• Clinical and Experimental Reproductive Medicine
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• v.24 no.3
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• pp.301-309
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• 1997

The objective of this study was to determine the microtubule assembly and chromatin configuration during the first cell cycle in bovine oocytes following injection of spermatozoon, sperm head and tail. The microtubule and chromatin configuration was imaged with fluorescent labeled monoclonal ${\alpha}$-tubulin antibody and propidium iodide under laser scanning confocal microscope. Microtubule and chromatin dynamics in bovine oocytes following intracytoplasmic sperm injection (ICSI) were not different from those observed during in vitro fertilization (IVF). Following ICSI, the microtubular aster was observed around sperm midpiece. During pronuclear formation, the sperm aster was enlarged and seen around male and female pronuclei. At mitotic metaphase, the microtubular spindle assemble astral poles and chromosomes were aligned on the spindle equator. At mitosis, asters were concentrated to each spindle pole and they filled the cytoplasm. After injection of the isolated sperm head, the microtubular aster was not seen around sperm head in any cases (0/18). Instead, microtubules were organized from the cytoplasm, which filled the whole cytoplasm during pronuclear apposition. These microtubules seem to move male and female pronuclei. These results suggest that isolated sperm head can develop into normal pronucleus in mature bovine oocytes, and competent to participate syngamy with the ootid chromatin. The functional microtubules following isolated sperm head injection in bovine oocytes appeared to be organized solely from maternal stores.

• Journal of Physiology & Pathology in Korean Medicine
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• v.33 no.2
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• pp.141-150
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• 2019

Despite the development of assisted reproduction technologies (ART) including in vitro fertilization (IVF), the poor ovarian response and endometrial receptivity remains clinically a major unmet need. Although these problems are difficulties to solve in infertility treatment, there are no good therapeutic option yet. Traditional herbal remedies and acupuncture, therefore are being proposed as alternative treatment. Our group found that traditional herbal medicines such as Paeonia lactiflora L.(PL, 芍藥), Cyperus rotundus L.(CR, 香附子), and Perilla frutescens (PF, 紫蘇葉) could improve endometrial receptivity. In this study, we found out Yeosin-san (如神散) as an optimal herbal formula via combination of the previously established herbal medicines. Yeosin-san is a traditional Korean medical formula which was established by Ziming Jin (陳自明) and recorded in Furendaiquanliangfang (婦人大全良方) at first. The formula traditionally used for treating abnormal uterine bleeding and leukorrhea. It showed a highest effect on leukemia inhibitory factor (LIF) expression and on the adhesion between trophoblastic cells and endometrial cells. In addition, it has been shown that the Yeosin-san not only increases the endometrial receptivity to improve the embryo implantation but also enhances the ovary function by expressing the angiogenesis-related genes. Here we suggest that Yeosin-san could be a novel and effective candidate for treating female infertility.

• Journal of Embryo Transfer
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• v.29 no.1
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• pp.13-19
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• 2014

This study was carried out to study the survival rate of thawed Hanwoo embryos frozen by the slow-rate freezing or the cryotop vitrification method. Hanwoo cumulus-oocyte complexes were recovered from ovaries at a slaughter house, matured for 20~22 hours, fertilized with Hanwoo semen for 5~6 hours, and cultured for 7~9 days in $38.5^{\circ}C$, 5% $CO_2$ incubator. For freezing, Day 7~9 blastocysts were collected. Embryos for the slow-rate freezing were equilibrated in 1.8 M ethylene glycol (EG) with Dulbecco's phosphate-buffered saline (D-PBS). Programmable cell freezer was precooled down to $-7^{\circ}C$, and the straw was seeded during 8 minutes-holding time, and was cooled to $-35^{\circ}C$ at the cooling rate of $0.3^{\circ}C/min$, and then was plunged and stored in liquid nitrogen. Embryos for the cryotop vitrification were treated in TCM199 with 0.5 M sucrose, 16% EG, 16% dimethylsulfoxide (DMSO). Embryos were then loaded individually onto cryotop and plunged directly into liquid nitrogen. The survival rates of embryos frozen by these two freezing methods were evaluated at 12 to 24h post-thawing. The survival rates of frozen/thawed Hanwoo embryos by the cryotop vitrification method ($56.86{\pm}26.53%$) were slightly higher than those by the slow-rate freezing method ($55.07{\pm}26.43%$) with no significant difference. Using the cryotop vitrification and the slow-rate freezing of Hanwoo blastocysts on Day 7 following in-vitro fertilization (IVF) treatment, the survival rates of frozen/thawed Hanwoo embryos were $72.65{\pm}18.3%$ and $79.06{\pm}17.8%$, respectively. The survival rates by the cryotop vitrification were higher than those by the slow-rate freezing on both Day 8 and 9 with significantly higher survival rate on Day 9 (p<0.05). Using the cryotop vitrification and the slow-rate freezing of Hanwoo embryos to compare between three different blastocyst stages, the survival rates of the blastocyst stage embryos were $66.22{\pm}18.8%$ and $45.76{\pm}12.8%$, respectively with higher survival rate by the vitrification method (p<0.05). And the survival rate of expanded blastocysts was higher than those of early blastocysts and blastocysts in two freezing methods with significantly higher survival rate by the slow-rate freezing method (p<0.05).

