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.

To allow selection of embryos for transfer after in vitro fertilization, ovarian stimulation is usually carried out with exogenous gonadotropins. To compensate for changes induced by stimulation, GnRH analog cotreatment, oral contraceptive pretreatment, late follicular phase human chorionic gonadotropin, and luteal phase progesterone supplementation are usually added. These approaches render ovarian stimulation complex and costly. The stimulation of multiple follicular development disrupts the physiology of follicular development, with consequences for the oocyte, embryo, and endometrium. In recent years, recombinant gonadotropin preparations have become available, and novel stimulation protocols with less detrimental effects have been developed. In this article, the scientific background to current approaches to ovarian stimulation for in vitro fertilization is reviewed. After a brief discussion of the relevant aspect of ovarian physiology, the development, application, and consequences of ovarian stimulation strategies are reviewed in detail.

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.

BACKGROUND Female cancer patients are offered 'banking' of gametes before starting fertility-threatening cancer therapy. Transplants of fresh and frozen ovarian tissue between healthy fertile and infertile women have demonstrated the utility of the tissue banked for restoration of endocrine and fertility function. Additional methods, like follicle culture and isolated follicle transplantation, are in development. METHODS Specialist reproductive medicine scientists and clinicians with complementary expertise in ovarian tissue culture and transplantation presented relevant published literature in their field of expertise and also unpublished promising data for discussion. As the major aims were to identify the current gaps prohibiting advancement, to share technical experience and to orient new research, contributors were allowed to provide their opinioned expert views on future research. RESULTS Normal healthy children have been born in cancer survivors after orthotopic transplantation of their cryopreserved ovarian tissue. Longevity of the graft might be optimized by using new vitrification techniques and by promoting rapid revascularization of the graft. For the in vitro culture of follicles, a successive battery of culture methods including the use of defined media, growth factors and three-dimensional extracellular matrix support might overcome growth arrest of the follicles. Molecular methods and immunoassay can evaluate stage of maturation and guide adequate differentiation. Large animals, including non-human primates, are essential working models. CONCLUSIONS Experiments on ovarian tissue from non-human primate models and from consenting fertile and infertile patients benefit from a multidisciplinary approach. The new discipline of oncofertility requires professionalization, multidisciplinarity and mobilization of funding for basic and translational research.

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This encapsulated 3D culture model permits further studies on the endocrine and local factors that influence primate follicle growth and oocyte maturation, with relevance to enhancing fertility preservation options in women.

In vitro ovarian follicle cultures may provide fertility-preserving options to women facing premature infertility due to cancer therapies. An encapsulated three-dimensional (3-D) culture system utilizing biomaterials to maintain cell-cell communication and support follicle development to produce a mature oocyte has been developed for the mouse. We tested whether this encapsulated 3-D system would also support development of nonhuman primate preantral follicles, for which in vitro growth has not been reported. Three questions were investigated: Does the cycle stage at which the follicles are isolated affect follicle development? Does the rigidity of the hydrogel influence follicle survival and growth? Do follicles require luteinizing hormone (LH), in addition to follicle-stimulating hormone (FSH), for steroidogenesis? Secondary follicles were isolated from adult rhesus monkeys, encapsulated within alginate hydrogels, and cultured individually for