The mitochondrial DNA 4977‐bp deletion and copy number alteration in Han Chinese samples with uterine fibroids

Uterine fibroids (UFs) are the most common benign neoplasms, but their pathogenesis is not completely understood. Thus far, alterations in the mitochondrial DNA (mtDNA) content and the mtDNA 4977‐bp deletion level in UFs, as well as the corresponding nontumorous tissue, have remained elusive. To test whether large mtDNA deletions and mtDNA content are involved in the pathogenesis of UFs, a total of 309 UF tissues and 28 paired adjacent myometrium from 270 UF patients were enrolled for the analysis of large mtDNA deletions and mtDNA content through the use of nested PCR and qPCR techniques, respectively. In our samples, a 4977‐bp deletion was identified: 36 out of 309 UF tissues (11.56%) and 15 out of 28 (53.57%) paired adjacent myometrium were detected to harbor the 4977‐bp deletion. In addition, a novel 4838‐bp mtDNA deletion was identified in three UF tissues, and other different sizes of deleted fragments (4910, 4926, 5135‐bp) were also found in UFs for the first time. Furthermore, older age was significantly associated with an mtDNA large deletion in the paired adjacent myometrium. We also found that increased mtDNA content and higher expression of ND1 occurred in solitary fibroids compared to adjacent myometrium. In conclusion, we identified a lower frequency of mtDNA large deletions and some novel large deletion in UFs for the first time. Furthermore, there was a general increase of mtDNA copy number during solitary UF development. Although the definite mechanism by which mtDNA was altered is supposed to be further confirmed, it will be helpful for further studies on the pathological mechanism of UFs.     

Mitochondrial DNA 4977bp Deletion Mutation in Peripheral Blood Reflects Atrial Remodeling in Patients with Non-Valvular Atrial Fibrillation

Purpose

Recently, mitochondrial DNA 4977bp deletion (mtDNA4977^-mut), a somatic mutation related to oxidative stress, has been shown to be associated with atrial fibrillation (AF). We hypothesized that patient age, as well as electroanatomical characteristics of fibrillating left atrial (LA), vary depending on the presence of mtDNA4977^-mut in peripheral blood among patients with non-valvular AF.

Materials and Methods

Analyzing clinical and electroanatomical characteristics, we investigated the presence of the mtDNA4977^-mut in peripheral blood of 212 patients (51.1±13.2 years old, 83.5% male) undergoing catheter ablation for non-valvular AF, as well as 212 age-matched control subjects.

Results

The overall frequency of peripheral blood mtDNA4977^-mut in patients with AF and controls was not significantly different (24.5% vs. 19.3%, p=0.197). When the AF patient group was stratified according to age, mtDNA4977^-mut was more common (47.4% vs. 20.0%, p=0.019) in AF patients older than 65 years than their age-matched controls. Among AF patients, those with mtDNA4977^-mut were older (58.1±11.9 years old vs. 48.8±11.9 years old, p<0.001). AF patients positive for the mtDNA mutation had greater LA dimension (p=0.014), higher mitral inflow peak velocity (E)/diastolic mitral annular velocity (Em) ratio (p<0.001), as well as lower endocardial voltage (p=0.035), and slower conduction velocity (p=0.048) in the posterior LA than those without the mutation. In multivariate analysis, E/Em ratio was found to be significantly associated with the presence of mtDNA4977^-mut in peripheral blood.

Conclusion

mtDNA4977^-mut, an age-related somatic mutation detected in the peripheral blood, is associated with advanced age and electro-anatomical remodeling of the atrium in non-valvular AF.     

Mitochondrial DNA deletions in rhesus macaque oocytes and embryos

Mitochondria are the most abundant organelles in mammalian oocytes and early embryos. Mitochondrial DNA (mtDNA) mutations, including the common deletion, have been found in skeletal muscle fibres from aged rhesus macaques. The specific aims of this study were to determine whether the mitochondrial common deletion is present in rhesus oocytes after hormonal stimulation and in embryos generated by in vitro production, or whether this deletion is already present in the immature oocyte. Using a nested primer PCR strategy, we found a significant increase in the proportion of mtDNA deletions in stimulated oocytes and embryos from rhesus macaques, compared with mtDNA deletions in immature, unstimulated oocytes derived from necropsied ovaries of age-matched monkeys. The common deletion is larger in the rhesus (5704 bp) than in humans (4977 bp). Accumulation of mtDNA deletions in oocytes may contribute to mitochondrial dysfunction and impaired ATP production. We propose the rhesus to be an excellent model to assess the quality of gametes and embryos and their developmental competence in primates, including humans.     

