• Clinical and Experimental Reproductive Medicine
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• v.28 no.2
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• pp.121-129
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• 2001

Objective: The aim of this study is to compare the efficiency of a method for the cryopreservation of mouse blastocyst.. Methods: Mouse embryos were obtained at 2-cell stage and cultured to blastocyst stage in T6 medium supplemented with 10% fetal bovine serum. Morphologically normal blastocysts were collected and randomly divided to one control and four experimental groups. In control group, blastocysts were cultured in vitro continuously for additional two days. In group 2, blastocysts were exposed to vitrification solution (ethylene glycol) only without cryopreservation (exposure only group). In group 3, 4 and 5, blastocysts were cryopreserved by slow-freezing procedure with glycerol (slow-fteezing group) or by vitrification procedure using EM grids (EM grids group) and cryoloop (cryoloop group), respectively. Frozen blastocysts were thawed and cultured for additional two days. Twenty four hours after thawing, some blastocysts were fixed and stained with Hoechst 33342 (bisbenzimide) and the number of nuclei in each blastocysts were counted to confirm the survival of bias to cysts in experimental groups. Results: Survival rate and hatching rate of the blastocysts in slow-freezing group (24 h: 72.4% and 66.0%, 48 h: 63.2% and 64.6%) and EM grids group (24 h: survival rate 77.3%, 48 h: 70.1% and 71.4%) were significantly lower ($X^2$-test p<0.05) than those of control group (24 h: 93.4% and 86.0%, 48 h: 88.5% and 90.7%). In contrast, the survival rate and hatching rate of the blastocysts in cryoloop group (24 h: 84.1% and 84.1%,48 h 79.3% and 87.7%) is well compared with those in the control group. The mean (${\pm}SD$) cell number of blastocyst in the exposure only ($89.2{\pm}11.5$), EM grids ($85.0{\pm}10.3$) and cryoloop ($89.0{\pm}11.0$) groups, except slow-freezing group ($79.0{\pm}10.0$), were not significantly different from that of control group ($93.1{\pm}13.9$) 24 h after thawing (Student's t-test). Conclusion: This study demonstrates that higher survival rate of vitrified-thawed mouse blastocyst can be obtained using cryoloop as the embryo container at freezing rather than slow-freezing or vitrification using EM grids. The results of this study suggest that vitrification using cryoloop (with ethylene glycol) may be a preferable procedure for mouse blastocyst cryopreservation and could be applied to the human blastocyst cryopreservation.

• Asian-Australasian Journal of Animal Sciences
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• v.23 no.9
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• pp.1152-1158
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• 2010

In differentiating human embryonic stem (d-hES) cells there are a number of types of cells which may secrete various nutrients and helpful materials for pre-implantation embryonic development. This study examined whether the d-hES could function as a feeder cell in vitro to support mouse embryonic development. By RT-PCR analysis, the d-hES cells revealed high expression of three germ-layered differentiation markers while having markedly reduced expression of stem cell markers. Also, in d-hES cells, LIF expression in embryo implantation-related material was confirmed at a similar level to undifferentiated ES cells. When mouse 2PN embryos were cultured in control M16 medium, co-culture control CR1aa medium or co-cultured with d-hES cells, their blastocyst development rate at embryonic day 4 (83.9%) were significantly better in the d-hES cell group than in the CR1aa group (66.0%), while not better than in the M16 group (90.7%)(p<0.05). However, at embryonic days 5 and 6, embryo hatching and hatched-out rates of the dhES cell group (53.6 and 48.2%, respectively) were superior to those of the M16 group (40.7 and 40.7%, respectively). At embryonic day 4, blastocysts of the d-hES cell group were transferred into pseudo-pregnant recipients, and pregnancy rate (75.0%) was very high compared to the other groups (M16, 57.1%; CR1aa, 37.5%). In addition, embryo implantation (55.9%) and live fetus rate (38.2%) of the d-hES cell group were also better than those of the other groups (M16, 36.7 and 18.3%, respectively; CR1aa, 23.2 and 8.7%, respectively). These results demonstrated that d-hES cells can be used as a feeder cell for enhancing in vitro and in vivo developmental potential of mouse pre-implantation embryos.

