-CD235a+ vesicles: the former contained significantly (p = 0.004) higher amount of mitochondrial DNA, and platelet enriched miR-21-5p (4-fold), miR-223-3p (38-fold) and miR-199a-3p (187-fold), but lower amount of erythrocyte enriched miR-451a (90-fold). CD41-CD235a+ and CD41-CD235a dim vesicles differed in levels of miR-451a (p = 0.016) and miR-21-5p (p = 0.031). Nuclear DNA was below the limit of detection in all EV subpopulations. The hs-FCM-based determination of the number of sorted EVs allowed the calculation of per single-event miRNA concentrations. It was demonstrated that the most abundant marker in CD41+ CD235a- subpopulation was miR-223-3p, reaching 38.2 molecules per event. In the CD41-CD235+ subpopulation, the most abundant marker was miR-451a, reaching 24.7 molecules per event. Taken together, our findings indicate that erythrocyte- and platelet-derived EVs carry different repertoires of nucleic acids, which were similar to the composition of their cellular sources.

The mitochondrial dna quantification in сumulus cells and implantation potential of embryos

The mitochondrial dna quantification in сumulus cells and implantation potential of embryos

The effect of cysteine oxidation on DJ-1 cytoprotective function in human alveolar type II cells

DJ-1 is a multifunctional protein with cytoprotective functions. It is localized in the cytoplasm, nucleus, and mitochondria. The conserved cysteine residue at position 106 (Cys106) within DJ-1 serves as a sensor of redox state and can be oxidized to both the sulfinate (-SO2−) and sulfonate (-SO3−) forms. DJ-1 with Cys106-SO2− has cytoprotective activity but high levels of reactive oxygen species can induce its overoxidation to Cys106-SO3−. We found increased oxidative stress in alveolar type II (ATII) cells isolated from emphysema patients as determined by 4-HNE expression. DJ-1 with Cys106-SO3− was detected in these cells by mass spectrometry analysis. Moreover, ubiquitination of Cys106-SO3− DJ-1 was identified, which suggests that this oxidized isoform is targeted for proteasomal destruction. Furthermore, we performed controlled oxidation using H2O2 in A549 cells with DJ-1 knockout generated using CRISPR-Cas9 strategy. Lack of DJ-1 sensitized cells to apoptosis induced by H2O2 as detected using Annexin V and propidium iodide by flow cytometry analysis. This treatment also decreased both mitochondrial DNA amount and mitochondrial ND1 (NADH dehydrogenase 1, subunit 1) gene expression, as well as increased mitochondrial DNA damage. Consistent with the decreased cytoprotective function of overoxidized DJ-1, recombinant Cys106-SO3− DJ-1 exhibited a loss of its thermal unfolding transition, mild diminution of secondary structure in CD spectroscopy, and an increase in picosecond–nanosecond timescale dynamics as determined using NMR. Altogether, our data indicate that very high oxidative stress in ATII cells in emphysema patients induces DJ-1 overoxidation to the Cys106-SO3− form, leading to increased protein flexibility and loss of its cytoprotective function, which may contribute to this disease pathogenesis.

Adjusted mitochondrial DNA quantification in human embryos may not be applicable as a biomarker of implantation potential.

To evaluate the feasibility of adjusted mitochondrial DNA quantification in human embryos as a biomarker for implantation potential. Double-blind, observational, prospective analysis of an Asian population in a single university-affiliated in vitro fertilization center. A total of 1617 embryos derived from 380 infertile couples were collected. The DNA from blastomere biopsy (n = 99) or trophectoderm biopsy (n = 1518) were analyzed with next-generation sequencing. The adjusted mtDNA quantification followed a non-normal distribution in both types of the embryos. When stratified by ploidy status, the adjusted mtDNA quantification was significantly higher in aneuploid trophectoderm than in euploid cells, but not in blastomeres. The adjusted mtDNA quantification of embryos showed significant but very weak positive correlation in total trophectoderm cells with maternal age (Spearman's correlation, r = 0.095, p = 0.0028) but neither in blastomeres nor stratified by ploidy status. The median adjusted mtDNA quantification was also significantly higher in aneuploid blastocysts than in euploid ones while corrected with embryo morphology. Viable embryos did not contain significantly different quantities of adjusted mtDNA compared with nonviable embryos (implanted n = 103, non-implanted n = 164; median 0.00097 vs. 0.00088, p = 0.21) in 267 transferred blastocysts. Quantification of adjusted mitochondria DNA in human embryos was significantly lower in euploid blastocysts than in aneuploid blastocysts. However, no statistically significant differences regarding implantation outcome were evident. To our best knowledge, this study provides the largest scale and the first correlation data between mitochondria copy number and human embryo implantation potential in Asians.