Effects of Micronucleus Frequencies and Mitochondrial DNA Copy Numbers among Benzene-Exposed Workers in China

To provide a more comprehensive understanding of genotoxic effects from benzene exposure, its effects on induction of mitochondrial DNA copy number (MtDNAcn) and of micronucleus (MN) were investigated using peripheral blood from workers in China. Changes in mtDNAcn and MN were determined using quantitative real-time polymerase chain reaction (PCR) and cytokinesis-block micronucleus assays (CBMN), respectively, in 58 control and 174 benzene-exposed workers in Shanghai, China. Among the exposed workers, relative mtDNAcn increased and then decreased with increasing doses of benzene exposure. Significant and dose-dependent increase in MN frequencies were observed among the different exposure groups. In addition, the relative mtDNAcn were significantly associated with the MN frequencies in the low-level exposure group (P = 0.046), but not in the high dose groups. Therefore, the mechanisms for induction of MtDNAcn and MN by benzene may be similar from exposure to low doses but different from high doses. Similar increase of MN frequencies and MtDNAcn may be due to oxidative stress induced by benzene at low concentrations, while higher concentrations may start to initiate the cell death pathway. The pathway may be associated with excessive MtDNAcn which can initiate apoptosis while MN can continue to be induced. However, the differential mechanisms need to be investigated because they may represent different levels of risk for different health consequences. On the other hand, our data indicate that induction of MtDNAcn may be a sensitive genotoxic biomarker for workers with exposure to low dose of benzene. Environ. Mol. Mutagen. 61:355–360, 2020. © 2020 Wiley Periodicals, Inc.     

Low oocyte mitochondrial DNA content in ovarian insufficiency

BACKGROUND: Mitochondrial biogenesis and bioenergetics play an important role in oocyte maturation and embryo development. We have investigated the relationship between defective mitochondrial biogenesis and the lack of oocyte maturity observed during IVF procedures with patients suffering from ovarian dystrophy and ovarian insufficiency. METHODS: We used real-time quantitative PCR to quantify mitochondrial DNA (mtDNA) in 116 oocytes obtained from 47 women undergoing the ICSI procedure. We compared the mtDNA content of oocytes from women with a normal ovarian profile with that of oocytes from women with ovarian dystrophy and ovarian insufficiency. RESULTS: We found an average of 256,000 +/- 213,000 mitochondrial genomes per cell. The mean mtDNA copy number was not significantly different in ovarian dystrophy compared with controls, but it was significantly lower in oocytes from women with ovarian insufficiency (100,000 +/- 99,000, P < 0.0001). CONCLUSIONS: Our results suggest that low mtDNA content is associated with the impaired oocyte quality observed in ovarian insufficiency.     

七个非综合征型耳聋家系患者的mtDNA A1555G突变性质与特点

目的 探讨非综合征型耳聋家系患者mtDNA A1555G突变性质及其特点,探索临床表型多样性的分子遗传学基础.方法 应用聚合酶链反应-限制性片段长度多态和实时荧光-扩增阻碍突变系统-定量PCR(real time-amplification refractory mutation system-quantitative PCR,RT-ARMS-qPCR)检测7个非综合征型耳聋家系71个成员的mtDNA A1555G突变,并收集、分析其临床资料.结果 7个家系中所有受检的母系成员mtDNA A1555G突变均为阳性,突变性质含同质性和异质性两种;非母系成员及配偶该突变为阴性.7个家系mtDNA A1555G同质性突变的拷贝数与耳聋轻重程度相关(R=0.341,P=0.022);mtDNA A1555G异质性突变的拷贝数与耳聋轻重程度相关(R=0.85,P=0.015).结论 mtDNA A1555G突变可导致非综合征型耳聋和氨基糖甙类抗生素致聋,其突变性质含同质性和异质性两种,且含mtDNA A1555G位点的突变型与野生型的比例与耳聋的严重程度密切相关.     

