Author Correction: Mitochondrial replacement in human oocytes carrying pathogenic mitochondrial DNA mutations.

Change history In this Letter, there are several errors regarding the assignments of mtDNA haplotypes for a subset of egg donors from our study. These errors have not been corrected online.

Structural properties and electron spin resonance (ESR) dose estimations of fossil cow tooth enamel from Kösk Höyük, Turkey.

In this work, two cow teeth collected from the Nigde-Kösk Höyük excavation site in Turkey were studied for characterization and dosimetric purposes. Each tooth sample was prepared by applying mechanical and chemical methods to obtain the enamel fractions. To do this, mineralogical and elemental concentration properties of the tooth enamels were investigated by performing X-ray diffraction (XRD) and scanning electron microscopy coupled with energy-dispersive X-ray measurements (SEM-EDX). It was found that the enamel structures contained a highly hydroxyapatite crystalline without any characteristic impurities. The dose response of the tooth enamels was determined by using the electron spin resonance (ESR) method. Absorbed radiation doses were calculated as (26.05 ± 0.15) Gy and (25.48 ± 0.18) Gy by the additive dose method using both natural radiation doses and artificial irradiation doses of the enamel samples. It is concluded that these samples could be used to reconstruct radiation doses. This result can be considered as a precursor for future ESR dosimetry/dating studies of other fossil teeth at this excavation site.

Mitochondrial replacement in human oocytes carrying pathogenic mitochondrial DNA mutations.

Maternally inherited mitochondrial (mt)DNA mutations can cause fatal or severely debilitating syndromes in children, with disease severity dependent on the specific gene mutation and the ratio of mutant to wild-type mtDNA (heteroplasmy) in each cell and tissue. Pathogenic mtDNA mutations are relatively common, with an estimated 778 affected children born each year in the United States. Mitochondrial replacement therapies or techniques (MRT) circumventing mother-to-child mtDNA disease transmission involve replacement of oocyte maternal mtDNA. Here we report MRT outcomes in several families with common mtDNA syndromes. The mother's oocytes were of normal quality and mutation levels correlated with those in existing children. Efficient replacement of oocyte mutant mtDNA was performed by spindle transfer, resulting in embryos containing >99% donor mtDNA. Donor mtDNA was stably maintained in embryonic stem cells (ES cells) derived from most embryos. However, some ES cell lines demonstrated gradual loss of donor mtDNA and reversal to the maternal haplotype. In evaluating donor-to-maternal mtDNA interactions, it seems that compatibility relates to mtDNA replication efficiency rather than to mismatch or oxidative phosphorylation dysfunction. We identify a polymorphism within the conserved sequence box II region of the D-loop as a plausible cause of preferential replication of specific mtDNA haplotypes. In addition, some haplotypes confer proliferative and growth advantages to cells. Hence, we propose a matching paradigm for selecting compatible donor mtDNA for MRT.

The impact of culture conditions on blastocyst formation and aneuploidy rates: a comparison between single-step and sequential media in a large academic practice.

PURPOSE: To compare a single-step medium with a sequential medium on human blastocyst development rates, aneuploidy rates, and clinical outcomes. METHODS: Retrospective cohort study of IVF cycles that used Sage advantage sequential medium (n = 347) and uninterrupted Sage 1-step medium (n = 519) from July 1, 2016, to December 31, 2017, in an academic fertility center. Main outcome measures are blastocyst formation rates per two-pronuclear (2PN) oocyte and aneuploidy rates per biopsy. RESULTS: Of all IVF cycles, single-step medium yielded higher blastocyst formation rate (51.7% vs 43.4%) but higher aneuploidy rate (54.0% vs 45.8%) compared with sequential medium. When stratified by maternal age, women under age 38 had no difference in blastocyst formation (52.2% vs 50.2%) but a higher aneuploidy rate (44.5% vs 36.4%) resulting in a lower number of euploid blastocysts per cycle (2.6 vs 3.3) when using single-step medium compared to sequential medium. In cycles used single-step medium, patients ≥ age 38 had higher blastocyst rate (48.0% vs 33.6%), but no difference in aneuploidy rate (68.8% vs 66.0%) or number of euploid embryos (0.8 vs 1.1). For patients reaching euploid embryo transfer, there was no difference in clinical pregnancy rates, miscarriage rates, or live birth rates between two culture media systems. CONCLUSIONS: Our study demonstrates an increase in aneuploidy in young women whose embryos were cultured in a single-step medium compared to sequential medium. This study highlights the importance of culture conditions on embryo ploidy and the need to stratify by patient age when examining the impact of culture conditions on overall cycle potential.

