Mitochondrial DNA mutations can influence the post-implantation development of human mosaic embryos.

Introduction: Several healthy euploid births have been reported following the transfer of mosaic embryos, including both euploid and aneuploid blastomeres. This has been attributed to a reduced number of aneuploid cells, as previously reported in mice, but remains poorly explored in humans. We hypothesized that mitochondrial function, one of the most critical factors for embryonic development, can influence human post-implantation embryonic development, including a decrease of aneuploid cells in mosaic embryos. Methods: To clarify the role of mitochondrial function, we biopsied multiple parts of each human embryo and observed the remaining embryos under in vitro culture as a model of post-implantation development (n = 27 embryos). Karyotyping, whole mitochondrial DNA (mtDNA) sequencing, and mtDNA copy number assays were performed on all pre- and post-culture samples. Results: The ratio of euploid embryos was significantly enhanced during in vitro culture, whereas the ratio of mosaic embryos was significantly reduced. Furthermore, post-culture euploid and culturable embryos had significantly few mtDNA mutations, although mtDNA copy numbers did not differ. Discussion: Our results indicate that aneuploid cells decrease in human embryos post-implantation, and mtDNA mutations might induce low mitochondrial function and influence the development of post-implantation embryos with not only aneuploidy but also euploidy. Analyzing the whole mtDNA mutation number may be a novel method for selecting a better mosaic embryo for transfer.

Horizontal mtDNA transfer between cells is common during mouse development.

Cells transmit their genomes vertically to daughter cells during cell divisions. Here, we demonstrate the occurrence and extent of horizontal mitochondrial (mt)DNA acquisition between cells that are not in a parent-offspring relationship. Extensive single-cell sequencing from various tissues and organs of adult chimeric mice composed of cells carrying distinct mtDNA haplotypes showed that a substantial fraction of individual cardiomyocytes, neurons, glia, intestinal, and spleen cells captured donor mtDNA at high levels. In addition, chimeras composed of cells with wild-type and mutant mtDNA exhibited increased trafficking of wild-type mtDNA to mutant cells, suggesting that horizontal mtDNA transfer may be a compensatory mechanism to restore compromised mitochondrial function. These findings establish the groundwork for further investigations to identify mtDNA donor cells and mechanisms of transfer that could be critical to the development of novel gene therapies.

Fertility preservation immediately after therapeutic abortion results in multiple normal follicular growth with the absence of mature oocytes due to early luteinization: a case report and literature review.

Cancer therapy has priority over fertility preservation. The time available for fertility preservation in patients with cancer is often very limited and depends on the condition of the underlying disease. This case report presents the results of two rounds of controlled ovarian stimulations (COSs) performed after an induced abortion. The patient had mixed phenotype acute leukemia diagnosed during early pregnancy and underwent a surgical abortion, followed by ovarian stimulation using urinary follicle-stimulating hormone (uFSH) and gonadotropin-releasing hormone (GnRH) agonists. Oocyte retrieval was subsequently performed for oocyte cryopreservation. Despite good hormonal and ultrasonic follicular growth, no oocytes were obtained. During a second COS performed at a low human chorionic gonadotropin (hCG) level (less than 100 IU/L), several mature oocytes were obtained, suggesting that higher hCG levels during COS induce the absence of mature oocytes during normal follicular growth. It is recommended to start COS post-abortion after confirming a low hCG level while considering the timing of cancer treatment.

Deleterious mtDNA mutations are common in mature oocytes.

Heritable mitochondrial DNA (mtDNA) mutations are common, yet only a few recurring pathogenic mtDNA variants account for the majority of known familial cases in humans. Purifying selection in the female germline is thought to be responsible for the elimination of most harmful mtDNA mutations during oogenesis. Here we show that deleterious mtDNA mutations are abundant in ovulated mature mouse oocytes and preimplantation embryos recovered from PolG mutator females but not in their live offspring. This implies that purifying selection acts not in the maternal germline per se, but during post-implantation development. We further show that oocyte mtDNA mutations can be captured and stably maintained in embryonic stem cells and then reintroduced into chimeras, thereby allowing examination of the effects of specific mutations on fetal and postnatal development.

