Mutations in mitochondrial DNA (mtDNA) are associated with serious human diseases and inherited from mother's eggs. Here we investigated the feasibility of mtDNA replacement in human oocytes by spindle transfer (ST). Of 106 human oocytes donated for research, 65 were subjected to reciprocal ST and 33 served as controls. Fertilization rate in ST oocytes (73%) was similar to controls (75%). However, a significant portion of ST zygotes (52%) displayed abnormal fertilization as determined by irregular number of pronuclei. Among normally fertilized ST zygotes, blastocyst development (62%) and embryonic stem cell (ESC) isolation (38%) rates were comparable to controls. All ESC lines derived from ST zygotes displayed normal euploid karyotypes and contained exclusively donor mtDNA. The mtDNA can be efficiently replaced in human oocytes. Although some ST oocytes displayed abnormal fertilization, remaining embryos were capable of developing to blastocysts and producing ESCs similar to controls.

SUMMARY Reprogramming somatic cells into pluripotent embryonic stem cells (ESCs) by somatic cell nuclear transfer (SCNT) has been envisioned as an approach for generating patient-matched nuclear transfer (NT)-ESCs for studies of disease mechanisms and for developing specific therapies. Past attempts to produce human NT-ESCs have failed secondary to early embryonic arrest of SCNT embryos. Here, we identified premature exit from meiosis in human oocytes and suboptimal activation as key factors that are responsible for these outcomes. Optimized SCNT approaches designed to circumvent these limitations allowed derivation of human NT-ESCs. When applied to premium quality human oocytes, NT-ESC lines were derived from as few as two oocytes. NT-ESCs displayed normal diploid karyotypes and inherited their nuclear genome exclusively from parental somatic cells. Gene expression and differentiation profiles in human NT-ESCs were similar to embryo-derived ESCs, suggesting efficient reprogramming of somatic cells to a pluripotent state.

We wish to correct a number of figure-related and typographical errors that appeared in the article above. None of these errors affect the conclusions of the paper.In Figures 2F and S5 (upper-right), we presented two phase-contrast photos of fields of cells, correctly labeled as SCNT-derived hESO-NT1 and IVF-derived hESO-7, respectively. These images are the same fields of cells shown in the top two images of Figure 6D; however, in Figure 6D, we inadvertently switched the labels on the images. This re-use of the images was intentional, but we should have indicated this in the original legend for Figure 6. We have corrected the labeling error in Figure 6D.In Figure S6, the scatterplot presenting a comparison between biological HDF-f replicates #2 and #3 is an inadvertent duplication of the scatterplot presenting the comparison of HDF-f replicates #1 and #3. This plot has been replaced in the figure online and is shown below.In Figure 1, the number of SCNT embryos for I/DMAP group (n = 51) has been corrected to 53.In Figure 5D, the numbers of plated blastocysts for agonist and antagonist were reversed and have been corrected to agonist (n = 4) and antagonist (n = 17).In the Experimental Procedures, the age range of oocyte donors in the paper was listed as 23-31; however, the range has been corrected to 23-33.In Table S2, percentages for fused oocytes in the 10 nM TSA for 24 hr group (95.4) and for compact morula (CM) in the 5 nM TSA for 12 hr group (26.0) have been corrected to 96.3 and 28.0, respectively.In Table S3, we incorrectly reported several figures due to errors that occurred in converting the raw patient data, from which these values are calculated, from a file created with Mac-based software to a file in the analogous Windows-based software. The following corrections have been made: number of oocytes in the antagonist group 11.7 ± 5.6 has been changed to 10.2 ± 4.9; number of oocytes in the agonist group, 20.5 ± 11.9 to 16.3 ± 5.2; AMH level in the antagonist group, 2.8 ± 0.5 to 2.5 ± 0.5; AFC in the antagonist group, 23.1 ± 7.2 to 23.2 ± 7.2; FSH dosage in antagonist group, 958.3 ± 241.7 to 966.7 ± 247.3; number of hMG ampoules in antagonist group, 8.5 ± 1.6 to 10.2 ± 4.2; number of hMG ampoules in agonist group, 8.8 ± 0.9 to 8.8 ± 1.0; stimulation days in antagonist group, 8.7 ± 1.6 to 8.7 ± 0.8; and stimulation days in agonist group, 9 ± 0.8 to 9.8 ± 1.0. We have confirmed that these differences do not affect any of the statistical conclusions originally reported.In Table S4, short tandem repeats (STR) readings for egg donor A in D6S291 and D6S276 loci were reversed and have been corrected to 199/209 for D6S291 and 245/249 for D6S276.

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Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) originating from the follicle or corpus luteum may be a physiological regulator of ovulation and neovascularization of luteinizing tissue, as well as a pathological factor in the development of ovarian hyperstimulation syndrome (OHSS). The objective of this study was to quantify VEGF production by human luteinized granulosa cells in vitro and to determine if gonadotrophin stimulates VEGF production directly and/or indirectly via enhanced synthesis of progesterone. In study 1, luteinized granulosa cells collected from women undergoing ovarian stimulation for in-vitro fertilization were cultured in the presence and absence of human chorionic gonadotrophin (HCG; 100 ng/ml) and/or low density lipoprotein (LDL; 100 microg protein/ml). In study 2, the progesterone synthesis inhibitor trilostane (250 ng/ml) and/or a progesterone receptor antagonist ZK137.316 (3.2 microM) were also added. Medium was harvested on days 1, 3, 5, 7 and 9 of culture and assayed for VEGF and progesterone. Results of study 1 were divided into two categories based on control concentrations of VEGF on day 1: 'low producers' (n = 6; <750 pg VEGF/ml) and 'high producers' (n = 5; >1000 pg VEGF/ml; P < 0.01). VEGF concentrations in cultures of both low and high producers increased (P < 0.01) from day 1 to maximal values on day 3, then steadily declined through to day 9. Chronic exposure to LDL or HCG increased (P < 0.05) VEGF concentrations in cultures of low producers by day 3 and day 5 respectively. In contrast, LDL did not alter VEGF concentrations in cultures of high producers and HCG did not increase VEGF concentrations until day 7. Nevertheless, acute exposure to HCG beginning on day 7 increased (P < 0.05) VEGF concentrations 3-fold in cultures of low or high producers. In study 2, trilostane treatment decreased (P < 0.05) progesterone concentrations by 91% on day 1 of culture but had no effect on VEGF concentrations on any day. ZK137.316 alone or with trilostane did not affect VEGF synthesis. These results suggest that VEGF production by luteinized granulosa cells is enhanced by gonadotrophin (HCG) independent of gonadotrophin-stimulated progesterone synthesis. These data are consistent with the hypothesis that the exacerbation of OHSS in early pregnancy is mediated by the CG stimulation of luteal VEGF production.