[Show abstract][Hide abstract] ABSTRACT: DNA methylation is globally reprogrammed during mammalian preimplantation development, which is critical for normal development. Recent reduced representation bisulfite sequencing (RRBS) studies suggest that the methylome dynamics are essentially conserved between human and mouse early embryos. RRBS is known to cover 5-10% of all genomic CpGs, favoring those contained within CpG-rich regions. To obtain an unbiased and more complete representation of the methylome during early human development, we performed whole genome bisulfite sequencing of human gametes and blastocysts that covered>70% of all genomic CpGs. We found that the maternal genome was demethylated to a much lesser extent in human blastocysts than in mouse blastocysts, which could contribute to an increased number of imprinted differentially methylated regions in the human genome. Global demethylation of the paternal genome was confirmed, but SINE-VNTR-Alu elements and some other tandem repeat-containing regions were found to be specifically protected from this global demethylation. Furthermore, centromeric satellite repeats were hypermethylated in human oocytes but not in mouse oocytes, which might be explained by differential expression of de novo DNA methyltransferases. These data highlight both conserved and species-specific regulation of DNA methylation during early mammalian development. Our work provides further information critical for understanding the epigenetic processes underlying differentiation and pluripotency during early human development.
[Show abstract][Hide abstract] ABSTRACT: Currently, in Japan, the most commonly used embryo vitrification method is “ultra-rapid vitrification” which is designed to achieve the maximum cooling rate by exposing embryos directly to LN2 in a tiny droplet of cryoprotectant. Though this method has yielded acceptable embryo viability rates, questions have been raised by various countries' regulatory agencies regarding the effects of LN2 exposure. The 0.25 ml plastic straw has long been used as an entirely closed vitrification system. However, the cooling rate is slower using the 0.25 ml straw than when using the ultra-rapid cooling method, resulting in injury to embryos exposed to a high concentration of cryoprotectant. Recently, a new system has been developed which addresses both the issues of rates of cooling and safeguards against LN2. This paper compares the clinical results of blastocyst vitrification and warming using the conventional 0.25 ml straw method with those of the Rapid-i™ method. The survival rate after warming the group cryopreserved using Rapid-i™ was significantly higher than that of the group of frozen-thawed cryopreserved using the 0.25 ml straw. Moreover, the on-going pregnancy rate after transfer blastocysts into recipients' uteri was higher after cryopreservation with the Rapid-i™ method. These results suggest that the new closed system is safe to use in cryopreservation.
[Show abstract][Hide abstract] ABSTRACT: The aim of this retrospective study was to investigate the relationship between the oxygen consumption rate of blastocysts before freezing and their viability after warming with respect to their re-expansion and blastomere loss after warming. A total of 41 blastocysts from 29 in vitro fertilization (IVF) treatment cycles, that were not scheduled for cryopreservation for the next cycle, were examined. Good quality blastocysts were defined those having as less than 20% of blastomere loss, and rapid re-expanded blastocysts were defined those having as more than 50% blastocoel re-expansion during post-warming culture of 2 h. We evaluated the oxygen consumption rates before freezing and after warming as well as their relationship with the morphological features of good-quality and rapid re-expanded blastocysts during the post-warming culture. Good-quality blastocysts had a significantly higher oxygen consumption rate after warming than damaged blastocysts; furthermore, rapid re-expanded blastocysts had a significantly higher oxygen consumption rate before freezing than slow or no re-expansion blastocysts. These observations suggest that measurements of the oxygen consumption rate of individual blastocysts before freezing provides important information regarding viability after warming from the viewpoint of blastocoel re-expansion.
[Show abstract][Hide abstract] ABSTRACT: Developmental competence of in vitro matured human oocytes is dependent on the morphology of the cumulus-oocyte complex (COC) just after collection from the follicle. We postulated that COCs categorized as having poor morphology (two or fewer less than two layers of cumulus cells) would not secrete a sufficient amount of matulation factors, resulting in low developmental competence of the matured oocytes. In the present study, the level of progesterone secreted from good morphology COCs with three or more layers of cumulus cells, 39.2 ± 12.8 ng/ml (n=31), was significantly higher than that secreted from poor morphology COCs (9.65 ± 1.34 ng/ml, n=22). The addition of 20 ng/ml progesterone to in vitro maturation culture of the poor morphology group significantly improved the fertilization ability of the oocytes. The rates of development to the morula and blastosyst stages were also increased by progesterone, however the differences were not significant. In conclusion, the secreted level of progesterone during in vitro maturation of human COCs was dependent on the number of cumulus cells attached to oocyte. When an oocyte is surrounded by two or fewer 2 layers of cumulus cells, the addition of progesterone to FSH- and hCG-containing medium appears to be a useful method for obtaining an oocyte with a high developmental competence.