A high-content, high-throughput siRNA screen identifies cyclin D2 as a potent regulator of muscle progenitor cell fusion and a target to enhance muscle regeneration.

Cell-mediated regenerative approaches using muscle progenitor cells hold promises for the treatment of many forms of muscle disorders. Their applicability in the clinic, however, is hindered by the low levels of regeneration obtained after transplantation and the large number of cells required to achieve an effect. To better understand the mechanisms that regulate the temporal switch of replicating muscle progenitor cells into terminally differentiated cells and to develop new strategies that could enhance muscle regeneration, we have developed and performed a high-throughput screening (HTS) capable of identifying genes that play active roles during myogenesis. Secondary and tertiary screens were used to confirm the effects of RNAi in vitro and in vivo and to select for candidate hits that significantly increase regeneration into skeletal muscles. Downregulation of cyclin D2 (CCND2) was shown to dramatically enhance myogenic differentiation of muscle progenitor cells and to induce a robust regeneration after cell transplantation into skeletal muscles of dystrophin-deficient mice. Protein interaction network and pathway analysis revealed that CCND2 directly interacts with the cyclin-dependent kinase Cdk4 to inhibit phosphorylation of the retinoblastoma protein (pRb), thus blocking the activation of the myogenic switch during fusion. These studies identify CCND2 as a new key regulator of terminal differentiation in muscle progenitor cells and open new possibilities for the treatment of many forms of muscle disorders characterized by impaired regeneration and loss of muscle mass.

Read-through compound 13 restores dystrophin expression and improves muscle function in the mdx mouse model for Duchenne muscular dystrophy.

Molecules that induce ribosomal read-through of nonsense mutations in mRNA and allow production of a full-length functional protein hold great therapeutic potential for the treatment of many genetic disorders. Two such read-through compounds, RTC13 and RTC14, were recently identified by a luciferase-independent high-throughput screening assay and were shown to have potential therapeutic functions in the treatment of nonsense mutations in the ATM and the dystrophin genes. We have now tested the ability of RTC13 and RTC14 to restore dystrophin expression into skeletal muscles of the mdx mouse model for Duchenne muscular dystrophy (DMD). Direct intramuscular injection of compound RTC14 did not result in significant read-through activity in vivo and demonstrated the levels of dystrophin protein similar to those detected using gentamicin. In contrast, significant higher amounts of dystrophin were detected after intramuscular injection of RTC13. When administered systemically, RTC13 was shown to partially restore dystrophin protein in different muscle groups, including diaphragm and heart, and improved muscle function. An increase in muscle strength was detected in all treated animals and was accompanied by a significant decrease in creatine kinase levels. These studies establish the therapeutic potential of RTC13 in vivo and advance this newly identified compound into preclinical application for DMD.

Arginine-rich cell-penetrating peptide dramatically enhances AMO-mediated ATM aberrant splicing correction and enables delivery to brain and cerebellum.