• Clinical and Experimental Reproductive Medicine
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• v.38 no.3
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• pp.148-152
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• 2011

Objective: Laser-assisted intracytoplasmic sperm injection (LA-ICSI), also known as micro-opening or thinning of the zona pellucida (ZP) prior to ICSI, may help to reduce mechanical damage to the oocyte during the procedure. The aim of the present study was to evaluate and analyze the efficacy of our institutional LA-ICSI program, which features laser-assisted ZP thinning prior to ICSI, in comparison with conventional ICSI (C-ICSI), performed on patients with different clinical characteristics. Methods: Patients undergoing a total of 212 ICSI cycles were randomly divided into an LA-ICSI group (106 cycles) and a conventional ICSI group (106 cycles). To reduce tissue damage, we thinned the ZP by approximately 70%, using a laser, before ICSI. Patients thus treated formed the LAICSI group. Comparisons included the morphological quality of transferred embryos, blastocyst development of the remaining embryos, and clinical pregnancy, in terms of ICSI method and patient characteristics. Results: Fertilization, development of remaining embryos, and pregnancy rate were significantly higher in the LA-ICSI group compared with the C-ICSI group. Fertilization, embryonic development, and the pregnancy rate were all improved in younger patients (<38 years of age) and in those who underwent a low number of IVF-ET attempts (<3 trials). In addition, the pregnancy rate was increased in older patients. Conclusion: LA-ICSI may be useful in improving the chance of pregnancy in all ICSI patients.

• Animal Bioscience
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• v.37 no.6
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• pp.1007-1020
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• 2024

Objective: We increased the nuclear maturation rate of antral follicle derived oocytes by using a pre-in vitro maturation (IVM) culture system and improved the developmental potential of these porcine pathenotes by supplementing with melatonin. Furthermore, we investigated the expression patterns of genes involved in cumulus expansion (HAS2, PTGS2, TNFAIP6, and PTX3) derived from small and medium antral follicles before and after oocyte maturation. Methods: Only the cumulus oocyte-complexes (COCs) derived from small antral follicles were induced with [Pre-SF(+)hCG] or without [Pre-SF(-)hCG] the addition of human chorionic gonadotropin (hCG) during the last 7 h of the pre-IVM period before undergoing the regular culture system. The mature oocytes were investigated on embryonic development after parthenogenetic activation (PA). Melatonin (10^-7 M) was supplemented during in vitro culture (IVC) to improve the developmental potential of these porcine pathenotes. Results: A pre-IVM culture system with hCG added during the last 7 h of the pre-IVM period [Pre-SF(+)hCG] effectively supported small antral follicle-derived oocytes and increased their nuclear maturation rate. The oocytes derived from medium antral follicles exhibited the highest nuclear maturation rate in a regular culture system. Compared with oocytes cultured in a regular culture system, those cultured in the pre-IVM culture system exhibited considerable overexpression of HAS2, PTGS2, and TNFAIP6. Porcine embryos treated with melatonin during IVC exhibited markedly improved quality and developmental competence after PA. Notably, melatonin supplementation during the IVM period can reduce and increase the levels of intracellular reactive oxygen species (ROS) and glutathione (GSH), respectively. Conclusion: Our findings indicate that the Pre-SF(+)hCG culture system increases the nuclear maturation rate of small antral follicle-derived oocytes and the expression of genes involved in cumulus expansion. Melatonin supplementation during IVC may improve the quality and increase the blastocyst formation rate of porcine embryos. In addition, it can reduce and increase the levels of ROS and GSH, respectively, in mature oocytes, thus affecting subsequent embryos.

• Clinical and Experimental Reproductive Medicine
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• v.38 no.1
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• pp.53-60
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• 2011

Objective: This study was carried out to compare the clinical outcome of elective single cleavage-embryo transfer (eSCET) to that of elective single blastocyst-embryo transfer (eSBET) in human IVF-ET. Methods: This study was a retrospective study which analyzed for 614 women who visited the Daegu Maria Clinic from August 2008 to December 2009. All were under 37 years old and had more than 8 mm of endometrial thickness on the day of hCG administration and at least one good quality embryo on day 3. The eSCETs were performed on day 3 (n=450) and the eSBETs were conducted on day 5 (n=164). Results: The numbers of retrieved oocytes, fertilized oocytes, and day 3 good quality embryos were significantly lower in the eSCET group (12.1${\pm}$6.0, 8.2${\pm}$4.6, and 4.2${\pm}$3.1, respectively) compared to the eSBET group (16.7${\pm}$7.2, 12.1${\pm}$5.0, and 8.5${\pm}$4.5, respectively; p<0.001). However, the clinical pregnancy, implantation, on-going pregnancy, and live birth rates of the eSCET group (46.7, 46.9, 40.0, and 36.7%, respectively) were not statistically different from those of the eSBET group (51.2, 51.8, 45.1, and 43.9%, respectively; p=0.318, 0.278, 0.254, and 0.103, respectively). Conclusion: These results suggested that elective single embryo transfer should be performed regardless of the developmental stage to women less than 37 years old who had more than 8 mm of endometrial thickness on the hCG administration day and at least one good quality embryo on day 3 in order to reduce the twin pregnancy rate without reducing the whole pregnancy rate.

