In vitro maturation (IVM) of immature oocytes is an important reproductive technology that generates mature oocytes that are capable of supporting preimplantation embryo development and full development to turm. The developmental competence of oocytes after in vitro fertilization (IVF) was supported in cumulus-cell density dependent manner. The developmental competence of oocytes matured under low oxygen (5% O2) tension was higher than that of oocytes matured under high oxygen tensinon (20% O2) due to the decrease of reactive oxygen content in the oocyte. To expand the availability of female gamete, an attempt to culture the growing oocytes have been made. We have focused attention on the mitochondrial activity of in vitro grown (IVG) oocytes and shown that L-Carnitine, which acts as a carrier of fatty acids across the inner mitochondrial membrane for subsequent β-oxidation, supported the viability of growing oocytes. Nowadays, IVM is gaining acceptance in assisted reproductive technology in cattle and human.

One of the most critical issues to be solved in reproductive medicine is the treatment of patients with multiple failures of assisted reproductive treatment caused by low-quality embryos. This study investigated whether mitochondrial transfer to human oocytes improves embryo quality and provides subsequent acceptable clinical results and normality to children born due to the use of this technology. We transferred autologous mitochondria extracted from oogonia stem cells to mature oocytes with sperm at the time of intracytoplasmic sperm injection in 52 patients with recurrent failures (average 5.3 times). We assessed embryo quality using the following three methods: good-quality embryo rates, transferable embryo rates, and a novel embryo-scoring system (embryo quality score; EQS) in 33 patients who meet the preset inclusion criteria for analysis. We also evaluated the clinical outcomes of the in vitro fertilization and development of children born using this technology and compared the mtDNA sequences of the children and their mothers. The good-quality embryo rates, transferable embryo rates, and EQS significantly increased after mitochondrial transfer and resulted in 13 babies born in normal conditions. The mtDNA sequences were almost identical to the respective maternal sequences at the 83 major sites examined. Mitochondrial transfer into human oocytes is an effective clinical option to enhance embryo quality in recurrent in vitro fertilization-failure cases.

Does the human embryo growth rate affect the outcome of vitrified–warmed blastocyst transfer? Following vitrification, the incidence of abnormal spindle morphology was increased and the implantation competence was decreased in growth-retarded embryos compared with normally developing embryos. Various types of spindle abnormality occur in human cleavage- and blastocyst-stage embryos. However, the incidence of abnormal spindle morphology in growth-retarded blastocysts is not known. Furthermore, there is conflicting data about the implantation potential of such blastocysts. This was a retrospective cohort study including 878 single vitrified–warmed blastocyst transfers between 9 January 2010 and 10 July 2012, and an experimental study using 121 vitrified–warmed blastocysts donated to research. A comparison on the implantation potential and spindle shape of vitrified–warmed blastocysts was made between normally developing and growth-retarded blastocysts. In the clinical study, we compared the implantation rates of vitrified–warmed embryos that developed to the blastocyst stage on Day 5 after insemination (normally developing embryos) with those that required culture to Day 6 (growth-retarded embryo). In the experimental study, donated vitrified–warmed blastocysts were immunostained with an anti-α-tubulin antibody to visualize microtubules, an anti-γ-tubulin antibody to image centrosomes and Hoechst 33342 or 4,6-diamidino-2-phenylindole to visualize DNA. Confocal image analysis captured a z-series stack of 0.5-µm-thick optical sections encompassing the entire blastocyst. Only spindles with fusiform poles and with chromosomes aligned at the equator were classified as normal. The implantation rate of growth-retarded embryos (47%, n = 270) was significantly lower (P < 0.05) than that of normally developing embryos (57%, n = 608). A total of 533 spindles were analyzed in Day 5 and 6 vitrified–warmed blastocysts. The incidence of abnormal spindles in the growth-retarded embryos (47%, n = 274) was significantly higher (P < 0.01) than in the normally developing embryos (30%, n = 259). Further studies are required to clarify the link between an increase in abnormal spindle formation and a decrease in embryonic implantation potential. This study provided new insights into the possible implications of abnormalities in spindle formation in growth-retarded human blastocysts. Part of this work was supported by a grant from the Japan Society for the Promotion of Science (JPS-RFTF 23 580 397 to S.H.). No other competing interests are declared.

In this study, we aimed to develop a model for embryo selection based on oxygen consumption following cryopreservation, the relationship between the developmental competence of blastocysts and their oxygen consumption was assessed. Oxygen consumption of vitrified-warmed human blastocysts was measured at 0, 1.5, 3, 4.5, 6, 7.5, 9 and 24 h after warming using scanning electrochemical microscopy. On the basis of their developmental stage at 24 h, blastocysts were classified into four groups (hatched, hatching, arrested and degenerated). Moreover, cytochrome c oxidase (CCO) activity in vitrified-warmed blastocysts was assessed at 0 and 24 h. The oxygen consumption rate of blastocysts just after warming was significantly lower than that of non-vitrified blastocysts (P< 0.05). The oxygen consumption rate of blastocysts was significantly higher in the hatched group than in the arrested and the degenerated groups after 1.5 h (P< 0.05) and than in the hatching group (P< 0.05) at 7.5 and 9 h. Moreover, CCO activity was absent in vitrified-warmed blastocysts at 0 h, but was detected at 24 h. The respiratory rate of vitrified blastocysts after warming was significantly lower than non-cryopreserved blastocysts. Oxygen consumption of blastocysts with high developmental potential was restored earlier than that of blastocysts with low developmental potential. The results of this study suggest that it is possible to select vitrified-warmed blastocysts with high developmental potential based on their respiratory activity.