Cryopreservation of mammalian animal embryos was first developed using mouse embryos in 1972 and has since been applied to human embryos to utilize surplus embryos in in-vitro fertilization programs: the slow freezing method in the first half of the 1980's and vitrification in the 1990's. Recently, the protocol of vitrification for human embryos has been improved by the ultra-rapid vitrification method in which the rate of cooling is highly improved. It is mainly used as an effective basic technique for cryopreservation of human embryos in current clinical IVF programs due to the extremely high embryonic survival rate after storage.

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When the first successful delivery following in vitro fertilization and embryo transfer was reported in 1978, oocyte aspiration was performed laparoscopically under general anesthesia. Since 1985, almost all IVF centers have collected oocytes using transvaginal ultrasound-directed methods for the good reason that these methods are the easiest, most accurate and most acceptable to patients. Color Doppler ultrasonography is recommended to decrease blood loss during oocyte aspiration. Embryos were gently, slowly and transcervically, expelled into the uterine cavity, with the patient in a lithotomy position. This basic method remained unchanged since the first description. Relatively important factors for successful embryo transfer include removal of hydrosalpinges, absence of blood or mucus on catheter, using soft catheter type, avoidance of fundus contact, avoiding tenaculum, removal of all mucus, ultrasonography of cavity before transfer, inject one embryo within 10 μL volume slowly 1.5 cm from fundus, trial transfer, ultrasonographic monitoring, and antiprostaglandins administration to prevent uterine contractions.

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Cryopreservation of mouse spermatozoa has been widely applied for maintenance of genetically modified mouse strains. Although cryopreservation of spermatozoa is simpler, less time-consuming, and less costly than of embryos for maintaining transgenic or gene-disrupted mouse strains, maintenance of cryopreserved spermatozoa still has high running costs because of the need for a constant supply of liquid nitrogen. It has been reported that freeze-dried mouse spermatozoa are capable of participating in normal embryonic development after injection into oocytes, and so they have attracted a great deal of attention as storable gene resources. However, it is particularly essential to assure long-term preservation for several decades or centuries. The application of the determination of accelerated degradation kinetics calculated by extrapolation of Arrhenius plots to the preservation of freeze-dried mouse spermatozoa is a possible solution. This theory also is being applied to long-term stability of drugs. In this issue, we introduce recent studies of freeze-dried mouse spermatozoa.

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In ART, once an ovum is successfully yielded, embryologists have to progress the treatments as arranged regardless of the quality of semen. Sperm qualities are, therefore, the most variable factor in each case. Human ejaculate contains heterogeneous sperm populations, which possess a variety of abnormalities at nuclear, cytoskeletal and organelle levels. Embryologists are certainly well informed about physiology and genetics of ova and embryos, moreover, it is also essential for them to study how to evaluate and prepare the sperm according to their physiology.

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This article reviews systematically relevant clinical data of rFSH for COS on ART, which were mainly obtained in the Cochrane library, PubMed, MEDLINE, and reference lists of articles. HMG and rFSH have both been used equally successful for COS in ART. However the other review has concluded a statistically significant increase in clinical pregnancy rate with rFSH compared to uFSH, when used for COS in standard IVF cycles and not in cycles in which ICSI was used. Recombinant FSH is a new treatment option for Japanese women undergoing COS for ART with several advantages over conventional urinary gonadotropin preparations. Since SC administration of rFSH is safe, efficacious, and acceptable, the availability of rFSH as a ready-for-use solution supplied in an injector system may make its administration, in particular self-administration by the patient or her partner. The current study indicates that the use of rFSH is not associated with a higher incidence of obstetrical and neonatal problems compared to urinary gonadotropins.

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The aim of the present study was to assess the efficacy of laser assisted hatching (LAH) in human embryos. Embryos were assigned to one of two groups: LAH group (n=24) or control group (n=19). In the LAH group, zona opening was performed at the day 3 embryo with the use of a 1.48-μm infrared diode laser (OCTAX Laser Shot^TM system, MTG Medical Technology, Altdorf, Germany). Following the LAH, embryos were cultured up to the hatching blastocyst stage to determine whether the LAH resulted in damage to subsequent development. The LAH was performed on 14 patients who experienced IVF/ICSI failures in two or more cycles in our clinic. Respective blastocyst development rates in the LAH and the control group were 37.5% (9/24) and 42.1% (8/19). There was no significant difference between the two groups. As rates of blastocyst hatching were 88.9% (8/9) and 12.5% (1/8), respectively, the LAH group showed a significant increase as compared to the control group (P<0.01). To date, five pregnancies have occurred following the transfer of zona opening day 3 embryos. LAH of human embryos does not have an adverse effect on subsequent development and increases the rate of blastocyst hatching.