• Asian-Australasian Journal of Animal Sciences
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• v.19 no.11
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• pp.1574-1579
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• 2006

This study was performed to determine the developmental potentials of nuclear transfer (NT) embryos using life-span extended cells transfected with a foreign gene as donor cells. A life-span extended bovine embryonic fibroblast cell line was transfected with an expression vector in which the human type II collagen (BOMAR) and ear fibroblasts were used as a donor cell. Cytogenetic analysis was performed to analyze the chromosomal abnormality of donor cells. The fusion rate of 1.8 kV/cm for $15{\mu}sec$ given twice was significantly higher than that of other groups (p<0.05) and the embryos lysed were significantly higher after 1.8 kV/cm for $20{\mu}sec$ given once compared to other groups (p<0.01). The blastocyst development in the ear cell group was statistically significant compared to both BOMAR groups (p<0.01). Both BOMAR groups cultured more than 40 passages (>40 passages) had a lower number of chromosomes; however, fresh granulosa cell (GC) and BOMAR groups cultured less than 20 passages had normal chromosome numbers. Both >40 passages BOMAR groups had numerous obscure debris in metaphase spreads. The transfected foreign gene was expressed in all BOMAR groups, but not in the GC group. Based on these results, the lower developmental potential of NT embryos using life-span extended donor cells transfected with a foreign gene might be a cause of chromosomal abnormality in donor cells.

• Journal of Embryo Transfer
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• v.31 no.1
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• pp.1-7
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• 2016

Xenotransplantation involves multiple steps of immune rejection. The present study was designed to produce nuclear transfer embryos, prior to the production of transgenic pigs, using fibroblasts carrying transgenes human complement regulatory protein hCD59 and interleukin-18 binding protein (hIL-18BP) to reduce hyperacute rejection (HAR) and cellular rejection in pig-to-human xenotransplantation. In addition to the hCD59-mediated reduction of HAR, hIL-18BP may prevent cellular rejection by inhibiting the activation of natural killer cells, activated T-cell proliferation, and induction of $IFN-{\gamma}$. Transgene construct including hCD59 and ILI-18BP was introduced into miniature pig fetal fibroblasts. After antibiotic selection of double transgenic fibroblasts, integration of the transgene was screened by PCR, and the transgene expression was confirmed by RT-PCR. Treatment of human serum did not affect the survival of double-transgenic fibroblasts, whereas the treatment significantly reduced the survival of non-transgenic fibroblasts (p<0.01), suggesting alleviation of HAR. Among 337 reconstituted oocytes produced by nuclear transfer using the double transgenic fibroblasts, 28 (15.3%) developed to the blastocyst stage. Analysis of individual embryos indicated that 53.6% (15/28) of embryos contained the transgene. The result of the present study demonstrates the resistance of hCD59 and IL-18BP double-transgenic fibroblasts against HAR, and the usefulness of the transgenic approach may be predicted by RT-PCR and cytolytic assessment prior to actual production of transgenic pigs. Further study on the transfer of these embryos to surrogates may produce transgenic clone miniature pigs expressing hCD59 and hIL-18BP for xenotransplantation.

• Korean Journal of Animal Reproduction
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• v.18 no.2
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• pp.87-94
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• 1994

The objective of this study is to evaluate the developmental ability of mouse embryo in the presence of human amniotic fluid (hAF), The highest development rate was found in the culture media supplemented with 20% mid-term hAF but this rate was concomitantly reduced with more than 20% hAF. Furthermore, mouse two-cell embryos cultured in 20% mid-term hAF were developed more consistently to the expanded and hatched blastocyst stages compared to those cultured in simple medium. However, no significant differences in the embryo development rates were observed among the supplemented effects of 20% mid-term hAF, 0.3% bovine serum albumin (BSA), and 10% fetal calf serum (FCS), Development rates of two-ceiI mouse embryos cultured in 20% full-term hAF were declined compared to 20% mid-term hAF. Outgrowth of hatched blastocysts were observed when the embryos were cultured in medium containing 20% mid-term hAF or 10% FCS. But two-cell mouse embryos cultured in the presence of 20% full-term hAF or O.3% BSA was not observed their outgrowth. The kinetics of outgrowth processes in the presence of hAF were similar to those with 10% FCS. However, embryos with FCS showed a considerably greater extents of trophetodermal cell proliferation and outgrowth. Taken together, these data suggest that mid-term hAF may have a suitability for the mammalian embryos and induce embryonic outgrowth.