Targeting reduced mitochondrial DNA quantity as a therapeutic approach in pediatric high-grade gliomas.

BackgroundDespite increased understanding of the genetic events underlying pediatric high-grade gliomas (pHGGs), therapeutic progress is static, with poor understanding of nongenomic drivers. We therefore investigated the role of alterations in mitochondrial function and developed an effective combination therapy against pHGGs.MethodsMitochondrial DNA (mtDNA) copy number was measured in a cohort of 60 pHGGs. The implication of mtDNA alteration in pHGG tumorigenesis was studied and followed by an efficacy investigation using patient-derived cultures and orthotopic xenografts.ResultsAverage mtDNA content was significantly lower in tumors versus normal brains. Decreasing mtDNA copy number in normal human astrocytes led to a markedly increased tumorigenicity in vivo. Depletion of mtDNA in pHGG cells promoted cell migration and invasion and therapeutic resistance. Shifting glucose metabolism from glycolysis to mitochondrial oxidation with the adenosine monophosphate–activated protein kinase activator AICAR (5-aminoimidazole-4-carboxamide ribonucleotide) or the pyruvate dehydrogenase kinase inhibitor dichloroacetate (DCA) significantly inhibited pHGG viability. Using DCA to shift glucose metabolism to mitochondrial oxidation and then metformin to simultaneously target mitochondrial function disrupted energy homeostasis of tumor cells, increasing DNA damage and apoptosis. The triple combination with radiation therapy, DCA and metformin led to a more potent therapeutic effect in vitro and in vivo.ConclusionsOur results suggest metabolic alterations as an onco-requisite factor of pHGG tumorigenesis. Targeting reduced mtDNA quantity represents a promising therapeutic strategy for pHGG.

Sirtuin 1 Regulates Mitochondrial Biogenesis and Provides an Endogenous Neuroprotective Mechanism Against Seizure-Induced Neuronal Cell Death in the Hippocampus Following Status Epilepticus.

Status epilepticus may decrease mitochondrial biogenesis, resulting in neuronal cell death occurring in the hippocampus. Sirtuin 1 (SIRT1) functionally interacts with peroxisome proliferator-activated receptors and γ coactivator 1α (PGC-1α), which play a crucial role in the regulation of mitochondrial biogenesis. In Sprague-Dawley rats, kainic acid was microinjected unilaterally into the hippocampal CA3 subfield to induce bilateral seizure activity. SIRT1, PGC-1α, and other key proteins involving mitochondrial biogenesis and the amount of mitochondrial DNA were investigated. SIRT1 antisense oligodeoxynucleotide was used to evaluate the relationship between SIRT1 and mitochondrial biogenesis, as well as the mitochondrial function, oxidative stress, and neuronal cell survival. Increased SIRT1, PGC-1α, and mitochondrial biogenesis machinery were found in the hippocampus following experimental status epilepticus. Downregulation of SIRT1 decreased PGC-1α expression and mitochondrial biogenesis machinery, increased Complex I dysfunction, augmented the level of oxidized proteins, raised activated caspase-3 expression, and promoted neuronal cell damage in the hippocampus. The results suggest that the SIRT1 signaling pathway may play a pivotal role in mitochondrial biogenesis, and could be considered an endogenous neuroprotective mechanism counteracting seizure-induced neuronal cell damage following status epilepticus.

PGC1α repression in IPF fibroblasts drives a pathologic metabolic, secretory and fibrogenic state

Idiopathic pulmonary fibrosis (IPF) is a fatal ageing-related disease linked to mitochondrial dysfunction. The present study aimed to determine whether peroxisome proliferator activated receptor gamma co-activator 1-alpha (PPARGC1A, encoding PGC1α),...