• Clinical and Experimental Reproductive Medicine
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• v.30 no.1
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• pp.31-37
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• 2003

연구목적: 일반적으로 IVF-ET에서 가장 높은 임신율을 얻는 방법은 5 day ET (배반포기 배아 이식)이지만 장기간 배양이 적절하지 못한 경우에는 $2{\sim}4$일째에 ET를 실시하고 나서 $5{\sim}7$일째에 배반포기에 도달한 배아를 재이식 (SET)하여, SET의 효용성에 대하여 조사하고자 실시하였다. 연구재료 및 방법: 48주기의 환자에서 회수한 난자와 수정란은 10%와 20% hFF가 첨가한 DMEM에서 이식 직전까지 각각 공배양하였다. 채란 2일 (group I, day 2 ET), 5일째 이식 (group II, day 5 ET) 또는 재이식 (group III, SET; 2-5, 2-6, 2-7, 3-5, 4-7일)을 실시하면서 수정률, 할구분할률 및 임신율을 각각 비교하였다. 결과에 대한 통계 분석을 SAS (version 6.2)를 이용한 Duncan's Multiple Range Test를 이용하여 p값이 0.05 보다 작을 때 통계적으로 유의차가 있는 것으로 하였다. 결 과: 수정률은 group II (90.5%)가 다른 군에 비하여 높게 (p<0.05) 나타났다 (group I: 80.6%; group III: 82.9%). 할구분할률은 군간에 차이가 없었다 (수정란 당 $93.3{\sim}99.1%$). 임상적 임신율은 group II와 III (각각 58.3%)가 group I (33.3%) 보다 높게 나타났다. 그러나 처리군이 적어서 통계적인 차이는 없었다. 결 론: 배반포기 배아를 단독 이식하는 것이 임신율을 높일 수 있는 최선의 방법으로 나타났지만, 채란수가 적거나 수정률이 저조한 경우에는 $2{\sim}4$일째에 ET를 실시한 후 여분의 배아를 배반포기까지 배양한 다음 $5{\sim}7$일에 재이식 (SET)하면 blastocyst ET에서 나타날 수 있는 이식 자체의 실패를 방지할 수 있으면서 임신율을 높일 수 있는 이식 기법이 될 것이다.

• Clinical and Experimental Reproductive Medicine
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• v.27 no.1
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• pp.47-57
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• 2000

