• Clinical and Experimental Reproductive Medicine
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• v.47 no.4
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• pp.293-299
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• 2020

Objective: The goal of the present study was to investigate the rate of chromosomal aneuploidies in surplus embryos after sex determination at the cleavage stage. Then, the same chromosomal aneuploidies were evaluated in blastocysts after extended culture. Methods: Sixty-eight surplus embryos were biopsied at the cleavage stage and incubated for an additional 3 days to allow them to reach the blastocyst stage. The embryos were reanalyzed via fluorescence in situ hybridization (FISH) to examine eight chromosomes (13, 15, 16, 18, 21, 22, X, and Y) in both cleavage-stage embryos and blastocysts. Results: Although the total abnormality rate was lower in blastocysts (32.35%) than in cleavage-stage embryos (45.58%), the difference was not significant (p=0.113). However, when we restricted the analysis to autosomal abnormalities, we observed a significant difference in the abnormality rate between the cleavage-stage embryos (44.11%) and the blastocysts (17.64%, p=0.008). A higher rate of sex chromosomal abnormalities was also observed in cleavage-stage embryos (29.4%) than in blastocysts (14.70%, p=0.038). Conclusion: The data indicated that embryo biopsy should be conducted at the blastocyst stage rather than the cleavage stage. The results also emphasized that examination of common chromosomal aneuploidies apart from sex selection cycles can be conducted in the blastocyst stage with the FISH method.

• Asian-Australasian Journal of Animal Sciences
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• v.16 no.1
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• pp.15-22
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• 2003

Experiments were conducted to evaluate the effect of blastomere diameters and cell cycle stages on the subsequent development of nuclear transplant rabbit embryos (NT-embryos) using nuclei derived from the 16- or 32-cell stage embryos. All blastomeres and NT-embryos were cultured individually in modified Ham's F-10 medium supplemented with 10% rabbit serum (RS) at $38^{\circ}C$ and 5% $CO_2$ in air. The diameter of blastomeres from 16-cell stage embryos was found twice of those from 32-cell stage (51 vs 27 ${\mu}m$). Significant differences were observed in cleavage rates ($\geq$3 divisions) in the isolated single blastomeres (54 vs 48 for 16-cell; 28 vs 14 for 32-cell, p<0.05), but the fusion rates of oocytes with transferred nuclei were similar between small and large single blastomeres derived from either 16-cell or 32-cell stage embryos. When 16-cell stage blastomeres were used as nuclear donors, cleavage rates ($\geq$3 divisions) of the NT-embryos were greater in the small nuclear donors than in the large donors (73 vs 55%, p<0.05). On the contrary, significantly higher cleavage (43 vs 6%, p<0.05) and developmental rates (14 vs 0%, p<0.05) were observed in the large blastomere nuclear donor group of the 32-cell stage embryos. When the cell cycle stages were controlled by a microtubule polymerization inhibitor (Demicolcine, DEM) or the combined treatment of DEM and Aphidicolin (APH), a DNA polymerase inhibitor, fusion rates were 88-96% for the 16-cell donor group (without DEM treatment), which were greater than the 32-cell donor group (54-58%). Cleavage rates were also greater in the transplants derived from G1 nuclear donor group (93-95%) than those from the DEM and APH combined treatment (73%) for the 16-cell donor group (p<0.05). No significant difference was detected in the morula/blastocyst rates in either donor cell stage (p>0.05). In conclusion, it appeared that no difference in the developmental competence between large and small isolated blastomeres was observed. When smaller 16-cell stage blastomeres were used as nuclear donor, the cleavage rate or development of NT-embryos was improved and was compromised when 32-cell stage blastomeres were used. Therefore, control nuclear stage of the donor cell at $G_1$ phase in preactivated nuclear recipients seemed to be beneficial for the cleavage rate of the reconstructed embryo in the 16-cell transplant, but not for subsequent morula or blastocyst development.

