Morphological evaluation has been widely used to evaluate embryo quality because it is non-invasive and useful in predicting pregnancy rate. However, morphological evaluations are subjective and categorization standards often vary between investigators. The respiration rate of embryos is a useful parameter for evaluating embryo quality. The scanning electrochemical microscopy (SECM) measuring system provides a non-invasive, simple, accurate, and consistent measurement of the respiration activity of human embryos. After morphological evaluation by Veeck's method, oxygen consumption by individual human embryos was quantified by SECM. Fundamentally, the maturation of mitochondria correlated with an increase in oxygen consumption during the development of embryos. The development of mitochondria may be an important factor in embryo quality, because mitochondria provide ATP for embryonic development by metabolism of nutrients in the cytoplasm. The respiration rates on the day 3 after in vitro fertilization (IVF) were measured and significant differences in oxygen consumption were registered even among embryos with the same morphological classification. There were no significant differences between the mean rates of oxygen consumption at each cleavage stage, however, there was considerable variation in respiration rate within embryos of the same morphological grade. The safety of SECM is assured as the embryos which were examined by SECM for oxygen consumption showed the same development levels as the control group. These results support the hypothesis that measuring embryonic respiration provides additional and valuable information about embryo quality.

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To enable improved success rates of IVF, we developed the technology to optimize embryo selection with the highest implantation potential while ensuring no damage to embryos using zona elasticity as the selection criterion. In this communication we outline the biomechanics and safety of zona pellucida (ZP) elasticity measurement using the micro tactile sensor (MTS) system and demonstrate the specific changes in ZP elasticity during oocyte maturation, fertilization, and early embryo development. Zona hardening was demonstrated mechanically following fertilization at the pronuclear (PN) stage, and Young's modulus decreased gradually as the embryo developed. Evaluation of the quality of expanded blastocysts (EPB) showed that the quality of EPBs could also be evaluated from elasticity parameters. Furthermore, the observations indicated that ZPs of embryos generated in vivo were significantly harder than those of embryos generated in vitro at each stage. Preliminary results also indicated that denuded oocytes matured in vitro did not show zona hardening following parthenogenetic activation by strontium chloride, suggesting that sufficient maturation and consequent oocyte activation may be evaluated by increases in ZP elasticity. We conclude that MTS-elective single embryo transfer can be applied to human assisted reproductive technology to enable embryo quality evaluation in both early embryos and EPBs.

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Soluble HLA-G (sHLA-G) has been reported as a biomarker for embryo quality in human in vitro fertilization (IVF). The first report showed that some embryos had secreted sHLA-G and that the secretion was necessary for implantation. If these data are true, sHLA-G could be a very useful marker for embryo selection. However, one major limitation of the report was a distinct lack of controls relevant to the methods of measurement. Therefore, we re-examined the detection of sHLA-G in 109 culture supernatants from embryos fertilized in vitro. We were unable to detect sHLA-G in any supernatant despite using a more sensitive method than that used in the previous report. We further explored other reports that had shown positive data of sHLA-G secretion from embryos, and found that neither standard curves to enable calculation of sHLA-G concentration correctly nor any data for calibration were available in these reports. In this review, we point out several problems with the detection of sHLA-G in the previous reports, and describe controls and methods that can be used to determine sHLA-G concentrations accurately.

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One of the major dilemmas surrounding assisted reproductive technologies (ART) is the inability to precisely estimate the reproductive potential of individual embryos. A number of new assessment techniques are currently being developed to address this dilemma, including techniques using genomic, proteomic and metabolomic approaches. Here we describe a metabolomic approach to embryo assessment that has the ability to distinguish specific biomarkers in embryo culture media. This technology assesses modifications of the chemical composition of the embryo's surrounding medium using a rapid Near Infra Red (NIR) spectroscopy approach and generates a value termed the "viability or Via Test-E™ score". We also report relationships between the algorithm generated Via Test-E™ score and morphology assessment on individual day 2 embryos selected for single embryo transfer and the implantation rates when a cut off score for the viability score is used in relation to implantation. The added armoury of metabolomic profiling by NIR to current morphological assessment techniques might allow a greater discrimination for selection of embryos for transfer and has the potential to improve IVF outcomes. Currently, additional studies are being performed to investigate the true value of metabolomic profiling by NIR spectroscopy.

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The objective of the present study was to compare the vitrification method for cryopreservation of canine oocytes. Canine cumulus-oocyte complexes (COCs) were collected from ovaries, and were vitrified by ethylene glycol based (E30S) or DMSO based (DAP213) methods. In the E30S method, COCs were exposed to the vitrification solution, composed of 30% ethylene glycol and 0.5 M sucrose, step-wise transferred onto a cryotop holder, then plunged directly into liquid nitrogen. In the DAP213 method, COCs were exposed to 1 M DMSO and DAP213 solution in a cryotube, and thereafter plunged directly into liquid nitrogen. Although vitrified-warmed COCs in the E30S method showed fewer morphological abnormalities, and higher viability than those in the DAP213 method, there was no significant difference in between. These results indicate that either method of vitrification is available and statistically comparable for cryopreservation of canine oocytes.