Objective: The effects of cryopreservation with or without coculture on the in vitro development of blastomere-biopsied 8-cell mouse embryos were investigated. This experimental study was originally designed for the setup of a preclinical mouse model for the preimplantation genetic diagnosis (PGD) in human. Methods: Eight-cell embryos were obtained after in vitro fertilization (IVF) from F1 hybrid mice (C57BL(표현불가)/CBA(표현불가)). Using micromanipulation, one to four blastomeres were aspirated through a hole made in the zona pellucida by zona drilling (ZD) with acid Tyrode's solution (ATS). A slow-freezing and rapid-thawing protocol with 1.5M dimethyl sulfoxide (DMSO) and 0.1M sucrose as cryoprotectant was used for the cryopreservation of blastomere- biopsied 8-cell mouse embryos. After thawing, embryos were cultured for 110 hours in Ham's F-10 supplemented with 0.4% bovine serum albumin (BSA). In the coculture group, embryos were cultured for 110 hours on the monolayer of Vero cells in the same medium. The blastocyst formation was recorded, and the embryos developed beyond blastocyst stage were stained with 10% Giemsa to count the total number of nuclei in each embryo. Results: The survival rate of embryos after cryopreservation was significantly lower in the blastomere-biopsied (7/8, 6/8, 5/8, and 4/8 embryos) groups than in the non-biopsied, zona intact (ZI) group. Without the coculture, the blastocyst formation rate of embryos after cryopreservation was not significantly different among ZI, the zona drilling only (ZD), and the balstomere-biopsied groups, but it was significantly lower than in the non-cryopreserved control group. The mean number of cells in embryos beyond blastocyst stage was significantly higher in the control group ($50.2{\pm}14.0$) than in 6/8 ($26.5{\pm}6.2$), 5/8 ($25.0{\pm}5.5$), and 4/8 ($17.8{\pm}7.8$) groups. With the coculture using Vero cells, the blastocyst formation rate of embryos after cryopreservation was significantly lower in 5/8 and 4/8 groups, compared with the control, 7/8, and 6/8 groups. The mean number of cells in embryos beyond blastocyst stage was also significantly lower in 4/8 group ($25.9{\pm}10.2$), compared with the control ($50.2{\pm}14.0$), 7/8 ($56.0{\pm}22.2$), and 6/8 ($55.3{\pm}25.5$) groups. Conclusion: After cryopreservation, blastomere-biopsied mouse embryos have a significantly impaired developmental competence in vitro, but this detrimental effect might be prevented by the coculture with Vero cells in 8-cell mouse embryos biopsied one or two blastomeres. Biopsy of mouse embryos after ZD with ATS is a safe and highly efficient preclinical model for PGD of human embryos.

• Journal of Embryo Transfer
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• v.30 no.2
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• pp.73-82
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• 2015

This study was designed to evaluate the possibility of increase through dairy female offspring's ratio by transfer of pre-selected transferrable blastocyst that was produced by pre-selected X-bearing semen with OPU derived oocytes. Elite dairy female cow is demanded strongly compared with male, the so called, farmer wants to produce only an elite female dairy offspring as a candidate female dairy cow for producing milk. In our study, we selected 2 elite dairy bull semen from National Agricultural Cooperative Federation to pre-select X-bearing semen and 5 elite dairy female cows as donor for collecting of OPU derived oocytes. OPU derived embryo production system was carried out an aspiration of immature oocytes from 5 donor cows 2 times per week, total 200 times for 2 to 7 months by an ultrasonographic guided follicular aspiration system and then produced in vitro-produced blastocysts by in vitro maturation, fertilization and culture. Dairy donor semen selected H-319, 320 bull in National Agricultural Cooperative federation was sorted X-bearing semen by flow-cytometer and frozen for using IVF with OPU derived oocytes. Donor cows were selected 5 elite dairy cows from Gyeongju Dairy Cow Community and then disease tests such as 4 kinds of disease before selecting was checked. Oocyte proportion of grade 1 to 3 from total collected oocytes was significantly lower in donor A and B than those in donor C, D and E (82.16 and 70.03% vs. 90.0, 91.78 and 93.57%), respectively (p<0.05). However, number of oocytes per session in donor A, C and E was significantly higher than those in donor B and D ($7.77{\pm}3.26$, $5.85{\pm}2.10$ and $7.03{\pm}2.14$ vs. $4.68{\pm}2.61$ and $5.21{\pm}1.97$ oocytes), but donor A was significantly higher than donor C (p<0.05). Development to blastocyst in donor B, C and E was significantly higher than those in donor A and D (31.0, 25.0 and 25.0% vs. 14.3 and 4.5%), but donor A was not different in donor C and E (p<0.05). Nine out of 10 blastocysts (90.0%) derived from OPU blastocysts were confirmed male embryos that was induced with Y-bearing semen to confirm sex ratio only. Total 96 blastocysts derived from female bearing semen were transferred into synchronized recipients and then confirmed 42 recipients (43.8%) pregnancy rate, 36 offspring (37.5%) and 91.7% female sex ratio (33 female vs. 3 male offspring). Taken together all data, elite dairy female offspring could be produced effectively by in vitro production system between pre-selected x-bearing semen and OPU derived oocytes that would be influential breeder in the breeding of dairy farm to increase effectively elite dairy offspring ratio as well as net income in the dairy farmer.