• Clinical and Experimental Reproductive Medicine
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• v.32 no.1
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• pp.47-54
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• 2005

Objective: Evaluation of embryos using early cleavage to 2-cell stage has been proposed, but a critical time-point for selecting embryos is unclear. The aim of the present study is to provide a guideline including critical time-point in the selection of early cleaving embryo for the reduction of multiple pregnancies as well as the increase of pregnancy rate in human IVF. Methods: This prospective study was performed in 116 cycles from 85 patients who underwent conventional IVF or ICSI at the infertility clinic of Good Moonhwa Hospital from January 2002 to December 2003. Early cleavage (EC) of embryos to 2-cell stage was assessed at 25 h and 27 h postinsemination/microinjection. Embryos that had early cleaved at each time point were designated as EC-1 and EC-2, respectively, while others were designated as non-early cleavage (NEC). Results: At least one early cleavage embryo was observed in 54 (46.6%) for the EC-1 and 84 (72.4%) for the EC-2 of the 116 cycles assessed. Clinical pregnancy rates (PR) were significantly higher in the EC-1 group (66.7%) compared to the EC-2 group (53.6%) or the NEC group (31.2%) (p<0.05). Significant improvement of the pregnancy rate was found when at least two or more embryos were early cleaved at 25 h postinsemination or when the proportion of early cleavage embryo at 25 h postinsemination was higher than 20% (p<0.05). Conclusion: The critical time-point for the selection of early cleavage embryos with high implantation potential is more effective in 25 h postinsemination/microinjection compared to 27 h. The proportion as well as number of early cleavage embryos is also an important factor for the prediction of pregnancy outcome and the chance of multiple pregnancies. These results demonstrated that the evaluation of early cleavage embryos to 2-cell stage is an easy, simple, and objective method for the selection of good quality embryos suitable for embryo transfer.

• Clinical and Experimental Reproductive Medicine
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• v.22 no.2
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• pp.191-201
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• 1995

Transcriptional activation of the embryonic genome initiates at 2-cell stage in mouse embryo and is characterized by the synthesis of TRC which is restricted to 2-cell stage. To investigate the roles of various protein kinases on the embryonic gene activation, the effects of protein kinase inhibitors on in vitro development and protein synthetic profiles of the early mouse embryos were examinded. None of ${\alpna}-amanitin$ which is a mRNA synthetic inhibitor, H8 which is a PKA inhibitor, and H7 which is a PKC inhibitor, affected on first cleavage of mouse 1-cell embryos in vitro. However, all of these drugs inhibited the second cleavage. When the drugs were removed following treatment for 6 hours, H8 or H7 treatment showed little inhibition on subsequent development of 1-cell embryos to 2-cell stage or further. In contrast, ${\alpna}-amanitin$ irreversibly inhibited the development of 1-cell embryos to 2-cell stage following removal of the drug. Genistein, a TPK inhibitor, inhibited both the first cleavage of 1-cell embryos and the second cleavage of 2-cell embryos, suggesting that TPK activity may be important during the early cleavages. All of the above four drugs inhibited TRC synthesis as shown by the fluorographic analysis of $[^{35}S]-Met$ labeled protein profiles. When late 1-cell embryos were treated with H7 and analyzed synthetic patterns of $[^{35}S]-Met$ labeled protein, the quantitative differences of protein synthesis on SDS-PAGE appeared on 77 kD and 33 kD region at $32{\sim}38$ hours post hCG. From these studies, transcriptional activation of embryonic genome is not essenting to the mouse 1-cell embryos to develop to 2-cell stage. Hawever, TPK activity is reguisite for both the first cleavage and second cleavage. Similarly, both PKC and PKA activities are required for the second cleavage of mouse embryos, but not for the first cleavage.

• Korean Journal of Veterinary Research
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• v.29 no.2
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• pp.123-128
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• 1989

Demi-embryos were successfully produced by bisection of ICR mouse embryos at preimplantation stages. They were microsurgically bisected using a microsurgical blade attached to a micromanipulator after pretreatment with 0.5% pronase in PBS for two minutes or not. Embryos with softened zona pellucida were more easily bisected and less damaged than intact embryos. The highest success rate in bisection has been achieved by selecting blastocysts(94.1% in success rate with intact blastocysts and 100% in success rate with zona softened blastocysts). Demi-embryos without zona pellucida were cultured in D-PBS or M-16 medium at $37^{\circ}C$, 5% $CO_2$ in air for 72 hours for 2-cell stage embryos, 48 hours for 4-to 8-cell stage embryos, 24 hours for morula stage embryos and 6~12 hours for blastocyst stage embryos. For the in vitro culture of 2-cell stage embryos, $100{\mu}M$ 2Na-EDTA was added to the media. M-16 medium was better for the in vitro development of mouse embryos than PBS, and PBS is not considered to be suitable for long-term culture of embryos, especially at early stage of cleavage. In M-16 medium, developing rate of demi-embryos of which pair underwent development to form eublastocysts was 15.8% at 2-cell stage, 16.8% at 4-cell stage, 38% at 8-cell stage, 89.6% at morula stage and 94.4% at blastocyst stage, respectively. The more rapid and efficient production of demi-embryos and higher viability after bisection can be expected by softening zona pellucida with pronase and by selecting morulae or blastocysts rather than embryos at early stage of cleavage.