Fertilization and embryology: The early development and DNA content of activated human oocytes and parthenogenetic human embryos

A total of 297 human oocytes that had failed to fertilize duringin-vitro fertilization (IVF) cycles were exposed to the calciumionophore A23187 to induce parthenogenetic activation. Of theseoocytes, 192 (65%) activated, the majority (63%) exhibitinga single pronucleus and extruding a second polar body. The appearanceof two pronuclei (18%) was generally associated with a failureto extrude the second polar body. Oocytes obtained from patientswho were 35 years had a significantly reduced activation rate(53%). The timing of developmental events, such as extrusionof the second polar body, appearance and disappearance of pronucleiand the first two cleavage divisions, is broadly similar tothat seen in fertilized oocytes. However, the developmentalpotential of human parthenogenetic embryos was reduced, as themajority of those allowed to continue in culture arrested betweenthe 2-cell and 8-cell stages. Measurements of cellular DNA contentusing a computerized image analysis system showed that activatedoocytes with one pronucleus had a DNA content compatible witha haploid number of chromosomes, while those with two pronucleiwere diploid. The ability of parthenogenetically activated oocytesto replicate their DNA was also demonstrated.     

线粒体DNA的改变与结直肠癌相关性的研究进展

结直肠癌(colorectal cancer,CRC)是最常见的胃肠道肿瘤之一,发病率与死亡率一直居高不下,因此其发病相关的分子机制的研究具有重要的临床意义。研究发现线粒体DNA(mitochondrial DNA, mtDNA)作为核外基因也参与肿瘤的生物学过程,为结直肠癌的发生发展机制的研究提供了新的思路。本文就线粒体DNA的改变在结直肠癌的最新研究进展进行概述。     

Development of a quantitative PCR (TaqMan) assay for relative mitochondrial DNA copy number and the common mitochondrial DNA deletion in the rat

Changes in mitochondrial DNA copy number and increases in mitochondrial DNA mutations, especially deletions, have been associated with exposure to mutagens and with aging. Common deletions that are the result of recombination between direct repeats in human and rat (4,977 and 4,834, bp, respectively) are known to increase in tissues of aged individuals. Previous studies have used long-distance PCR and Southern blot or quantitative PCR to determine the frequency of deleted mitochondrial DNA. A quantitative PCR (TaqMan) assay was developed to detect both mitochondrial DNA copy number and deletion frequency in the rat. This methodology allows not only the determination of changes in the amount of mitochondrial DNA deletion relative to total mitochondrial DNA but also to determine changes in total mitochondrial DNA relative to genomic DNA. As a validation of the assay in rat liver, the frequency of the common 4,834 bp deletion is shown to increase with age, while the relative mitochondrial DNA copy number rises at a young age (3-60 days), then decreases and holds fairly steady to 2 years of age.     

Familial childhood onset neuropathy and cirrhosis with the 4977bp mitochondrial DNA deletion

The common 4977 base pair mitochondrial deletion has been identified in association with a number of distinct clinical phenotypes. These include the Kearns-Sayre syndrome, the Pearson marrow-pancreas syndrome, and chronic progressive external ophthalmoplegia. We report the clinical and pathological findings in two siblings in whom the 4977 base pair mitochondrial DNA deletion was identified in muscle-derived mitochondrial DNA. One sibling manifested early onset liver and renal failure, and both developed prominent peripheral sensorimotor neuropathy. These clinical findings have not been previously described in association with the 4977bp mtDNA deletion and thus represent a further expansion of the spectrum of mitochondrial disease.     