Antisense morpholino oligonucleotides (AMOs) can reprogram pre-mRNA splicing by complementary binding to a target site and regulating splice site selection, thereby offering a potential therapeutic tool for genetic disorders. However, the application of this technology into a clinical scenario has been limited by the low correction efficiency in vivo and inability of AMOs to efficiently cross the blood brain barrier and target brain cells when applied to neurogenetic disorders such as ataxia-telangiecatasia (A-T). We previously used AMOs to correct subtypes of ATM splicing mutations in A-T cells; AMOs restored up to 20% of the ATM protein and corrected the A-T cellular phenotype. In this study, we demonstrate that an arginine-rich cell-penetrating peptide, (RXRRBR)(2)XB, dramatically improved ATM splicing correction efficiency when conjugated with AMOs, and almost fully corrected aberrant splicing. The restored ATM protein was close to normal levels in cells with homozygous splicing mutations, and a gene dose effect was observed in cells with heterozygous mutations. A significant amount of the ATM protein was still detected 21 days after a single 5 µm treatment. Systemic administration of an fluorescein isothiocyanate-labeled (RXRRBR)(2)XB-AMO in mice showed efficient uptake in the brain. Fluorescence was evident in Purkinje cells after a single intravenous injection of 60 mg/kg. Furthermore, multiple injections significantly increased uptake in all areas of the brain, notably in cerebellum and Purkinje cells, and showed no apparent signs of toxicity. Taken together, these results highlight the therapeutic potential of (RXRRBR)(2)XB-AMOs in A-T and other neurogenetic disorders.

Site-directed gene repair of the dystrophin gene mediated by PNA-ssODNs.

Permanent correction of gene defects is an appealing approach to the treatment of genetic disorders. The use of single-stranded oligodeoxynucleotides (ssODNs) has been demonstrated to induce single-point mutations in the dystrophin gene and to restore dystrophin expression in the skeletal muscle of models of Duchenne muscular dystrophy (DMD). Here we show that ssODNs made of peptide nucleic acids (PNA-ssODNs) can achieve gene repair frequencies more than 10-fold higher than those obtained using an older generation of targeting oligonucleotides. Correction was demonstrated in muscles cells isolated from mdx(5cv) mice and was stably inherited over time. Direct intramuscular injection of PNA-ssODNs targeting the mdx(5cv) mutation resulted in a significant increase in dystrophin-positive fibers when compared with muscles that received the ssODNs designed to correct the dystrophin gene but made of unmodified bases. Correction was demonstrated at both the mRNA and the DNA levels using quantitative PCR and was confirmed by direct sequencing of amplification products. Analysis at the protein level demonstrated expression of full-length dystrophin in vitro as well as in vivo. These results demonstrate that oligonucleotides promoting strand invasion in the DNA double helix can significantly enhance gene repair frequencies of the dystrophin gene. The use of PNA-ssODNs has important implications in terms of both efficacy and duration of the repair process in muscles and may have a role in advancing the treatment of DMD.

Criteria to evaluate patterns of segmental and complete aneuploidies in preimplantation genetic testing for aneuploidy results suggestive of an inherited balanced translocation or inversion.

OBJECTIVE: To define criteria for determining when preimplantation genetic testing for aneuploidy (PGT-A) results are suggestive of a potential balanced chromosomal rearrangement in the egg or sperm source and warrant karyotyping. DESIGN: Performance evaluation of criteria developed to assess PGT-A results for patterns of imbalances suggestive of a balanced chromosomal rearrangement in the egg or sperm source. SETTING: A single PGT-A laboratory and multiple in vitro fertilization centers. PATIENTS: Reproductive couples who underwent routine PGT-A testing. INTERVENTIONS: Karyotyping of reproductive couples for whom patterns of imbalances observed in PGT-A results suggested a balanced chromosomal rearrangement in the egg or sperm source. MAIN OUTCOME MEASURES: Correct or incorrect flagging of predicted translocation in either the egg or sperm source based on chromosome analysis. RESULTS: Proposed criteria correctly predicted a balanced reciprocal translocation in 97% of cases (n = 33), a (13;14) Robertsonian translocation in all cases (n = 3), and an inversion in all cases (n = 2). Other criteria evaluated were determined to be ineffective because of relatively low occurrences that met the criteria and/or low predictive value. CONCLUSIONS: Our results showed that the proposed criteria were effective for evaluating patterns of imbalances observed in PGT-A results suggestive of a potential chromosomal rearrangement in the egg or sperm source. Our proposed criteria can be employed by clinicians in the in vitro fertilization setting in combination with a patient's reproductive history to identify PGT-A patients who are likely carriers of balanced chromosomal rearrangements.

Aneuploidy Screening using Next Generation Sequencing.

