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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. CRISPR–Cas9 genome editing is used to induce a DNA repair response and correct a disease-causing heterozygous mutation in human embryos with reduced mosaicism and preferential repair using the wild-type copy of the gene. CRISPR-corrected mutation Genome editing could be applied to correct disease-causing mutations in human embryos, but concerns about efficacy and safety are paramount. Shoukhrat Mitalipov and colleagues use CRISPR–Cas9 to correct a heritable cardiomyopathy mutation in human embryos. By optimizing the experimental conditions, the authors show very reduced mosaicism, and report that for this heterozygous mutation, CRISPR–Cas9-induced breaks seem to be preferentially repaired using the wild-type allele as a template in human embryos. The results advance our understanding of the promises and challenges of editing the human germline.

The sperm of infertile men have higher rates of chromosomal abnormalities than those of fertile men. Miscarriage rate is also higher following testicular sperm extraction combined with intracytoplasmic sperm injection (TESE-ICSI). Sperm chromosomal abnormalities are assumed to be the cause of miscarriages. Previous testicular sperm karyotyping studies have only examined a few selected chromosomes using fluorescence in situ hybridization. The aim of this study was to provide a more detailed analysis of sperm karyotyping by analyzing all chromosomes using next-generation sequencing (NGS) in clinically usable testicular sperm. Sperm discarded after clinical use was collected for NGS. Additionally, sperm were individually collected by micromanipulation from patients with obstructive azoospermia (OA) and non-obstructive azoospermia (NOA) who underwent TESE-ICSI. For comparison, ejaculated sperm from control and balanced translocation (BT) carriers were examined. Karyotyping was performed on individual sperm cells using NGS. The number of normal and aberrant sperm was compared. Seventeen patients participated in this study: control (n = 4), BT (n = 3), OA (n = 5), and NOA (n = 5). Ten sperm samples per patient were analyzed. The total acquisition rate for single sperm karyotyping was 85% (145/170). Karyotyping of sperm from the BT group revealed sperm with unbalanced chromosomes derived from carrier translocations. Among the NOA group, 7/41 (17%) sperm samples exhibited aberrant karyotypes, whereas no aberrant sperm were identified in the control and OA groups. Individual differences were observed in the frequency of sperm chromosomal abnormalities among patients with NOA. In conclusion, sperm chromosomal abnormalities are frequently observed in patients with NOA even after sperm selection for clinical use. As the frequency of chromosomal abnormalities varies among patients with NOA, single sperm sequencing may help identify patients with NOA most likely to benefit from PGT-A.

IntroductionBisphosphonates and denosumab increase bone mineral density (BMD) for osteoporosis treatment in patients with aromatase inhibitor-associated bone loss (AIBL). This study aimed to directly compare bisphosphonates with denosumab in treating patients with AIBL and to determine the effect of denosumab on the trabecular bone score (TBS).Materials and methodsThirty-nine patients with AIBL receiving osteoporosis treatment (21 in the bisphosphonates group and 18 in the denosumab group) were retrospectively evaluated for changes in lumbar spine and femoral BMD, lumbar spine bone quality (assessed by TBS), and blood bone metabolic markers. The Mann–Whitney and Wilcoxon tests were used for statistical evaluation.ResultsAfter 24 months of treatment, the lumbar spine BMD change rate was 5.82 ± 1.10% with bisphosphonates and 10.49 ± 1.20% with denosumab, with the change rate of denosumab significantly increasing over that of bisphosphonates. The change rate in femoral BMD was 2.69 ± 1.16% with bisphosphonates and 2.95 ± 1.26% with denosumab, with no significant difference between the two groups. The rate of decrease in tartrate-resistant acid phosphatase isoform 5b was significantly higher in the denosumab group. The change rate in TBS at 24 months of treatment was 0.53 ± 1.26% in the bisphosphonates group and 1.08 ± 1.33% in the denosumab group, with no significant difference between the two groups. After 24 months, TBS remained stable.ConclusionBoth bisphosphonates and denosumab may increase BMD, improve bone metabolism, and inhibit bone quality loss in patients with AIBL.

