Derivation of novel human ground state naive pluripotent stem cells.

Mouse embryonic stem (ES) cells are isolated from the inner cell mass of blastocysts, and can be preserved in vitro in a naive inner-cell-mass-like configuration by providing exogenous stimulation with leukaemia inhibitory factor (LIF) and small molecule inhibition of ERK1/ERK2 and GSK3ß signalling (termed 2i/LIF conditions). Hallmarks of naive pluripotency include driving Oct4 (also known as Pou5f1) transcription by its distal enhancer, retaining a pre-inactivation X chromosome state, and global reduction in DNA methylation and in H3K27me3 repressive chromatin mark deposition on developmental regulatory gene promoters. Upon withdrawal of 2i/LIF, naive mouse ES cells can drift towards a primed pluripotent state resembling that of the post-implantation epiblast. Although human ES cells share several molecular features with naive mouse ES cells, they also share a variety of epigenetic properties with primed murine epiblast stem cells (EpiSCs). These include predominant use of the proximal enhancer element to maintain OCT4 expression, pronounced tendency for X chromosome inactivation in most female human ES cells, increase in DNA methylation and prominent deposition of H3K27me3 and bivalent domain acquisition on lineage regulatory genes. The feasibility of establishing human ground state naive pluripotency in vitro with equivalent molecular and functional features to those characterized in mouse ES cells remains to be defined. Here we establish defined conditions that facilitate the derivation of genetically unmodified human naive pluripotent stem cells from already established primed human ES cells, from somatic cells through induced pluripotent stem (iPS) cell reprogramming or directly from blastocysts. The novel naive pluripotent cells validated herein retain molecular characteristics and functional properties that are highly similar to mouse naive ES cells, and distinct from conventional primed human pluripotent cells. This includes competence in the generation of cross-species chimaeric mouse embryos that underwent organogenesis following microinjection of human naive iPS cells into mouse morulas. Collectively, our findings establish new avenues for regenerative medicine, patient-specific iPS cell disease modelling and the study of early human development in vitro and in vivo.

Total fertilization failure in intra-cytoplasmic sperm injection cycles--classification and management.

In this retrospective cohort study we intended to propose a classification and preliminary management strategy for couples exhibiting total fertilization failure (TFF) in intra-cytoplasmic sperm injection (ICSI) cycles. Sixteen couples with a total of 27 cycles exhibiting TFF, age <40 and/or more than four M2 oocytes aspirated were enrolled. While TFF occurred in 4.3% of all 3723 ICSI cycles, in women younger than 40 with at least 5 M2 oocytes the TFF rate was 0.7%. Indications for ICSI were severe male factor and unexplained infertility. Of the 16 couples with TFF, 4 demonstrated a single episode of TFF, with either subsequent or former normal fertilizations, thus implying possible sporadic faulty laboratory conditions. Ten couples demonstrated repeated total or very low fertilization rates, hinting at a gamete defect not overcome by ICSI. Two couples experienced TFF in the first and only cycle performed at our unit. We conclude that initial and repeated TFF hints at severe gamete defects for which only donor gametes may prove successful while sporadic TFF events could simply imply a technical modifiable condition.

Effects of laser polar-body biopsy on embryo quality.

OBJECTIVE: To evaluate the effect of laser polar-body biopsy (PBB) for preimplantation genetic diagnosis on embryo quality. STUDY DESIGN: Retrospective case-control analysis. The quality of 145 embryos after PBB was compared to 276 embryos of the same group of women without biopsy. SETTING: University-based tertiary-care medical center. PATIENT(S): Women with inherited genetics disease. INTERVENTION(S): Laser PBB of IVF embryos for genetic diagnosis. MAIN OUTCOME MEASURE(S): The study and control embryos were compared for fertilization rate, pronuclear grading, and cleavage-stage parameters on days 1, 2, and 3 after oocyte retrieval. RESULT(S): The study embryos demonstrated higher rates of cleavage arrest (3.6% vs. 0.7%), higher rate of significant fragmentation on day 2 (9.5% vs. 3.0%), and lower rate of good cleavage embryos on day 2 (69.1% vs. 78.4%) compared with control embryos. On day 3, the study embryos had lower cleavage rates (six or more blastomeres; 56.5% vs. 74.5%), higher fragmentation (11.7% vs. 3.9%), higher rate of embryos presenting inferior cleavage pattern (57.2% vs. 38.5%), and lower mean blastomere number (5.8 ± 2.1 vs. 6.6 ± 1.9) compared with control embryos. CONCLUSION(S): Polar-body biopsy may have a negative effect on embryo quality.

Retrieval of immature oocytes after chemotherapy for Hodgkin's disease and prolonged ovarian down-regulation with gonadotropin-releasing hormone agonist.

OBJECTIVE: To describe isolation and in vitro maturation of primary oocytes from the ovarian cortex in the presence of hypothalamic pituitary down-regulation. DESIGN: Case report. SETTING: Tertiary care university-affiliated hospital. PATIENT(S): An 18-year-old patient was given treatment with the ABVD (Adriamycin, bleomycin, vinblastine, and dacarbazine) protocol for Hodgkin's disease. She underwent ovarian tissue cryopreservation while being cotreated with GnRH agonist because of disease relapse. INTERVENTION(S): Laparoscopic oophorectomy, ovarian tissue cryopreservation, and in vitro maturation of primary oocytes. MAIN OUTCOME MEASURE(S): Maturation of primary oocytes isolated from the medium used for preparation of ovarian tissue. RESULT(S): Twenty-one immature germinal vesicle-stage oocytes were isolated from the medium of dissection. All were incubated in in vitro maturation medium, and five were maturated and frozen. CONCLUSION(S): The fact that germinal vesicle-stage oocytes were present in our patient's medium despite hormonal down-regulation demonstrates that GnRH agonist does not completely inhibit antral follicle development.

Elucidation of abnormal fertilization by single-cell analysis with fluorescence in situ hybridization and polymorphic marker analysis.

OBJECTIVE: To analyze the genetic composition of oocytes and embryos presenting abnormal fertilization. DESIGN: Case report. SETTING: In vitro fertilization unit of a university-affiliated hospital. PATIENT(S): A couple with unexplained infertility with abnormal fertilizations in nine failed IVF-intracytoplasmic sperm injection cycles characterized by the presence of embryos with only one pronucleus and nonextrusion of the second polar body. INTERVENTION(S): All first polar bodies and blastomeres were analyzed by fluorescence in situ hybridization for chromosomes 13, 18, 21, X, and Y. Because some of the one-pronucleus embryos were found to be diploid, they were subjected further to single-cell polymerase chain reaction analysis with use of a panel of highly polymorphic markers from chromosomes 6, 7, 17, 19, X, and Y. MAIN OUTCOME MEASURE(S): Fluorescence in situ hybridization analysis of polar bodies and blastomeres and polymorphic marker analysis of day 5 embryos. RESULT(S): Fluorescence in situ hybridization analysis of the first polar body demonstrated normal segregation of chromosomes in meiosis I. Fluorescence in situ hybridization analysis of two aspirated blastomeres demonstrated three diploid and two mosaic triploid embryos. Polymorphic marker analysis, however, demonstrated that all embryos, including the diploid ones, had two sets of maternal alleles. CONCLUSION(S): Single-cell genetic analysis routinely used for preimplantation genetic diagnosis may provide insight into the genetic composition of oocytes and embryos. These data may be used in cases of abnormal fertilization or impaired embryo development for evidence-based fertility counseling regarding prognosis and treatment options.