Endovascular hypothermia improves post-resuscitation myocardial dysfunction by increasing mitochondrial biogenesis in a pig model of cardiac arrest

The aim of the study was to investigate the effects of endovascular hypothermia on mitochondrial biogenesis in a pig model of prolonged cardiac arrest (CA). Ventricular fibrillation was electrically induced, and animals were left untreated for 10 min; then after 6min of cardiopulmonary resuscitation (CPR), defibrillation was attempted. 25 animals that were successfully resuscitated were randomized into three groups: Sham group (SG, 5, no CA), normal temperature group (NTG, 5 for 12 h observation and 5 for 24 h observation), and endovascular hypothermia group (EHG, 5 for 12 h observation and 5 for 24 h observation). The core temperatures (Tc) in the EHG were maintained at 34 ± 0.5 °C for 6 h by an endovascular hypothermia device (Coolgard 3000), then actively increased at the speed of 0.5 °C per hour during the next 6 h to achieve a normal body temperature, while Tc were maintained at 37.5 ± 0.5 °C in the NTG. Cardiac and mitochondrial functions, the quantification of myocardial mitochondrial DNA (mtDNA), peroxisome proliferator-activated receptor coactivator-1α (PGC-1α), nuclear respiratory factor (NRF)-1, and NRF-2 were examined. Results showed that myocardial and mitochondrial injury and dysfunction increased significantly at 12 h and 24 h after CA. Endovascular hypothermia offered a method to rapidly achieve the target temperature and provide stable target temperature management (TTM). Cardiac outcomes were improved and myocardial injuries were alleviated with endovascular hypothermia. Compared with NTG, endovascular hypothermia significantly increased mitochondrial activity and biogenesis by amplifying mitochondrial biogenesis factors’ expressions, including PGC-1α, NRF-1, and NRF-2. In conclusions, endovascular hypothermia after CA alleviated myocardial and mitochondrial dysfunction, and was associated with increasing mitochondrial biogenesis.

Ketogenic treatment reduces the percentage of a LHON heteroplasmic mutation and increases mtDNA amount of a LHON homoplasmic mutation

BackgroundThe vision loss in Leber hereditary optic neuropathy patients is due to mitochondrial DNA mutations. No treatment has shown a clear-cut benefit on a clinically meaningful end-point. However, clinical evidences suggest two therapeutic approaches: the reduction of the mutation load in heteroplasmic patients or the elevation of mitochondrial DNA amount in homoplasmic patients.ResultsHere we show that ketogenic treatment, in cybrid cell lines, reduces the percentage of the m.13094 T > C heteroplasmic mutation and also increases the mitochondrial DNA levels of the m.11778G > A mitochondrial genotype.ConclusionsThese results suggest that ketogenic diet could be a therapeutic strategy for Leber hereditary optic neuropathy.

SD quants—Sensitive detection tetraplex-system for nuclear and mitochondrial DNA quantification and degradation inference

Measuring the quantity of DNA present in a forensic sample is relevant in a number of ways. First, it informs the analyst about the general DNA content to adjust the volume of DNA extract used for the genotyping assay to the optimal conditions (when possible). Second, quantification values can serve as plausibility checks for the performance of the DNA extraction method used as extraction positive and negative controls demand expected values. Third and relevant to highly compromised specimens, DNA quantification can inform about the degradation state of the DNA extracted from the unknown biological sample and aid the choice of downstream genotyping assays. While there are different, commercial products for the quantification of nuclear DNA available, commercial mitochondrial DNA (mtDNA) quantification systems are rare. Even more so, the simultaneous quantification of nuclear and mtDNA that is of relevance in highly degraded forensic specimens has rarely been described. We present here a novel real-time qPCR based tetraplex system termed SD quants that targets two different-sized mtDNA and a nuclear DNA region and includes an internal positive control to monitor potential inhibition. SD quants was compared to other existing quantification systems and subjected to analysis of severely degraded DNA present in ancient DNA and aged forensic specimens. This study complies with the MIQE (Bustin et al., 2009) guidelines (when applicable).