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The relationship between morphologic criteria including zona pellucida thickness (ZPT) and the ET outcomes was studied for day-4 fresh morula transfer (n=306). Pregnancy rate (PR) was significantly correlated with the proportion of compaction (P<0.0001), the morphology (P<0.0001) and the amount of fragmentation (P=0.0004) of the best morula. In cycles with single morula transfer (n=121), only the morphology was significantly correlated with PR (P=0.03). The average ZPT of the best morula was significantly thinner (P<0.0001) in conception cycles (7.9 ± 2.1 μm) than in non-conception cycles (9.3 ± 1.8 μm). Also in cycles with single morula transfer, the average ZPT was significantly thinner (P=0.004) in conception cycles (8.5 ± 1.9 μm) than in non-conception cycles (9.7 ± 2.0 μm). Factors influencing conception were assessed by logistic-regression analysis. The average ZPT (P<0.0001), the morphology (P=0.0003) and the amount of fragmentation (P=0.02) of the best morula were significantly related to the conception, whereas only the average ZPT (P=0.01) was related to the conception in single morula transfer cycles. We concluded that the average ZPT and the morphology of embryo seem to be strong predictors of pregnancy in day-4 morula transfer and thus important indicators of good embryo quality.

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It has been reported that the rate of blastocyst formation after ICSI is lower than that after conventional in vitro fertilization IVF. The aim of this study is to assess relationship between maturity of chromatin structure of spermatozoa and embryo development. Samples; Ejaculated sperm was collected from 21 patients treated with ICSI and 22 patient treated with IVF from January to October in our hospital. N-ethylmaleimide (NEM) was used to prevent natural oxydation. Centrifugated and washed sperm was mixed with 5 mM NEM and stored at -80°C until analysis. Two hundred μl of thawed sperm aliquot was suspended in same dose of low pH detergent for 30 sec followed by suspension with 1.2 ml of acridine orange (AO) working solution for 3 min. FACScan equipped with CELL QUEST (BECTON DICKINSON) was used for analysis. Cells outside the main population (COMP; %) was measured for SCSA parameter. Although there are no corelation in conventional IVF (r=0.03, p=0.899, n=21), COMP in ICSI has positive corelation with the rate of blastocyst formation (r=0.477, p=0.025, n=22). In ICSI, the number of disulfide bond in sperm chromatin might effect on dyssynchrony during process of pronuclear formation and embryo development.

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Recently, with the acetylation of histone and the modification of chromatin structure, the methylation of cytosine residue within CpG dinucleotides in genomic DNA sequence attracts many researchers' attention as one of major epigenetic regulation systems of gene expression. There are several methods (immunofluorescence with 5-methylcytosine specific antibody and methylation-sensitive restriction enzyme-PCR method, etc.) to analyze the methylation of cytosine residue. Bisulfite-sequencing method, which is one of methods for analyzing the methylation of cytosine residue in genomic DNA sequence, has advantages of high sensitivity and analyzing the methylation of cytosine residue directly. In this note, the detailed procedure of bisulfite-sequencing method for mouse early pre-implantation embryos is described.

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The procedure of the mouse in-vitro fertilization method which can obtain high rates of fertilization and development to the blastocyst stage was explained in detail using several figures. Some factors affecting the successful rates were also described.

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With the rapid development of advanced reproductive technology (ART), embryologists have risen in stature. The basic ART techniques were promoted in Japan by a number of physicians who studied this new technology abroad; however, since the first successful intracytoplasmic sperm injection in 1992, the performance of these techniques abruptly changed from physicians to embryologists. In Japan, although we recognize that embryologists are essential members of an ART team, their aspects of background is limited. Therefore, here is described some states for embryologists in regard to factors such as regulation, certification, and salary.

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In the United States, assisted reproduction devices are categorized as "medical devices" and regulated by FDA. Most of assisted reproduction devices are classified in Class II based on level of control necessary to assure the safety and effectiveness as well as on risk to patient and/or user. Manufactures must submit a 510(k) to FDA before introducing a device to the U. S. market. Assisted Reproduction devices should be manufactured in accordance with Good Manufacturing Practice (GMP) and manufactures must demonstrate that the device to be marketed is as safe and effective, that is, substantially equivalent, to a legally marketed device. The status of regulations and the contents required to applicants in the U. S. are reported.

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