Mitochondrial DNA deletions and nuclear DNA fragmentation in testicular and epididymal human sperm

BACKGROUND: There are still concerns about the safety of intracytoplasmic sperm injection (ICSI) due to its brief clinical record and lack of animal testing. Testicular and epididymal sperm are now used routinely for ICSI in patients with obstructive azoospermia. The use of such immature sperm compounds fears, since little is known of their mitochondrial and nuclear DNA quality. METHODS: A modified long polymerase chain reaction (LPCR) was employed to study mitochondrial DNA (mtDNA) and a modified alkaline Comet assay to determine nuclear DNA (nDNA) fragmentation in testicular and epididymal sperm from men with obstructive azoospermia (n = 25) attending the Regional Fertility Centre. RESULTS: Testicular sperm displayed significantly more wild-type mtDNA (45% of patients) than epididymal sperm (16% of patients). They also had a lower incidence of multiple deletions and smaller mtDNA fragments. Epididymal sperm harboured more large-scale deletions (P < 0.05). There was a strong correlation between nuclear DNA fragmentation, the number of mtDNA deletions (r = 0.48, r = 0.50, P < 0.001) and their size (r = 0.58, r = 0.60, P < 0.001) in both epididymal and testicular sperm. CONCLUSION: This study suggests that mtDNA and nDNA of testicular sperm have fewer mutations and fragmentation than epididymal sperm and should be used in preference for ICSI in clinical treatment.     

Aberrant DNA methylation of imprinted loci in superovulated oocytes

BACKGROUND: There is an increased incidence of rare imprinting disorders associated with assisted reproduction technologies (ARTs). The sex-specific epigenetic modifications that are imposed during gametogenesis act as a primary imprint to distinguish maternal and paternal alleles. The most likely candidate for the gametic mark is DNA methylation. However, the timing of DNA methylation acquisition in adult oocytogenesis and the effects of superovulation are unknown. METHODS: We examined the maternal methylation of PEG1(MEST), LIT1(KCNQ1OT1) and ZAC(PLAGL1) and the paternal methylation of H19 in adult growing oocytes of humans and mice and compared them with the methylation status of mouse neonatal growing oocytes by using bisulphite sequencing. Furthermore, we examined the effects of superovulation in the human and mouse. RESULTS: Maternal methylation of these genes has already been initiated to some extent in adult human and mouse non-growing oocytes but not in mouse neonates. In addition, the methylation dynamics during adult human and mouse oocyte development changed more gradually than those during neonatal oocyte development. Furthermore, we found the demethylation of PEG1 in growing oocytes from some ART-treated infertile women and a gain in the methylation of H19. We also detected methylation changes in superovulated mice. CONCLUSION: Our studies in the human and mouse suggest that superovulation can lead to the production of oocytes without their correct primary imprint and highlight the need for more research into ARTs.     

Mitochondrial DNA deletion in human oocytes and embryos

Mitochondrial DNA (mtDNA) deletions are present in both human oocytes and embryos. It has been found that these tissues contain a mtDNA mutation which is present in high amounts in patients with Kearns-Sayre syndrome (KSS) and progressive external ophthalmoplegia. In the present study, the frequency of this KSS deletion was investigated in human oocytes and embryos. Using a nested primer polymerase chain chian reaction (PCR) strategy, the frequency of the KSS deletion in 74 human oocytes and 137 embryos was found to be 32.8 and 8.0% respectively. Using a 'long PCR-short PCR' nested primer strategy, the frequency of the KSS deletion in 181 human oocytes and 104 embryos was found to be 47.0 and 20.2% respectively. There was no statistical correlation between the age of the patients at the time of oocyte retrieval and the presence of the deleted molecules. There was a statistical difference between the presence of the deleted molecules in oocytes versus embryos using either technique (P < 0.0001). The relevance of these findings to the accumulation of low levels of deleted mtDNA in both oocytes and embryos is discussed in this study.      