Non-invasive mitochondrial DNA quantification on Day 3 predicts blastocyst development: a prospective, blinded, multi-centric study.

In ART, embryo quality evaluation is routinely based on morphological criteria. We previously demonstrated that the mitochondrial DNA (mtDNA)/genomic DNA (gDNA) ratio in culture medium was significantly associated with embryo quality and viability potential. The purpose of this prospective, blinded, multi-centric study was to validate the use of mtDNA/gDNA ratio in Day 3 spent medium as a predictor of human embryo developmental competence. The mtDNA/gDNA ratio was assessed in Day 3 culture media (n=484) of embryos from 143 patients by quantitative PCR. A mixed effect logistic regression model was applied. We found that mtDNA/gDNA ratio in Day 3 culture medium combined with embryo morphology improves the prediction upon blastulation compared to morphology alone (P< 0.0001), independent of patient and cycle characteristics. With regard to routine use in clinics, we evaluated the ability of the novel, combined grading score to improve selection of developmentally competent embryos of a single cohort. Including embryos from 44 patients, the sensibility and specificity of the scoring system based on Day 3 morphological stage were 92% and 13%, respectively. Integration with the culture medium mtDNA/gDNA ratio increased the performance of the method (sensibility: 95%; specificity: 65%). The results of this study suggest the possibility of carrying out a non-invasive evaluation of embryonic mtDNA content through the culture medium. When combined with embryo morphology, it has the potential to help embryologists rank embryos and choose which embryo(s) has the greater development potential, and thus should be transferred on Day 3, among sibling embryos with the same morphological grade.

Characterization of long living yeast deletion mutants that lack mitochondrial metabolism genes DSS1, PPA2 and AFG3

Molecular mechanisms of aging and longevity are still mostly unknown. Mitochondria play central roles in cellular metabolism and aging. In this study, we identified three deletion mutants of mitochondrial metabolism genes (ppa2∆, dss1∆, and afg3∆) that live longer than wild-type cells. These long-lived cells harbored significantly decreased amount of mitochondrial DNA (mtDNA) and reactive oxygen species (ROS). Compared to the serpentine nature of wild-type mitochondria, a different dynamics and distribution pattern of mitochondria were observed in the mutants. Both young and old long-lived cells produced relatively low but adequate levels of ATP for cellular activities. The status of the retrograde signaling was checked by expression of CIT2 gene and found activated in long-lived mutants. The mutant cells were also profiled for their gene expression patterns, and genes that were differentially regulated were determined. All long-lived cells comprised similar pleiotropic phenotype regarding mitochondrial dynamics and functions. Thus, this study suggests that DSS1, PPA2, and AFG3 genes modulate the lifespan by altering the mitochondrial morphology and functions.

Births from embryos with highly elevated levels of mitochondrial DNA.

Conflicting data exist on the utility of quantification of mitochondrial DNA (mtDNA) levels as a predictor of blastocyst implantation in the IVF clinic. The current study determined whether blastocysts with highly elevated mtDNA levels could result in healthy pregnancies and births, and whether mitochondrial functional output might be a readout of cell stress in the embryo. mtDNA levels were determined in 109 blastocysts used in clinical transfers into 100 women, noting their clinical outcomes. In a separate set of embryos, mitochondrial function was quantified in a model of embryo stress, aneuploidy. Measurement of mtDNA levels made use of surplus material from the process of preimplantation genetic testing for aneuploidies, and followed recently proposed unifying guidelines for mtDNA quantification. Unusually high mtDNA levels did not preclude blastocyst implantation and healthy births. An analysis of 109 blastocysts showed no significant difference between mtDNA levels in implanted (n = 55) versus non-implanted (n = 54) blastocysts. No obvious differences in the degree of mitochondrial functional output were detected in a model of embryo stress. Measurement of mtDNA copy number might not provide any advantage in embryo prioritization and could lead to a deselection of blastocysts that would result in healthy pregnancies and births. Furthermore, the quantification of mitochondrial functional output in a model of cellular stress might suggest that mitochondria are not clear targets for biomarker identification as it relates to blastocyst viability. Any suggested link between mtDNA levels, mitochondria or their output with blastocyst transfer outcome requires further validation.