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.

Generation of Vascular Endothelial Cells and Hematopoietic Cells by Blastocyst Complementation.

In the case of organ transplantation accompanied by vascular anastomosis, major histocompatibility complex mismatched vascular endothelial cells become a target for graft rejection. Production of a rejection-free, transplantable organ, therefore, requires simultaneous generation of vascular endothelial cells within the organ. To generate pluripotent stem cell (PSC)-derived vascular endothelial cells, we performed blastocyst complementation with a vascular endothelial growth factor receptor-2 homozygous mutant blastocyst. This mutation is embryonic lethal at embryonic (E) day 8.5-9.5 due to an early defect in endothelial and hematopoietic cells. The Flk-1 homozygous knockout chimeric mice survived to adulthood for over 1 year without any abnormality, and all vascular endothelial cells and hematopoietic cells were derived from the injected PSCs. This approach could be used in conjunction with other gene knockouts which induce organ deficiency to produce a rejection-free, transplantable organ in which all the organ's cells and vasculature are PSC derived.

Germline and somatic mtDNA mutations in mouse aging.

The accumulation of acquired mitochondrial genome (mtDNA) mutations with aging in somatic cells has been implicated in mitochondrial dysfunction and linked to age-onset diseases in humans. Here, we asked if somatic mtDNA mutations are also associated with aging in the mouse. MtDNA integrity in multiple organs and tissues in young and old (2-34 months) wild type (wt) mice was investigated by whole genome sequencing. Remarkably, no acquired somatic mutations were detected in tested tissues. However, we identified several non-synonymous germline mtDNA variants whose heteroplasmy levels (ratio of normal to mutant mtDNA) increased significantly with aging suggesting clonal expansion of inherited mtDNA mutations. Polg mutator mice, a model for premature aging, exhibited both germline and somatic mtDNA mutations whose numbers and heteroplasmy levels increased significantly with age implicating involvement in premature aging. Our results suggest that, in contrast to humans, acquired somatic mtDNA mutations do not accompany the aging process in wt mice.

Correction of a pathogenic gene mutation in human embryos.

Genome editing has potential for the targeted correction of germline mutations. Here we describe the correction of the heterozygous MYBPC3 mutation in human preimplantation embryos with precise CRISPR-Cas9-based targeting accuracy and high homology-directed repair efficiency by activating an endogenous, germline-specific DNA repair response. Induced double-strand breaks (DSBs) at the mutant paternal allele were predominantly repaired using the homologous wild-type maternal gene instead of a synthetic DNA template. By modulating the cell cycle stage at which the DSB was induced, we were able to avoid mosaicism in cleaving embryos and achieve a high yield of homozygous embryos carrying the wild-type MYBPC3 gene without evidence of off-target mutations. The efficiency, accuracy and safety of the approach presented suggest that it has potential to be used for the correction of heritable mutations in human embryos by complementing preimplantation genetic diagnosis. However, much remains to be considered before clinical applications, including the reproducibility of the technique with other heterozygous mutations.

Mitochondrial genome inheritance and replacement in the human germline.

Mitochondria, the ubiquitous power packs in nearly every eukaryotic cell, contain their own DNA, known as mtDNA, which is inherited exclusively from the mother. The number of mitochondrial genomes varies depending on the cell's energy needs. The mature oocyte contains the highest number of mitochondria of any cell type, although there is little if any mtDNA replication after fertilization until the embryo implants. This has potential repercussions for mitochondrial replacement therapy (MRT; see description of currently employed methods below) used to prevent the transmission of mtDNA-based disorders. If only a few mitochondria with defective mtDNA are left in the embryo and undergo extensive replication, it might therefore thwart the purpose of MRT In order to improve the safety and efficacy of this experimental therapy, we need a better understanding of how and which mtDNA is tagged for replication versus transcription after fertilization of the oocyte.

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.

Concise Review: Embryonic Stem Cells Derived by Somatic Cell Nuclear Transfer: A Horse in the Race?