Chromosomal aneuploidy is recognized to be a significant contributing factor in implantation failure and spontaneous miscarriage Hellani et al. (Reprod Biomed Online 17:841-847, 2008), Vanneste et al. (Nat Med 15:577-583, 2009) and is likely to be responsible for the majority of IVF failure [Baltaci et al. (Reprod Biomed Online 12:77-82, 2006), Munne (Placenta 24:S70-76, 2003)]. Preimplantation genetic testing for aneuploidy (PGT-A) screening, formerly termed preimplantation genetic screening (PGS), enables the assessment of the numeric chromosomal constitution in blastomere and/or trophectoderm biopsy before embryo transfer.Preimplantation genetic testing for aneuploidy (PGT-A) has been proven to improve the selection of embryos for transfer and therefore also assisted reproductive technology (ART) cycles. In this chapter we describe the current gold standard platform for PGT-A, next generation sequencing (NGS) protocol used in our laboratory.

Postmitotic tissue selenium and manganese levels in alpha-lipoic acid-supplemented aged rats.

Redistribution of selenium and manganese in postmitotic tissues of alpha-lipoic acid-supplemented aged rats has been proposed to contribute to metal-catalyzed protein oxidation. DL-Alpha-lipoic acid (LA) (100 mg/[kg body wt.day]) was administered intraperitoneally to the Sprague-Dawley rats for 14 days. Serum selenium levels were lowered in the aged rats with LA supplementation compared with those of the rats without LA supplementation. Similarly, the selenium levels of the heart, brain and muscle were found to be significantly lower in LA-supplemented rats when compared to control rats. On the other hand, serum manganese levels were not changed in the aged rats with LA supplementation compared with those of the rats without LA supplementation. The heart manganese levels detected in LA-supplemented rats were significantly lower than controls. Manganese levels of the brain and muscle tissues were increased in the aged rats with LA supplementation compared with those of the rats without LA supplementation. Based on the findings of our study, we conclude that LA may exhibit pro-oxidant effect depending on the altered selenium and manganese homeostasis. Thus, our results stress the importance of monitoring the dose of LA supplementation and serum selenium levels, duration of treatment and its potential harmful pro-oxidant effects in the postmitotic tissues of aged rats.

Relation of plasma protein oxidation parameters and paraoxonase activity in the ageing population.

The incidence of atherosclerosis increases with age. Oxidative changes in proteins and lipids are considered to be among the molecular mechanisms leading to endothelial dysfunction. Paraoxonase (PON1) is exclusively associated with high-density lipoprotein (HDL) and protects both HDL and low-density lipoprotein (LDL) from oxidation. PON1 has two cysteine residues for its antioxidant function. We investigated the relation between PON1 activity and protein oxidation parameters such as protein hydroperoxides (P-OOH), protein carbonyl (PCO), total thiol (T-SH) and advanced oxidation protein products (AOPP). Our study also covered other oxidative stress parameters such as oxidised LDL (oxLDL) and superoxide dismutase activity in the plasma of young, middle-aged and elderly individuals. PON1 activity of elderly and middle-aged individuals was decreased significantly compared with that in the young group. oxLDL levels of elderly individuals were increased significantly compared with those of both the young and middle-aged individuals. P-OOH, PCO and AOPP levels of the elderly and middle aged individuals were higher compared with those of the young. On the other hand, T-SH levels of the elderly and middle-aged individuals were lower compared with those of the young. Side by side with the decrease in the T-SH levels in the middle-aged and elderly groups as compared to the young, the increase we have observed in other protein oxidation parameters in the groups leading to decreasing PON1 activity might, we think, create a predisposition to atherosclerosis.

Male rats exhibit higher oxidative protein damage than females of the same chronological age.