• Clinical and Experimental Reproductive Medicine
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• v.38 no.3
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• pp.126-134
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• 2011

Preimplantation genetic diagnosis (PGD) is gradually widely used in prevention of gene diseases and chromosomal abnormalities. Much improvement has been achieved in biopsy technique and molecular diagnosis. Blastocyst biopsy can increase diagnostic accuracy and reduce allele dropout. It is cost-effective and currently plays an important role. Whole genome amplification permits subsequent individual detection of multiple gene loci and screening all 23 pairs of chromosomes. For PGD of chromosomal translocation, fluorescence $in-situ$ hybridization (FISH) is traditionally used, but with technical difficulty. Array comparative genomic hybridization (CGH) can detect translocation and 23 pairs of chromosomes that may replace FISH. Single nucleotide polymorphisms array with haplotyping can further distinguish between normal chromosomes and balanced translocation. PGD may shorten time to conceive and reduce miscarriage for patients with chromosomal translocation. PGD has a potential value for mitochondrial diseases. Preimplantation genetic haplotyping has been applied for unknown mutation sites of single gene disease. Preimplantation genetic screening (PGS) using limited FISH probes in the cleavage-stage embryo did not increase live birth rates for patients with advanced maternal age, unexplained recurrent abortions, and repeated implantation failure. Polar body and blastocyst biopsy may circumvent the problem of mosaicism. PGS using blastocyst biopsy and array CGH is encouraging and merit further studies. Cryopreservation of biopsied blastocysts instead of fresh transfer permits sufficient time for transportation and genetic analysis. Cryopreservation of embryos may avoid ovarian hyperstimulation syndrome and possible suboptimal endometrium.

• Journal of Yeungnam Medical Science
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• v.12 no.1
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• pp.124-134
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• 1995

It is the most important to select optimal culture conditions to promote safe embryo growth in the technique of human in vitro fertilization and embryo transfer. It has been shown that the addition of biologic fluids, such as blood serum, of various origins, improved fertilization and early cleavage rates in numerous species. The purpose of this study is to attempt to measure developmental potential of mouse eggs fertilized and cleaved in Ham's F10 culture medium containing a chelating agent, EDTA and fetal cord serum. In this study, we selected 40 female mice and 20 male mice, and investigated optimal serum concentrations for mouse embryo growth. Two cell stage mouse embryos were cultured in Ham's F-10 medium, Ham's F-10 medium with various concentrations of EDTA, or Ham's F-10 medium with EDTA and 10% human cord serum. Developmental ratios to morula in Ham's F-10 medium containing various concentrations of EDTA and/or 10% fetal cord serum were significantly higher than in unsupplemented Ham's F-10 medium (p<0.05). Developmental ratios to blastocyst in Ham's F-10 containing 10% fetal cord serum and $50{\mu}M$ or $100{\mu}M$ EDTA were significanltly higher than in unsupplemented Ham's F-10 medium (p<0.05). Developmental ratios to morula in Ham's F-10 containing 10% fetal cord serum and $100{\mu}M$ EDTA were significanltly higher than in Ham's F-10 with 10% fetal cord serum used commonly in many human IVF centers(p<0.05). Developmental ratio to blastocyst in Ham's F-10 containing 10% fetal cord serum and $100{\mu}M$ EDTA was significanlty higher than in Ham's F-10 with $200{\mu}M$ EDTA(P<0.05). In summary, embryo development to morula and blastocyst was significanlty higher in the presence of human cord serum or EDTA than in the unsupplemented medium. The most significanly development to morula and blastocyst was obtained at Ham's F-10 medium with $100{\mu}M$ concentration of EDTA and 10% fetal cord serum. These results suggest that Ham's F-10 medium containing 10% fetal cord serum and optimal concentrations of EDTA significantly promoted early cleavage of mouse zygotes, and these will be useful as basic data for the selection of culture medium in human in vitro fertilization.