• Clinical and Experimental Reproductive Medicine
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• v.32 no.4
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• pp.315-324
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• 2005

Objectives: To compare the efficacy of GnRH antagonist multiple dose protocol (MDP) with that of GnRH agonist long protocol (LP) in controlled ovarian hyperstimulation for in vitro fertilization in patients with high basal FSH (follicle stimulating hormone) level or old age, a retrospective analysis was done. Methods: Two hundred ninety four infertile women (328 cycles) who were older than 41 years of age or had elevated basal FSH level (> 8.5 mIU/mL) were enrolled in this study. The patients had undergone IVF-ET after controlled ovarian hyperstimulation using GnRH antagonist multiple dose protocol (n=108, 118 cycles) or GnRH agonist long protocol (n=186, 210 cycles). The main outcome measurements were cycle cancellation rate, consumption of gonadotropins, the number of follicles recruited and total oocytes retrieved. The number of fertilized oocytes and transferred embryos, the clinical pregnancy rates, and the implantation rates were also reviewed. And enrolled patients were divided into three groups according to their age and basal FSH levels; Group A - those who were older than 41 years of age, Group B - those with elevated basal FSH level (> 8.5 mIU/mL) and Group C - those who were older than 41 years of age and with elevated basal FSH level (> 8.5 mIU/mL). Poor responders were classified as patients who had less than 4 retrieved oocytes, or those with $E_2$ level <500 pg/mL on the day of hCG injection or those who required more than 45 ampules of exogenous gonadotropin for stimulation. Results: The cancellation rate was lower in the GnRH antagonist group than in GnRH agonist group, but not statistically significant (6.8% vs. 9.5%, p=NS). The amount of used gonadotropins was significantly lower in GnRH antagonist group than in agonist group ($34.8{\pm}11.3$ ampules vs. $44.1{\pm}13.4$ ampules, p<0.001). The number of follicles > 14 mm in diameter was significantly higher in agonist group than in antagonist group ($6.7{\pm}4.6$ vs. $5.0{\pm}3.4$, p<0.01). But, there were no significant differences in clinical pregnancy rate (24.5% in antagonist group vs. 27.4% in agonist group, p=NS) and implantation rate (11.4% in antagonist group vs. 12.0% in agonist group, p=NS) between two groups. Mean number of retrieved oocytes was significantly higher in GnRH agonist LP group than in GnRH antagonist MDP group ($5.4{\pm}3.5$ vs. $6.6{\pm}5.0$, p<0.0001). But, the number of mature and fertilized oocytes, and the number of good quality (grade I and II) and transferred embryos were not different between two groups. In each group A, B, and C, the rate of poor response did not differ according to stimulation protocols. Conclusions: In conclusion, for infertile women expected poor ovarian response such as who are old age or has elevated basal FSH level, a protocol including a controlled ovarian hyperstimulation using GnRH antagonist appears at least as effective as that using a GnRH agonist, and may offer the advantage of reducing gonadotropin consumption and treatment period. However, much work remains to be done in optimizing the GnRH antagonist protocols and individualizing these to different cycle characteristics.