The basis of the difference in life expectancy between males and females is still unknown. Previous studies have provided compelling evidence for the presence of oxidized proteins, and lipids in advanced human atherosclerotic lesions. The gender factor responsible for such protein oxidation is unknown and controversial. Our aim was to reveal the difference between protein oxidation parameters of male and female rats of the same chronological age to understand the protein oxidation mechanisms enabling females live longer than males. In the current study, we investigated the relation between protein hydroperoxide levels (P-OOH) and other protein oxidation parameters such as protein carbonyl (PCO), total thiol (T-SH), advanced oxidation protein products (AOPP), and nitrotyrosine (NT). Our study also covered other oxidative stress parameters such as lipid hydroperoxides (L-OOH), and superoxide dismutase (SOD) activity in the plasma of male and female aged rats. Plasma P-OOH and AOPP levels of male rats were significantly higher compared with those of the female rats. T-SH levels were significantly lower in the aged male rats compared with those of the female rats. On the other hand, PCO, NT, and L-OOH levels, and SOD activity were all found to be not different. These data support the hypothesis that elevated levels of P-OOH and AOPP contribute to the extent of protein, but not lipid, oxidation in plasma of aged male rats. Furthermore, the results presented here may also rationalize studies, which have shown that protein oxidation is modulated on the basis of gender dependency.

Effect of alpha-lipoic acid supplementation on trace element levels in serum and in postmitotic tissue in aged rats.

Redistribution of redox-active divalent metal ions (e.g. copper, zinc, and iron) in postmitotic tissues of lipoic acid supplemented aging rats has been proposed to contribute to metal-catalyzed protein oxidation. DL-alpha lipoic acid (LA) (100 mg/kg body wt/day) was administered intraperitoneally to the Sprague-Dawley rats for 14 days. Serum copper levels lowered in the aged rats with LA supplementation compared to the rats without LA supplementation. On the other hand, serum zinc and iron levels increased in the aged rats with LA supplementation compared to the rats without LA supplementation. Copper levels of the postmitotic tissues were not changed in the aged rats with LA supplementation compared to the controls. The heart zinc levels detected in LA supplemented rats were significantly lower than controls. Similarly, the iron levels of the heart were found to be significantly lower in LA supplemented rats when compared to control rats. LA supplementation did not affect brain and muscle iron levels. The brain and muscle zinc levels remained the same in both group of rats. Based on the findings of our study, we have concluded that LA may exhibit prooxidant effect depending on the altered trace element homeostasis. Therefore, our results emphasize the importance of monitoring the dose of LA supplementation, duration of treatment and its potential harmful effects in the postmitotic tissues of aged rats.

Functional Human Oocytes Generated by Transfer of Polar Body Genomes.

Oocyte defects lie at the heart of some forms of infertility and could potentially be addressed therapeutically by alternative routes for oocyte formation. Here, we describe the generation of functional human oocytes following nuclear transfer of first polar body (PB1) genomes from metaphase II (MII) oocytes into enucleated donor MII cytoplasm (PBNT). The reconstructed oocytes supported the formation of de novo meiotic spindles and, after fertilization with sperm, meiosis completion and formation of normal diploid zygotes. While PBNT zygotes developed to blastocysts less frequently (42%) than controls (75%), genome-wide genetic, epigenetic, and transcriptional analyses of PBNT and control ESCs indicated comparable numbers of structural variations and markedly similar DNA methylation and transcriptome profiles. We conclude that rescue of PB1 genetic material via introduction into donor cytoplasm may offer a source of oocytes for infertility treatment or mitochondrial replacement therapy for mtDNA disease.

The evaluation of altered redox status in plasma and mitochondria of acute and chronic diabetic rats.