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In normal development, a limited number of cells in the proximal epiblast differentiate into germ cells. In this process, the interaction between primordial germ cells (PGCs) and surrounding somatic cells in the gonad is essential for germ cell development. In the present study, we demonstrated a novel method for inducing mouse embryonic stem cells (ESCs) to differentiate into PGCs using co-culture with undifferentiated gonadal cells or with their extracts. After embryoid bodies (EBs) formed, they were cultured for 10 days. Mvh and Dazla gene expressions increased to a peak, and also the number of Mvh-positive cells increased and were distributed in the peripheral region of EBs co-cultured with undifferentiated gonadal cells, whereas for EBs cultured in the medium supplemented with cell extracts, Mvh-positive cells were scattered within the EBs. Our results demonstrate that growth factors derived from undifferentiated gonadal cells may induce differentiation of ESCs into potential PGCs.

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In mouse oocytes soon after collection, perivitelline space could be seen in about half of them and its mean size was 0.28 μm. After 6 hrs of in vivo and in vitro maturation, perivitelline space appeared in all oocytes. In oocytes in the process of maturation in vivo, the mean size of perivitelline space significantly increased 6 hrs after hCG injection and reached 7.00 μm at 14 hrs after the hCG injection. On the other hand, perivitelline space in cultured oocytes enlarged significantly at 2 hrs culture. Thereafter, perivitelline space in oocytes continued to enlarge with culture time and reached 5.40 μm at 14 hrs of maturation culture. The size of perivitelline space in oocytes 14 hrs after the hCG injection was significantly larger than that in oocytes cultured for 14 hrs. The fertilization rates of oocytes matured in vivo (85%) did not differ from that of oocytes matured in vitro (80%). However, the incidence of polyspermy in oocytes matured in vivo was 4%, which was significantly lower than the 16% of oocytes matured in vitro. These findings suggest there could be a relationship between the size of the perivitelline space and the incidence of polyspermy in mouse oocytes.

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The local circumstances of the ovary may influenced by the presence of corpus luteum (CL). We investigated the cleavage rate and blastocyst development of bovine oocytes collected from ovaries with or without CL. Oocytes were incubated with brilliant cresyl blue (BCB) for 90 min and separated on the basis of low (BCB+) or high (BCB-) activity of glucose-6-phosphate dehydrogenase. For both types of ovaries, the embryo cleavage rates in the case of BCB+ oocytes were significantly higher than those in the case of BCB- oocytes, but were not higher than the rates in the control group. The percentage of blastocysts developing from BCB+ and BCB- oocytes from the ovaries with CL did not significantly differ. However, in the case of ovaries without CL, a significantly higher (P<0.05) percentage of blastocysts developed from BCB+ oocytes than from BCB- and control group oocytes. The presence or absence of CL did not significantly influence the cleavage rate and blastocyst development. Based on our results, we conclude that BCB staining facilitates the selection of competent oocytes that will develop into blastocysts for IVF better than conventional morphological selection methods. We also conclude that the CL does not significantly influence blastocyst development.

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It has been established that mouse oocytes grow under appropriate culture conditions. To be able to utilize a broad range of growing oocytes, a way of estimating the optimal culture period is required. In the present study, a detailed analysis was conducted of the relationship between the initial oocyte size and necessary length of culture period to achieve full growth. Oocyte-granulosa cell complexes were obtained from juvenile mice, and classified into three groups according to oocyte size: 50.0-54.5, 55.0-59.5, and 60.0-64.5 μm. The complexes were cultured for various periods up to 12 days. Mean oocyte size increased in a linear fashion, eventually reaching the full size for mouse oocytes. Meiotic competence was acquired during the last 4 days of culture, when oocytes approached full size in all size classes. During this period, the rate of oocytes incompetent to resume meiosis decreased sharply, while that of oocytes having competence to progress to metaphase II increased. This relationship was particularly clear cut in the large- and middle-size classes. Thus, appropriate length of culture period is mostly determined by the oocyte size at the start of culture. Considerable attention should be paid to the oocyte size as well as to the duration of culture period.

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The aim of the present study was to examine the rate of activation and time-dependent changes in p34^cdc2 kinase and MAP kinase activities in porcine oocytes after injection of sperm extracts (SE) or treatment with Ca²⁺ ionophore to clarify whether SE injection is useful for porcine oocyte activation. SE was prepared from miniature pig sperm by non-ionic surfactant. Oocytes that were treated with Ca²⁺ ionophore and injected with SE were activated at rates of 41% and 46%, respectively. The activities of p34^cdc2 kinase and MAP kinase in each culture period (4, 8 and 12 h) in the Ca²⁺ ionophore group and SE injection group were significantly lower than those in the control group. These results suggest that SE injection induces decrease of p34^cdc2 kinase and MAP kinase activity, and induces oocyte activation, as well as Ca²⁺ ionophore treatment, but the induction was comparatively limited.

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