Dynamic changes in mitochondrial distribution in human oocytes during meiotic maturation.

PURPOSE: The change of mitochondrial distribution in human oocytes during meiotic maturation was assessed using 223 human oocytes donated from patients undergoing fertility treatment between June 2013 and February 2016. METHODS: Live cell images of fluorescence-labelled mitochondria in human oocytes were analysed to investigate dynamic changes in mitochondrial distribution during meiotic maturation using a confocal microscope combined with an incubator in the presence or absence of colchicine and cytochalasin B, inhibitors for tubulin and actin filament, respectively. Subcellular distribution of mitochondria in human oocytes was also assessed at various stages using a transmission electron microscope (TEM). RESULTS: Live cell imaging analysis revealed that the mitochondria-occupied cytoplasmic area decreased from 83 to 77 % of the total cytoplasmic area around 6 h before germinal vesicle breakdown (GVBD) and that mitochondria accumulated preferentially close to the perinuclear region. Then, the mitochondria-distributed area rapidly increased to 85 % of total cytoplasm at the time of GVBD. On the other hand, there was no significant change in mitochondrial distribution before and after polar body extrusion. Such changes in mitochondrial localization were affected differently by colchicine and cytochalasin B. Most of mitochondria in the cytoplasm formed cluster-like aggregates before GVBD while they distributed homogeneously after GVBD. CONCLUSIONS: Most mitochondria localized predominantly in the non-cortical region of the cytoplasm of GV stage-oocytes, while the mitochondria-occupied area decreased transiently before GVBD and increased rapidly to occupy the entire area of the cytoplasm at GVBD by some cytoskeleton-dependent mechanism.

Neonatal outcomes after the implantation of human embryos vitrified using a closed-system device.

PURPOSE: Closed vitrification poses a risk of adversely affecting embryo development, while it may minimize the risk of contamination. We assessed the effects of closed-system human embryo vitrification on fetal development after implantation, neonatal outcome, and clinical safety. METHODS: This was a retrospective cohort study conducted at a private fertility clinic. A total of 875 vitrified-warmed blastocysts that were single-transferred under hormone-replacement cycles between November 2011 and December 2013 were randomly divided into two groups (closed vitrification, n 313; open vitrification, n 562) after receiving the patients' consent forms. Developmental competence after implantation, including gestational age, birth weight, sex, Apgar score, and anomalies of newborns, after the transfer of blastocysts vitrified by closing vitrification was compared with that obtained in the case of open vitrification. RESULTS: There were no significant differences between the use of closed and open vitrification systems in embryo development after implantation, gestational age, birth weight, sex ratio, Apgar score, and congenital anomalies of newborns. CONCLUSION: Human embryos can be vitrified using a closed vitrification system without impairment of neonatal development.

Cabergoline administration prevents development of moderate to severe ovarian hyperstimulation syndrome and it contributes to reduction in ovarian volume.

Purpose: The aim of this study was to determine the prophylactic effects of cabergoline on ovarian hyperstimulation syndrome (OHSS) after oocyte retrieval. Methods: A total of 187 women underwent controlled ovarian stimulation using gonadotropin releasing hormone (GnRH) agonist long protocol or flexible GnRH antagonist protocol for in vitro fertilization. They responded excessively to ovulation induction, and fresh embryo transfers were canceled. Sixty-one patients in the intervention group were administered oral cabergoline (0.5 mg) three times after oocyte retrieval (day 0, 2, and 4 following the oocyte retrieval). Ultrasonography and blood examination were performed on the seventh day following oocyte retrieval. The main outcomes measured were the incidence of OHSS, estimated ovarian volumes, ascites, hematocrits, and white blood cell counts. Results: The incidence of moderate to severe OHSS was lower after cabergoline administration (9.8 vs. 23.0 %, p = 0.03). The ovarian volumes reduced after intervention (96.2 vs. 145.5 cm3, p = 0.008). The reduction was evident in the patients with agonist long protocol (92.1 vs. 167.5 cm3, p = 0.0005). No significant differences were observed for other factors. Conclusions: Cabergoline has a favorable effect on the prevention of moderate to severe OHSS affiliated with ovarian volume reduction.

Oral melatonin supplementation improves oocyte and embryo quality in women undergoing in vitro fertilization-embryo transfer.

