Human germline nuclear transfer to overcome mitochondrial disease and failed fertilization after ICSI.

PURPOSE: Providing additional insights on the efficacy of human nuclear transfer (NT). Here, and earlier, NT has been applied to minimize transmission risk of mitochondrial DNA (mtDNA) diseases. NT has also been proposed for treating infertility, but it is still unclear which infertility indications would benefit. In this work, we therefore additionally assess the applicability of NT to overcome failed fertilization. METHODS: Patient 1 carries a homoplasmic mtDNA mutation (m.11778G > A). Seventeen metaphase II (MII) oocytes underwent pre-implantation genetic testing (PGT), while five MII oocytes were used for spindle transfer (ST), and one in vitro matured (IVM) metaphase I oocyte underwent early pronuclear transfer (ePNT). Patients 2-3 experienced multiple failed intracytoplasmic sperm injection (ICSI) and ICSI-assisted oocyte activation (AOA) cycles. For these patients, the obtained MII oocytes underwent an additional ICSI-AOA cycle, while the IVM oocytes were subjected to ST. RESULTS: For patient 1, PGT-M confirmed mutation loads close to 100%. All ST-reconstructed oocytes fertilized and cleaved, of which one progressed to the blastocyst stage. The reconstructed ePNT-zygote reached the morula stage. These samples showed an average mtDNA carry-over rate of 2.9% ± 0.8%, confirming the feasibility of NT to reduce mtDNA transmission. For patient 2-3 displaying fertilization failure, ST resulted in, respectively, 4/5 and 6/6 fertilized oocytes, providing evidence, for the first time, that NT can enable successful fertilization in this patient population. CONCLUSION: Our study showcases the repertoire of disorders for which NT can be beneficial, to overcome either mitochondrial disease transmission or failed fertilization after ICSI-AOA.

Cardiogenic shock in a woman with a mitochondrial cardiomyopathy: a case report.

Background: Mitochondrial cardiomyopathy (MCM) is an alteration in cardiac structure and function caused by gene mutations or deletions affecting components of the mitochondrial respiratory chain. We report a case of MCM presenting as cardiogenic shock, ultimately requiring left ventricular assist device (LVAD) placement. Case summary: A 35-year-old woman with chronic weakness and non-ischaemic cardiomyopathy, on home dobutamine, was referred to our institution for heart transplantation evaluation. She was admitted to the hospital for suspected cardiogenic shock after laboratory tests revealed a lactate level of 5.4 mmol/L (ref: 0.5-2.2 mmol/L). Her hospital course was complicated by persistently undulating lactate levels (0.2-8.6 mmol/L) that increased with exertion and did not correlate with mixed venous oxygen saturation measurements obtained from a pulmonary artery catheter. Electrodiagnostic testing demonstrated a proximal appendicular and axial myopathy. A left deltoid muscle biopsy was performed that demonstrated evidence of a mitochondrial disease on light and electron microscopy. Muscle genetic testing revealed two large-scale mitochondrial deoxyribonucleic acid sequence deletions, confirming the diagnosis of MCM. She subsequently underwent LVAD placement, which was complicated by significant right ventricular failure requiring early mechanical support. She was ultimately discharged home with chronic inotropic support. Discussion: Mitochondrial cardiomyopathy in adults is a diagnostic and therapeutic challenge. Prompt diagnosis should be made in patients with unknown causes of heart failure via skeletal muscle histopathology guided by electrodiagnostic studies, and targeted genetic testing in affected tissue. Outcomes in adult MCM patients who receive an LVAD are unknown and warrant further investigation.

Clinical application of sequencing-based methods for parallel preimplantation genetic testing for mitochondrial DNA disease and aneuploidy.