Nuclear and mitochondrial DNAs microsatellite instability and mitochondrial DNA copy number in adenocarcinoma and squamous cell carcinoma of lung: a pilot study

Mitochondrial genetic changes are considered as a key molecular step of mutations in various cancers. To clarify the role of genetic instability in lung cancer, we analyzed clinicopathological characteristics and frequencies of nuclear and mitochondrial microsatellite instability (nMSI and mtMSI), and alteration of mitochondrial DNA copy number (mtCN) in adenocarcinoma (ADC) and squamous cell carcinoma (SCC) of lung. DNA was isolated from 48 patients with ADC and 42 with SCC. Markers for nMSI, BAT 25 and 26, and markers for mtMSI, (C)n and (CA)n in mitochondrial D‐loop region, were utilized. The mtCN were measured by real‐time polymerase chain reaction. The nMSI was found in two patients (4.2%) of ADC and 6 (14.3%) of SCC. The mtMSI was detected in 10 patients (20.8%) of ADC and 8 (19.0%) of SCC. Mean mtCN was 5.05 ± 8.17 and 3.34 ± 5.14 in ADC and SCC respectively. The mtCN was increased in 35 patients (72.9%) of ADC and 30 (71.4%) of SCC. The mtMSI more frequently appeared in more advanced pathologic T stage in ADC (p = 0.003). Alterations of mtCN and a high frequency of mtMSI in our patient samples indicate that mitochondrial DNA is a potential molecular marker in lung cancers (ADC and SCC) correlating with their histological classification.     

Micromorphometry and spermatozoa binding patterns of fertilized and unfertilized human oocytes after in-vitro fertilization

Human oocytes (n = 380) from 71 in-vitro fertilization patientswere measured 18 h after insemination to find out if certainparameters of oocyte morphology could be related to fertilization.In addition, the number and distribution patterns of spermatozoabound to or within the zona pellucida of 534 oocytes were analysed.The mean diameter of the human oocyte was 167.7 ± 9.5µm and its mean volume was 2.5 x 10⁶ µm³. Therewere no significant differences in diameter between 112 fertilizedand 168 unfertilized oocytes, although they displayed differencesin the size of the perivitelline space and the zona pellucida.The age of the patients had no significant effect on the morphometryof the oocytes. Sperm binding patterns did not correlate withfertilization. The number and distribution of the spermatozoaon the surface of the zona pellucida was extremely heterogeneousand was not related to the occurrence of fertilization. Allpossible binding and distribution patterns were in the samerange in both fertilized and unfertilized oocytes. In conclusion,the micromorphometry of human oocytes and their sperm bindingpatterns were not related to the occurrence of fertilization.     

A simple method for the morphological analysis of human ova: zona penetration in oocytes failing to fertilize or fertilizing abnormally in vitro

A simple and rapid technique is described whereby several unfertilized oocytes and embryos can be processed simultaneously for embedding and sectioning for histological analysis. Oocytes are encased in a serum matrix and thereafter can be handled with ease. A total of 112 unfertilized oocytes and abnormal embryos following insemination in vitro have been examined. Only 31% of oocytes showed pyknotic nuclei after a mean of 4.0 +/- 1.24 days in culture and 37% had recognizable metaphase II chromosomes. Twenty per cent showed evidence of fertilization, but spermatozoa had failed entirely to penetrate the zonae pellucidae of 34% of oocytes. The mean percentages of penetrating sperm were not significantly different between oocytes which did and did not fertilize. Under these in-vitro conditions, approximately 1-2% of spermatozoa reaching the oocyte penetrated into the zona pellucida and a lesser number to the perivitelline space. Polar bodies were also observed and the incidence of divisions, retention and parthenogenesis estimated. This technique may advance our understanding of the reasons for the success or failure of fertilization in vitro.     

Morphological and cytogenetic observations of unfertilized human oocytes and abnormal embryos obtained after ovarian stimulation with pure follicle stimulating hormone following pituitary desensitization

Morphological and cytological observations of 189 unfertilizedoocytes and 40 abnormal embryos obtained from 32 patients ina routine in-vitro fertilization programme were performed. Boththe oocytes and the embryos were mounted whole to preserve theoriginal topology of all the structural elements. With the appliedprotocol of ovarian stimulation associating pituitary desensitizationand follicle stimulating hormone stimulation, a high degreeof immaturity of the unfertilized eggs was observed in comparisonwith previous reports. This immaturity was deduced from thehigher incidence of unfertilized eggs arrested at the germinalvesicle or metaphase I stage, as well as metaphase II oocyteswith multiple metaphase plates. Nine triploid and four tetraploidembryos were analysed: except for one tetraploid embryo, allthe polyploid embryos cleaved. The percentages of mononucleatedblastomeres in these polyploid embryos were 57 and 27% respectively.We also analysed 21 cleaving diploid embryos which exhibiteda high degree of fragmentation.No more than 40% of the blastomerescontained single nucleus. Moreover, in only one of the 21 diploidembryos could all the blastomeres be considered normal.     