Journal of Mammalian Ova Research 04/2012; 29(1):41-47. DOI:10.1274/jmor.29.41
[Show abstract][Hide abstract] ABSTRACT: To assess the clinical value of bisulfite polymerase chain reaction Luminex (BPL), an automated, high-throughput procedure for the detection of alterations in DNA methylation.
Experimental prospective study.
University research laboratory and private in vitro fertilization (IVF) clinic.
A total of 337 men, 61 with severe oligozoospermia, 67 with moderate oligozoospermia, and 209 with microscopically normozoospermia.
The ejaculated sperm samples after the routine semen analysis with patients' consent.
Examination of the methylation patterns of eight imprinted loci in sperm DNA, and confirmation with combined bisulfite PCR restriction analysis (COBRA).
A total of 47 cases (13.9%) showed abnormal methylation at one or more imprinted loci (18 paternal, 18 maternal, and 11 cases with alterations of both maternal and paternal imprints).
The relative ease of the BPL method provides a practical method within a clinical setting to reduce the likelihood of abnormal samples being used in assisted reproduction treatments.
[Show abstract][Hide abstract] ABSTRACT: PurposeSeveral recent reports have discussed refrozen and thawed embryo transfer; however, the process may cause a degree of chromosomal
damage and subtle genomic mutation. In view of this possibility, the purpose of this study was to investigate the incidence
of aneuploidy in refrozen embryos.
MethodsIn order to investigate the incidence of aneuploidy and mosaicism observed in chromosome 1, fluorescent in situ hybridization
(FISH) was used on surviving embryos that first underwent one freeze-thaw cycle, then were allowed to develop to the blastocyst
stage, and subsequently survived a second freeze-thaw cycle.
ResultsOf 1,132 blastomeric nuclei analyzed from 15 refrozen embryos, disomy was found in 82.9%. In contrast, for the 11 blastocysts
subjected to only one freeze-thaw cycle, disomy was noted in 78.4%. Of the 197 blastomeric nuclei analyzed in all arrested
embryos, disomy was found in 51.8%.
ConclusionsThe refreezing process did not increase aneuploidy. The good and fair morphology groups demonstrated a higher percentage of
disomy than the poor morphology group regardless of whether they were frozen once or twice.
Reproductive Medicine and Biology 09/2009; 8(3):103-106. DOI:10.1007/s12522-009-0016-y
[Show abstract][Hide abstract] ABSTRACT: There is an increased prevalence of imprinting disorders, such as Beckwith-Wiedemann syndrome, associated with human assisted reproductive technologies (ART). Work on animal models suggests that in vitro culture may be the source of these imprinting errors. However, in this study we report that, in some cases, the errors are inherited from the father. We analyzed DNA methylation at seven autosomal imprinted loci and the XIST locus in 78 paired DNA samples. In seven out of seventeen cases where there was abnormal DNA methylation in the ART sample (41%), the identical alterations were present in the parental sperm. Furthermore, we also identified DNA sequence variations in the gene encoding DNMT3L, which were associated with the abnormal paternal DNA methylation. Both the imprinting errors and the DNA sequence variants were more prevalent in patients with oligospermia. Our data suggest that the increase in the incidence of imprinting disorders in individuals born by ART may be due, in some cases, to the use of sperm with intrinsic imprinting mutations.
European journal of human genetics: EJHG 06/2009; 17(12):1582-91. DOI:10.1038/ejhg.2009.68 · 4.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of Mammalian Ova Research 04/2008; 25(1):2-7. DOI:10.1274/jmor.25.2
[Show abstract][Hide abstract] ABSTRACT: To confirm the clinical benefits of recryopreserved, twice-thawed embryo transfer (ET).
Private fertility clinic.
Forty-nine women whose embryos had been refrozen after a previous frozen-thawed ET.
Comparison of implantation and pregnancy rates of twice-cryopreserved, twice-thawed embryos versus once-cryopreserved, once-thawed embryos.
The pregnancy rate per ET cycle was 27.8% in the refrozen group and 25.9% in the control group (no statistically significant difference). The implantation rate was 25.0% in the refrozen group and 19.3% in the control group (no statistically significant difference).
The refreezing of supernumerary embryos can prevent ovarian hyperstimulation syndrome in stimulated patients and in those who have experienced repeated failed pregnancies. If unexpected supernumerary embryos are available for recryopreservation after frozen-thawed ET, these embryos may be revitrified for a future transfer.
Fertility and sterility 03/2008; 91(2):383-6. DOI:10.1016/j.fertnstert.2007.11.079 · 4.59 Impact Factor