• Clinical and Experimental Reproductive Medicine
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• v.37 no.4
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• pp.349-359
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• 2010

Objective: This study was performed to compare the clinical outcome of elective single embryo transfer (eSET) performed at the cleavage stage to that of elective double embryo transfer (eDET). Methods: Of the women less than 36 years old who visited Daegu Maria from January 2008 to April 2009, the only women (n=330) with more than 8 mm of endometrial thickness and at least one good quality embryo, who were treated with GnRH agonist long protocol, were included in this study. After information about complications that can arise by multiple embryo transfer, either eSET or eDET was conducted by their request (167 and 163, respectively).Results: The implantation rate of eSET group was significantly higher than that of eDET group (53.9% vs. 40.2%, p<0.01). The twin pregnancy rate of eSET group was significantly lower than that of eDET group (1.1% vs. 32.3%, p<0.001). However, there were no significant differences between two groups in the clinical pregnancy (53.3% vs. 60.7%, p=0.172), ongoing pregnancy (47.3% vs. 54.6%, p=0.185) and live birth rates (44.9% vs. 50.9%, p=0.275). The number of the surplus embryos which developed to the blastocyst stage and cryopreserved at that stage was significantly higher in eSET group than that of eDET group ($3.2{\pm}2.6$ vs. $2.1{\pm}2.4$, p<0.001). Conclusion: These results suggest that eSET should reduce significantly the multiple baby pregnancy without decreasing the whole pregnancy rate in women with less than 36 years old.

• Journal of Embryo Transfer
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• v.14 no.1
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• pp.17-22
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• 1999

To investigate the maturational and development competece of porcine oocytes of different diameter groups, oocytes were obtained by aspiration from slaughterdhouse ovaries. After washing three times in NCSU23 medium, each cumulus-oocyte complex was transferred into a $8{mu}ell$ drop of the maturation medium (one oocyte per drop) under paraffin oil. The diameter without zona pellucida of oocytes was measured with micor-calibrator (Mikrometer, E. Leitz) on a screen connected to a VCR on an inverted microscope $(200\times)$. After being measured, the oocytes were divided into 6 groups according to their diameter size : <105, 105 to < 110, 110 to < 115, 115 to < 120, 120 to < 125 and > $125{\mu}{\textrm}{m}$, and in vitro maturation (IVM), fertillzation (IVF) and production (IVP) of oocytes / embryo was performed. The rates of in vitro maturation on oocytes in the greater 105 ${\mu}{\textrm}{m}$ size groups(91.8~100%) were significantly (P<0.05) higher than in the < 105 ${\mu}{\textrm}{m}$ group(66.7%). The rates of sperm penetration were significantly (P<0.05) low in < $105{\mu}{\textrm}{m}$ group (50.0%) than others groups (81.6~85.5%). But the plyspermic fertilization rate was significantly (P<0.05) higher in < $110{\mu}{\textrm}{m}$ oocytes groups than in the $110\leq{\mu}{\textrm}{m}$ size groups. The rates of cleavage and development to blastocysts rose as oocytes diameter increased, however, while oocytes over $120{\mu}{\textrm}{m}$ in diameter failed to develop to blastocysts. There results suggest that porcine oocytes have acquired full meiotic competece at a diameter of $105{\mu}{\textrm}{m}$ but not yet attended full development competence to blastocyst and that oocytes have acquired full development competence at a diameter of $110{\mu}{\textrm}{m}$.

• Korean Journal of Animal Reproduction
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• v.24 no.1
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• pp.97-108
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• 2000

The objectives of this study were performed to increase the efficiency of the culture conditions of embryos produced in vitro, and to assess the developmental potential after transfer of those embryos into recipients. The mean number of folliclular oocytes recovered from an ovary was 10.7. The rates of maturation and fertilization in Grade I oocytes were significantly (P＜0.05) higher than Graden and III. Developmental rate into blastocyst in the culture group of TCM-199 with BOEC were significantly higher (P＜0.05) than the groups of TCM-199 and conditioned medium (24.7% vs. 12.4% and 18.2%). The survivability of post-thawed blastocysts equilibrated for 3 min in EFS solution was significantly (P＜0.05) lower than l0 for 1 and 2 min (32.1% vs. 82.9% and 73.3%). Significantly higher (P＜0.05) survival rate in blastocysts was seen after freezing than in morulae stage embryos. Out of all 105 recipients, 49 (46.7%) were confirmed in pregnant. On pregnancy of cattle, 48 calves were born from 40 recipients. The ratio of twin and single calves was 30.5% (32/40 and 7.6% (8/40), respectively. However, the others composed of abnormal, as judging as 6 (12.2%) for abortion and 3 (6.1%) for stillbirth during the pregnant period.