弱精子症病人精子线粒体DNA4977—bp缺失研究

本文采用聚合酶链反应（ＰＣＲ） 技术对４１ 例标本,其中２０ 例弱精子症病人和２１ 例精子活力正常人进行了精子线粒体ＤＮＡ（ｍｔＤＮＡ）缺失的研究。结果发现２０ 例弱精子症病人中１８ 例有缺失,而２１ 例精子活力正常人中仅有两例有缺失。说明线粒体ＤＮＡ４９７７ｂｐ 缺失在弱精子症的发病中起重要作用。     

Fertilization and early embryology: Intracytoplasmic single sperm injection of 1-day-old unfertilized human oocytes

Although the average fertilization rate in most in-vitro fertilization(IVF) centres is 60–70%, there are cases of complete orvirtually complete fertilization failures. The aim of our workwas to study the fertilization and the subsequent cleavage characteristicsof 1-day-old human oocytes treated by intracytoplasmic singlesperm injection (ICSI) after failing to fertilize during thestandard IVF procedure. A total of 115 metaphase II 1-day-oldunfertilized oocytes were collected from 23 patients. No additionaltreatment was applied to the oocytes or to the semen sample.A single spermatozoon from the patient's husband was injectedinto the cytoplasm of each of these oocytes 21–33 h afterovum retrieval. Injected oocytes were observed at 16–18h and again 42–44 h after the ICSI procedure. Of the injectedoocytes, 92% (n = 106) were intact after ICSI, 38% (n = 44)had two distinct pronuclei and there was no difference in thefertilization rate of oocytes when andrological and non-andrologicalpatients were compared. Similarly, there was no difference inthe fertilization rate after ICSI where patients with acceptableor good (> 15%) fertilization after standard IVF were comparedto patients who had poor (<15%) fertilization after IVF.There was no significant difference in the sperm concentrationor in the progressive forward motility (a + b motility) in thesegroups except where a + b motility of andrological and non-adrologicalpatients was compared. The majority (84%) of the normally fertilizedoocytes cleaved and most (77%) of these embryos showed <20%fragmentation 2 days after the ICSI procedure. From this studyit can be concluded that 1-day-old metaphase II oocytes whichhave failed to be fertilized after standard IVF procedure canbe fertilized and cleave when ICSI is performed on them theday after oocyte retrieval.     

Increased sperm mitochondrial DNA content in male infertility

BACKGROUND: There is increasing evidence that mitochondrial DNA (mtDNA) anomalies in sperm may lead to infertility. Point mutations, deletions and the presence of a specific mtDNA haplogroup have been associated with poor sperm quality, but little attention has been paid to the role of mtDNA content. METHODS: Using density gradient separation and swim-up methods, we selected motile sperm from 32 normal and 35 abnormal sperm samples. The mtDNA/beta-globin gene ratio was determined by real-time quantitative PCR. RESULTS: The average mtDNA/beta-globin ratio of sperm collected from 100% density layers was 1.4 for normal sperm, 6.1 for sperm samples presenting at least one abnormal criterion [among the three criteria established by World Health Organization (1999), i.e. sperm count, motility and morphology], and 9.1 for sperm samples presenting two or more of these abnormal criteria. These differences are very highly significant (P < 0.0001). The mtDNA numbers were also much greater in sperm collected from the 40% density gradient layers (mean: 17.1, P < 0.001), known to contain the most abnormal sperm of the sperm samples, than in those collected from the 100% layers known to contain sperm with the best fertilizing ability. CONCLUSION: Our results showed significant mtDNA amplification in sperm collected from abnormal sperm samples.