The aim of this study was to evaluate the efficacy of oral melatonin supplementation on oocyte and embryo quality in patients in an assisted reproductive technologies program. All patients were treated for at least 2 weeks with melatonin (3 mg/day). To evaluate the cumulative effect of melatonin supplementation, we compared cycle outcomes between the first (no supplementation) and second cycles (melatonin supplementation) of patients who completed two treatment cycles. There were no significant differences in maturation rates (p = 0.50), blastocyst rates (p = 0.75), and the rate of good quality blastocysts (p = 0.59) between the first and second cycles. The fertilization rate of ICSI was higher in the second cycle than that in the first cycle (69.3 versus 77.5%). Being limited to patients with a low fertilization rate in the first cycle (<60%), the fertilization rate dramatically increased after melatonin treatment (35.1 versus 68.2%). The rate of good quality embryos also increased (48.0 versus 65.6%). An important finding in our study was that oral melatonin supplementation can have a beneficial effect on the improvement of fertilization and embryo quality and this may have occurred due to a reduction in oxidative damage.

Growth retardation in human blastocysts increases the incidence of abnormal spindles and decreases implantation potential after vitrification.

STUDY QUESTION: Does the human embryo growth rate affect the outcome of vitrified-warmed blastocyst transfer? SUMMARY ANSWER: 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. WHAT IS KNOWN ALREADY: 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. STUDY DESIGN, SIZE, DURATION: 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. PARTICIPANTS/MATERIALS, SETTING, METHODS: 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. MAIN RESULTS AND THE ROLE OF CHANCE: 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). LIMITATIONS, REASONS FOR CAUTION: Further studies are required to clarify the link between an increase in abnormal spindle formation and a decrease in embryonic implantation potential. WIDER IMPLICATIONS OF THE FINDINGS: This study provided new insights into the possible implications of abnormalities in spindle formation in growth-retarded human blastocysts.

Reproducibility of up-flow column percolation tests for contaminated soils.

Up-flow column percolation tests are used at laboratory scale to assess the leaching behavior of hazardous substance from contaminated soils in a specific condition as a function of time. Monitoring the quality of these test results inter or within laboratory is crucial, especially if used for Environment-related legal policy or for routine testing purposes. We tested three different sandy loam type soils (Soils I, II and III) to determine the reproducibility (variability inter laboratory) of test results and to evaluate the difference in the test results within laboratory. Up-flow column percolation tests were performed following the procedure described in the ISO/TS 21268-3. This procedure consists of percolating solution (calcium chloride 1 mM) from bottom to top at a flow rate of 12 mL/h through softly compacted soil contained in a column of 5 cm diameter and 30 ± 5 cm height. Eluate samples were collected at liquid-to-solid ratio of 0.1, 0.2, 0.5, 1, 2, 5 and 10 L/kg and analyzed for quantification of the target elements (Cu, As, Se, Cl, Ca, F, Mg, DOC and B in this research). For Soil I, 17 institutions in Japan joined this validation test. The up-flow column experiments were conducted in duplicate, after 48 h of equilibration time and at a flow rate of 12 mL/h. Column percolation test results from Soils II and III were used to evaluate the difference in test results from the experiments conducted in duplicate in a single laboratory, after 16 h of equilibration time and at a flow rate of 36 mL/h. Overall results showed good reproducibility (expressed in terms of the coefficient of variation, CV, calculated by dividing the standard deviation by the mean), as the CV was lower than 30% in more than 90% of the test results associated with Soil I. Moreover, low variability (expressed in terms of difference between the two test results divided by the mean) was observed in the test results related to Soils II and III, with a variability lower than 30% in more than 88% of the cases for Soil II and in more than 96% of the cases for Soil III. We also discussed the possible factors that affect the reproducibility and variability in the test results from the up-flow column percolation tests. The low variability inter and within laboratory obtained in this research indicates that the ISO/TS 21268-3 can be successfully upgraded to a fully validated ISO standard.

Visualizing microbial dechlorination processes in underground ecosystem by statistical correlation and network analysis approach.

Microbial ecosystems are typified by diverse microbial interactions and competition. Consequently, the microbial networks and metabolic dynamics of bioprocesses catalyzed by these ecosystems are highly complex, and their visualization is regarded as essential to bioengineering technology and innovation. Here we describe a means of visualizing the variants in a microbial community and their metabolic profiles. The approach enables previously unidentified bacterial functions in the ecosystems to be elucidated. We investigated the anaerobic bioremediation of chlorinated ethene in a soil column experiment as a case study. Microbial community and dechlorination profiles in the ecosystem were evaluated by denaturing gradient gel electrophoresis (DGGE) fingerprinting and gas chromatography, respectively. Dechlorination profiles were obtained from changes in dechlorination by microbial community (evaluated by data mining methods). Individual microbes were then associated with their dechlorination profiles by heterogenous correlation analysis. Our correlation-based visualization approach enables deduction of the roles and functions of bacteria in the dechlorination of chlorinated ethenes. Because it estimates functions and relationships between unidentified microbes and metabolites in microbial ecosystems, this approach is proposed as a control-logic tool by which to understand complex microbial processes.