• Reproductive and Developmental Biology
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• v.28 no.1
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• pp.13-20
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• 2004

This study was conducted to investigate the developmental ability of caprine embryos after somatic cell interspecies nuclear transfer. Donor cells were obtained from an ear-skin biopsy of a caprine, digested with 0.25% trypsin-EDTA in PBS, and primary fibroblast cultures were established in TCM-199 with 10% FBS. After maturation, expanded cumulus cells were removed by vigorous pipetting in the presence of 0.3% hyaluronidase. The matured oocytes were dipped in D-PBS plus 10% FBS＋7.5 $\mu\textrm{g}$/ml cytochalasin B and 0.05 M sucrose. The reconstructed oocytes were electrically fused with donor cells in 0.3 M mannitol fusion medium. After the electofusion, embryos were activated by electric stimulation. Interspecies nuclear transfer embryos with bovine cytoplasts were cultured in TCM-199 medium supplemented with 10% FBS including bovine oviduct epithelial cells for 7∼9 day. On the other hand, the NT embryos with porcine cytoplasts were cultured in NCSU-23 medium supplemented with 10% FBS for 6∼8 day at $39^{\circ}C, 5% CO_2$ in air. In caprine-bovine NT embryos, the cleavage(2-cell) rate was 36.8% in confluence and 43.8% in serum starvation. The developmental rate of morula- and blastocyst-stage embryos was 0.0% in confluence and 18.8% in serum starvation. In caprine-porcine NT embryos, the cleavage(2-cell) rate was 76.7% in confluence and 66.7% in serum starvation. The developmental rate of morula and blastocyst stage embryos was 3.3% in confluence and 3.0% in serum starvation, and no significant difference was observed in synchronization treatment between donor cells. In caprine-bovine NT embryos, the cleavage(2-cell) rate of cultured donor cells was 30.8% and 17.6% in 5∼9 and 10∼14 passage(P＜0.05). The developmental rate of morula and blastocyst stage embryos were significantly higher(P＜0.05) in 5∼9 passage(23.1%) than in 10∼14 passage(0.0%) of cultured donor cells. In caprine-porcine NT embryos, the cleavage rate was significantly higher(P＜0.05) in 5∼9 passage(86.7%) than in 10∼14 passage(50.0%) of cultured donor cells. The developmental rate of morula and blastocyst stage embryos were 3.3 and 0.0% in 5∼9 and 10∼14와 passage of cultured donor cells. In caprine-bovine NT embryos, the developmental rate of morula and blastocyst stage embryos were 22.6% in interspecies nuclear transfer, 33.9% in in vitro fertilization and 28.1% in parthenotes, which was no significant differed. The developmental rate of morula and blastocyst stage embryos with caprine-porcine NT embryos were lower(P＜0.05) in interspecies nuclear transfer(5.1%) than in vitro fertiltzation(26.9%) and parthenotes(37.4%).

• Journal of Embryo Transfer
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• v.12 no.2
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• pp.161-169
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• 1997