Embryonic stem cells (ESC) hold promise for the treatment of human medical conditions but are allogeneic. Here, we consider the differences between autologous pluripotent stem cells produced by nuclear transfer (NT-ESCs) and transcription factor-mediated, induced pluripotent stem cells (iPSCs) that impact the desirability of each of these cell types for clinical use. The derivation of NT-ESCs is more cumbersome and requires donor oocytes; however, the use of oocyte cytoplasm as the source of reprogramming factors is linked to a key advantage of NT-ESCs-the ability to replace mutant mitochondrial DNA in a patient cell (due to either age or inherited disease) with healthy donor mitochondria from an oocyte. Moreover, in epigenomic and transcriptomic comparisons between isogenic iPSCs and NT-ESCs, the latter produced cells that more closely resemble bona fide ESCs derived from fertilized embryos. Thus, although NT-ESCs are more difficult to generate than iPSCs, the ability of somatic cell nuclear transfer to replace aged or diseased mitochondria and the closer epigenomic and transcriptomic similarity between NT-ESCs and bona fide ESCs may make NT-ESCs superior for future applications in regenerative medicine. Stem Cells 2017;35:26-34.

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.

Incompatibility between Nuclear and Mitochondrial Genomes Contributes to an Interspecies Reproductive Barrier.

Vertebrate cells carry two different genomes, nuclear (nDNA) and mitochondrial (mtDNA), both encoding proteins involved in oxidative phosphorylation. Because of the extensive interactions, adaptive coevolution of the two genomes must occur to ensure normal mitochondrial function. To investigate whether incompatibilities between these two genomes could contribute to interspecies reproductive barriers, we performed reciprocal mtDNA replacement (MR) in zygotes between widely divergent Mus m. domesticus (B6) and conplastic Mus m. musculus (PWD) mice. Transfer of MR1 cybrid embryos (B6nDNA-PWDmtDNA) supported normal development of F1 offspring with reduced male fertility but unaffected reproductive fitness in females. Furthermore, donor PWD mtDNA was faithfully transmitted through the germline into F2 and F3 generations. In contrast, reciprocal MR2 (PWDnDNA-B6mtDNA) produced high embryonic loss and stillborn rates, suggesting an association between mitochondrial function and infertility. These results strongly suggest that functional incompatibility between nuclear and mitochondrial genomes contributes to interspecies reproductive isolation in mammals.

Age-Related Accumulation of Somatic Mitochondrial DNA Mutations in Adult-Derived Human iPSCs.

The genetic integrity of iPSCs is an important consideration for therapeutic application. In this study, we examine the accumulation of somatic mitochondrial genome (mtDNA) mutations in skin fibroblasts, blood, and iPSCs derived from young and elderly subjects (24-72 years). We found that pooled skin and blood mtDNA contained low heteroplasmic point mutations, but a panel of ten individual iPSC lines from each tissue or clonally expanded fibroblasts carried an elevated load of heteroplasmic or homoplasmic mutations, suggesting that somatic mutations randomly arise within individual cells but are not detectable in whole tissues. The frequency of mtDNA defects in iPSCs increased with age, and many mutations were non-synonymous or resided in RNA coding genes and thus can lead to respiratory defects. Our results highlight a need to monitor mtDNA mutations in iPSCs, especially those generated from older patients, and to examine the metabolic status of iPSCs destined for clinical applications.

Practical selection methods for rat and mouse round spermatids without DNA staining by flow cytometric cell sorting.

Round spermatid injection (ROSI) into unfertilized oocytes enables a male with a severe spermatogenesis disorder to have children. One limitation of the application of this technique in the clinic is the identification and isolation of round spermatids from testis tissue. Here we developed an efficient and simple method to isolate rodent haploid round spermatids using flow cytometric cell sorting, based on DNA content (stained with Hoechst 33342 or Dye Cycle Violet) or by cell diameter and granularity (forward and side scatter). ROSI was performed with round spermatids selected by flow cytometry, and we obtained healthy offspring from unstained cells. This non-invasive method could therefore be an effective option for breeding domestic animals and human male infertility treatment. Mol. Reprod. Dev. 83: 488-496, 2016. © 2016 Wiley Periodicals, Inc.