OBJECTIVES: An increase in plasma oxidative stress and decreased mitochondrial lipid hydroperoxides may contribute to the imbalance in the redox status between intramitochondrial and extramitochondrial milieu in chronic experimental diabetic rats. DESIGN AND METHODS: To determine the effect of hyperglycemia in promoting redox imbalance, we determined lipid hydroperoxides (LHP), protein carbonyl (PCO), total antioxidant activity (ferric reducing/antioxidant power; FRAP) and albumin as markers of redox status of plasma, and mitochondrial lipid hydroperoxide levels as a marker of lipid peroxidation in liver, pancreas and kidney tissue of acute and chronic diabetic male Sprague-Dawley rats and their controls. The levels of the studied markers were determined by colorimetric methods. RESULTS: Plasma and mitochondrial oxidative stress parameter levels of acute diabetic rats were not significantly different from their controls. Plasma LHP and PCO levels of chronic diabetic rats were increased significantly as compared to those of both acute diabetic rats and the controls. Plasma FRAP levels of chronic diabetic animals were decreased significantly as compared to those of the controls. On the other hand, LHP levels in liver, pancreas and kidney mitochondria of chronic diabetic rats were decreased significantly as compared to those of both acute diabetic rats and the controls. We observed a negative correlation between LHP levels in liver mitochondria of chronic diabetic rats, and PCO and fructosamine levels in plasma of chronic diabetic rats were correlated. LHP levels in the pancreatic mitochondria of chronic diabetic rats and plasma oxidative stress parameters of chronic diabetic rats were not significantly correlated. LHP levels in kidney mitochondria of chronic diabetic rats were significantly correlated with serum albumin. There was no correlation between LHP levels in kidney mitochondria and other plasma oxidative stress parameters in chronic diabetic rats. CONCLUSIONS: Our data suggest that redox imbalance between plasma and liver mitochondria might become a major threat to chronic diabetic rats.

Prooxidant activities of alpha-lipoic acid on oxidative protein damage in the aging rat heart muscle.

In the present study, we investigated whether alpha-lipoic acid (ALA) supplementation could have prooxidant or antioxidant effects on protein oxidation parameters such as protein carbonyl (PCO), nitrotyrosine (NT), advanced oxidation protein products (AOPP), and protein thiol (P-SH), as well as oxidative stress parameters such as total thiol (T-SH), non-protein thiol (Np-SH), and lipid hydroperoxide (LHP) in the heart muscle tissue of aged rats. ALA (100 mg/kg body wt/day) was administered intraperitoneally to the experimental animals for 14 days. PCO, NT, AOPP, and P-SH levels were increased, T-SH and Np-SH levels were not changed, and only LHP levels were decreased in the heart muscle tissue of aged rats with ALA supplementation. When compared with non-supplemented aged rats, increasing levels of protein oxidation markers such as PCO, NT, and AOPP in ALA-supplemented aged rats may suggest that oxidative protein damage is increased in ALA-supplemented aged rats. We assume that an explanation for our findings regarding ALA supplementation on protein oxidation markers in the heart muscle tissue of aged rats may be due to the prooxidant effects of ALA. The prooxidant effects of ALA supplementation should be considered in future studies.

Relation of aging with oxidative protein damage parameters in the rat skeletal muscle.

OBJECTIVES: An increase in oxidative stress may contribute to the development of oxidative protein damage in the aging rat skeletal muscle. Our aim was to reveal protein carbonyl (PCO), advanced oxidation protein products (AOPP), a novel marker of oxidative stress, and protein thiol (P-SH) levels as markers of protein oxidation, as well as lipid hydroperoxide (LHP) levels as a marker of lipid peroxidation, and relation of nitrotyrosine (NT) levels with these markers in skeletal muscle tissue of young, adult, and old male Wistar rats. DESIGN AND METHODS: In the present study, we investigated the relation between aging and oxidative protein damage parameters such as PCO, NT, AOPP, and P-SH, as well as oxidative stress parameters such as total thiol, nonprotein thiol, and LHP in the skeletal muscle tissue of young, adult, and old Wistar rats. RESULTS: PCO and NT levels of old rats were significantly increased compared with those of young and adult rats. Skeletal muscle AOPP levels were significantly increased in old rats compared with those of adult rats. P-SH levels were significantly decreased in old rats compared with those of young rats. CONCLUSIONS: The finding that the increase in PCO levels of young vs. old group was more significant than that of adult vs. old group may suggest that PCO formation is an early specific marker of aging process in skeletal muscle. In addition, increased levels of nitrotyrosine in the skeletal muscle of the old rat group may be a novel specific marker of oxidative protein damage in the aging muscle. The absence of correlation between oxidative protein damage markers mentioned above and LHP levels may indicate that protein oxidation and lipid peroxidation in the aging rat skeletal tissue are two distinct mechanisms.