OBJECTIVE: To validate and apply a strategy permitting parallel preimplantation genetic testing (PGT) for mitochondrial DNA (mtDNA) disease and aneuploidy (PGT-A). DESIGN: Preclinical test validation and case reports. SETTING: Fertility centers. Diagnostics laboratory. PATIENTS: Four patients at risk of transmitting mtDNA disease caused by m.8993T>G (Patients A and B), m.10191T>G (Patient C), and m.3243A>G (Patient D). Patients A, B, and C had affected children. Patients A and D displayed somatic heteroplasmy for mtDNA mutations. INTERVENTIONS: Embryo biopsy, genetic testing, and uterine transfer of embryos predicted to be euploid and mutation-free. MAIN OUTCOME MEASURES: Test accuracy, treatment outcomes, and mutation segregation. RESULTS: Accuracy of mtDNA mutation quantification was confirmed. The test was compatible with PGT-A, and half of the embryos tested were shown to be aneuploid (16/33). Mutations were detected in approximately 40% of embryo biopsies from Patients A and D (10/24) but in none from Patients B and C (n = 29). Patients B and C had healthy children following PGT and natural conception, respectively. The m.8993T>G mutation displayed skewed segregation, whereas m.3243A>G mutation levels were relatively low and potentially impacted embryo development. CONCLUSIONS: Considering the high aneuploidy rate, strategies providing a combination of PGT for mtDNA disease and aneuploidy may be advantageous compared with approaches that consider only mtDNA. Heteroplasmic women had a higher incidence of affected embryos than those with undetectable somatic mutant mtDNA but were still able to produce mutation-free embryos. While not conclusive, the results are consistent with the existence of mutation-specific segregation mechanisms occurring during oogenesis and possibly embryogenesis.

Mitochondrial donation - hope for families with mitochondrial DNA disease.

In 2015, the UK became the first country to approve the use of mitochondrial donation. This novel in vitro fertilisation treatment was developed to prevent transmission of mitochondrial DNA (mtDNA) disease and ultimately give more reproductive choice to women at risk of having severely affected offspring. The policy change was a major advance that surmounted many scientific, legislative and clinical challenges. Further challenges have since been addressed and there is now an NHS clinical service available to families with pathogenic mtDNA mutations that provides reproductive advice and options, and a research study to look at the outcome at 18 months of children born after mitochondrial donation.

Clinical features of the pathogenic m.5540G>A mitochondrial transfer RNA tryptophan gene mutation.

Mitochondrial DNA disease is one of the most common groups of inherited neuromuscular disorders and frequently associated with marked phenotypic and genotypic heterogeneity. We describe an adult patient who initially presented with childhood-onset ataxia without a family history and an unremarkable diagnostic muscle biopsy. Subsequent multi-system manifestations included basal ganglia calcification, proteinuria, cataract and retinitis pigmentosa, prompting a repeat muscle biopsy that showed features consistent with mitochondrial myopathy 13 years later. She had a stroke with restricted diffusion change in the basal ganglia and internal capsule at age 44 years. Molecular genetic testing identified a previously-reported pathogenic, heteroplasmic mutation in the mitochondrial-encoded transfer RNA tryptophan (MT-TW) gene which based on family studies was likely to have arisen de novo in our patient. Interestingly, we documented an increase in the mutant mtDNA heteroplasmy level in her second biopsy (72% compared to 56%), reflecting the progression of clinical disease.

Preventive SNP-SNP interactions in the mitochondrial displacement loop (D-loop) from chronic dialysis patients.

Chronic dialysis association study involving individual single nucleotide polymorphisms (SNPs) in the mitochondrial displacement loop (D-loop) has previously been reported. However, possible SNP-SNP interactions for SNPs in the D-loop which could be associated with a reduced risk for chronic dialysis were not investigated. The purpose of this study was to propose an effective algorithm to identify protective SNP-SNP interactions in the D-loop from chronic dialysis patients. We introduce ISGA that uses an initialization strategy for genetic algorithms (GA) to improve the computational analysis for protective SNP-SNP interactions. ISGA generates genotype patterns with combined SNPs (SNP barcodes) for chronic dialysis. Using our previously reported 77 SNPs in the D-loop, the algorithm-generated protective SNP barcodes for chronic dialysis were evaluated. ISGA provides the SNP barcodes with the maximum frequency differences of occurrence between the cases and controls. The identified SNP barcodes with the lowest odds ratio (OR) values were regarded as the best preventive SNP barcodes against chronic dialysis. The best ISGA-generated SNP barcodes (two to nine SNPs) are more closely associated with the prevention of chronic dialysis when more SNPs are chosen (OR=0.64 to 0.32; 95% confidence interval=0.882 to 0.198). The cumulative effects of SNP-SNP interactions were more dominant in ISGA rather than in GA without the initialization strategy. We provide a fast identification of chronic dialysis-associated protective SNP barcodes and demonstrate that the SNP-SNP interactions may have a cumulative effect on prediction for chronic dialysis.