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. Summary sentence Our studies show that high heteroplasmy deleterious mtDNA mutations presenting in mature mouse oocytes are eliminated during post-implantation development. The occurrence of purifying selection against deleterious mtDNA mutations during fetal development plays an important role in preventing the accumulation of pathogenic mutations that would cause consequences to species survival.

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.

After the reflected light is captured and processed, the information is digitally projected onto the surface of the skin. [...]it provides a real time, accurate image of the patient's venous pattern. [...]the conventional method of distinguishing between veins and arteries by checking the presence or absence of pulsation of blood vessels using Doppler ultrasonography is time consuming; however, this device can facilitate distinction on direct visualisation. Because identification of blood vessels is repeated 30–40 times in microscopic varicocelectomy, this device can be expected to shorten operating times. [...]the effect of assisted reproductive techniques on the improvement of sperm quality and outcomes was not evaluated.

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.

Fair comparison of reprogramming efficiencies and in vitro differentiation capabilities among induced pluripotent stem cell (iPSC) lines has been hampered by the cellular and genetic heterogeneity of de novo infected somatic cells. In order to address this problem, we constructed a single cassette all-in-one inducible lentiviral vector (Ai-LV) for the expression of three reprogramming factors (Oct3/4, Klf4 and Sox2). To obtain multiple types of somatic cells having the same genetic background, we generated reprogrammable chimeric mice using iPSCs derived from Ai-LV infected somatic cells. Then, hepatic cells, hematopoietic cells and fibroblasts were isolated at different developmental stages from the chimeric mice, and reprogrammed again to generate 2nd iPSCs. The results revealed that somatic cells, especially fetal hepatoblasts were reprogrammed 1200 times more efficiently than adult hepatocytes with maximum reprogramming efficiency reaching 12.5%. However, we found that forced expression of c-Myc compensated for the reduced reprogramming efficiency in aged somatic cells without affecting cell proliferation. All these findings suggest that the Ai-LV system enables us to generate a panel of iPSC clones derived from various tissues with the same genetic background, and thus provides an invaluable tool for iPSC research.

Analysis of mitochondrial replacement therapy shows, even with efficient mutant mitochondrial DNA replacement and maintenance in embryonic stem cells, a gradual loss of donor mitochondrial DNA in some lines owing to a polymorphism in the D-loop, potentially causing preferential replication of specific mitochondrial DNA haplotypes. A new mitochondrial replacement technique Mitochondrial replacement techniques (MRT) could potentially be used to avoid mother-to-child transmission of mitochondria carrying disease-causing mutations. Shoukhrat Mitalipov and colleagues have investigated the outcome of MRT using oocytes from women from families with common mtDNA-associated syndromes and by transferring meiotic spindle from patient oocytes to healthy donor oocytes. Although donor mtDNA replaced the patient mtDNA efficiently and was stably maintained in embryonic stem cells (ES cells) derived from most embryos, some ES cell lines lost donor mtDNA. The authors' analysis suggests that polymorphisms in mtDNA could be associated with preferential replication and could be cause the amplification of specific maternal haplotype. Maternally inherited mitochondrial (mt)DNA mutations can cause fatal or severely debilitating syndromes in children 1 , 2 , 3 , with disease severity dependent on the specific gene mutation and the ratio of mutant to wild-type mtDNA (heteroplasmy) in each cell and tissue 4 . Pathogenic mtDNA mutations are relatively common, with an estimated 778 affected children born each year in the United States 5 . Mitochondrial replacement therapies or techniques (MRT) circumventing mother–to–child mtDNA disease transmission involve replacement of oocyte maternal mtDNA 6 , 7 , 8 . 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 8 , 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.