This study was conducted to compare the insemination time of bovine oocytes and determine the effects of glucose(1.5 mM) on the development of bovine embryos at early cleavage stage. Oocytes were matured for 24 h, followed by exposure to sperm and cultured in modified Tyrode's media drops or with bovine oviduct epithelial cell monolayer prepared in TCM199(BOECM). Insemination time and culture system were varied in each experiment. In experiment 1, to investigate the developmental capacity of bovine embryos after different time of exposure to sperm, bovine ova and sperm were co-incubated for 18, 30 or 54 h, respectively. The development to blastocysts of 30 and 54 h insemination groups were significantly higher(P<0.05) than 18 h group, and in case of blastocysts of cleaved embryos, 30 h group were significantly higher(P<0.05) than other groups. In experiment 2, we investigated the effect of glucose on early bovine embryos. After 18 h insemination, in vitro fertilized oocytes were separated following 3 groups ; G+0, C+24 and C+48. Oocytes of G+0 group were cultured in glucose added Tyrode's medium after fertilization, oocytes in C+24 and C+48 groups were cultured in glucose free Tyrode's medium after fertilization. After 24 h culture, G+24 group was moved to glucose added medium. All oocytes of 3 groups were moved to BOECM after 48 h culture. The rates of cleavage and development to blastocysts in G+0 group were significantly lower than other groups. In experiment 3, we determined the effects of glucose exposure from 8 to 20 h after insemination on the cleavage and development of oocytes. The oocytes in glucose added group had high capacity of cleavage and further development. This study shows that in bovine oocytes, the optimal exposure to sperm is 30 h and glucose exposure to bovine one-cell embryos is detrimental to their first cleavage and further development in vitro but there has no evidence of detrimental effect of glucose(1.5 mM) exposure to bovine embryos over the two-cell stage in vitro.

• Journal of Embryo Transfer
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• v.14 no.1
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• pp.23-29
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• 1999

The objectives of the study were to establish sperm separation method and duration of insemination for bovine IVF. Oocytes from slaughterhouse ovaries were matured and fertilized using general protocol. After 18 or 42 h of insemination, six to ten embryos were placed into a 30${mu}ell$ drop of each medium, and the embryos were examined 7~10d post in semination without medium renewal. First, we compared Percoll gradient will swim-up technique for sperm separation. There was no difference in cleavage rates between them, but the development rates over morula stage of oocytes fertilized with sperm separated by Percoll gradient was significantly higher than that sperm selected by swim-up technique (p<0.05). Second, we evaluated development of bovine embryos derived from the IVF procedure with different durations(18 vs 42 h) of fertilization. There was also no difference in cleavage rates, but the development to blastocyst stage of oocytes exposed in cleavage rates, but the development to blastocyst stage of oocytes exposed to sperm for 42 h was significantly higher than that exposed for 18 h (p<0.05). In conclusion, Percoll gradient can be used for sperm selecton, improving of embryonic development. Also, 42h of IVF may improve the development of bovine embryos.

• Asian-Australasian Journal of Animal Sciences
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• v.13 no.7
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• pp.894-896
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• 2000

In vitro fertilization can be used for salvaging superior buffalo germplasm which otherwise goes waste after the slaughter of animals. This technology has also increased our basic understanding of growth of germ cells and embryos. The requirement of growing embryos is peculiar and stage specific. In the present study the cleavage and development of buffalo embryos were studied with homologous (buffalo) and heterologous (goat) oviductal cell co-culture systems. The cleavage rate improved significantly (p<0.01) in both homologous and heterologous co-culture as compared to control (55.3, 46.8 and 11.4%). The morula formation using homologous and heterologous oviductal cells also increased significantly as compared to control group (43.6, 21.9 & 1.9%). There was no blastula formation in control group, but addition of oviductal cells either from homologous or heterologous species significantly increased the blastula formation (9.5, 12.5%). The cleavage rate and embryo development was slightly better (non significant) in homologous as compared to heterologous oviductal cell culture. It was concluded that the use of oviductal cell co-culture (homologous and heterologous species) have significantly improved cleavage and development of buffalo embryos in vitro.

• Clinical and Experimental Reproductive Medicine
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• v.43 no.3
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• pp.181-184
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• 2016

The aim was to report a healthy live birth using re-vitrified-warmed cleavage-stage embryos derived from supernumerary warmed embryos after frozen embryo transfer (ET) in a patient with recurrent implantation failure (RIF). The case was a 39-year-old female with a history of polycystic ovarian syndrome and adenomyosis, along with RIF. After ovarian hyperstimulation, 33 cumulus-oocyte complexes were retrieved and fertilized with conventional in vitro fertilization and intracytoplasmic sperm injection. Because of the risk of ovarian hyperstimulation syndrome, 16 grade B and C embryos were vitrified. After 3 and 6 months, 3 and 4 B-C warmed embryos were transferred to the uterus, respectively. However, implantation did not take place. Ten months later, four embryos were warmed, two grade B 8-cell embryos were transferred, and two embryos were re-vitrified. One year later, the two re-vitrified cleavage-stage embryos were warmed, which resulted in a successful live birth. This finding showed that following first warming, it is feasible to refreeze supernumerary warmed embryos for subsequent ET in patients with a history of RIF.