• Clinical and Experimental Reproductive Medicine
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• v.34 no.3
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• pp.159-166
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• 2007

Objective: To investigate whether semen parameters in infertile couples who undergone intrauterine insemination (IUI) change in the subsequent IUI cycle and the subsequent in vitro fertilization (IVF) cycle. Methods: Fifty-three infertile couples who had failed to become pregnant after the first IUI cycle with computer-assisted semen analysis (CASA) were included. After the first IUI, thirty-eight couples underwent the second IUI (Group 1), and fifteen underwent IVF-ET procedure (Group 2). All semen parameters including semen volume, concentration, motility and total motile sperm count were analyzed in the second IUI or IVF-ET procedure for comparison with the result of first IUI. Results: There were no significant differences in husband age, interval between the first and second procedure and cause of infertility. In Group 1, only sperm motility at the time of the latter IUI was significantly decreased when compared to the former IUI irrespective of the first semen parameters. In Group 2, sperm concentration, motility and total motile sperm count at the time of subsequent IVF were lower than the former IUI. By sub-analyses of Group 2, in the group of optimal semen parameter at IUI cycle, sperm concentration and total motile sperm count at the time of subsequent IVF were lower than the former IUI, while in the group of suboptimal semen parameter at IUI cycle, only sperm motility at the time of subsequent IVF were lower than the former IUI. Conclusion: The semen parameters in couples converted to IVF cycle were more adversely affected than those remained in IUI cycle. Further study on psychological stress should be necessary to explain the reason.

• Clinical and Experimental Reproductive Medicine
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• v.24 no.3
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• pp.325-333
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• 1997

The use of hormonal stimulation in human in vitro fertilization and embryo transfer (IVF-ET) leads to increased production of embryos for ET. So to avoid high pregnancies and to allow conception in future, unstimulated cycles, cryopreservation of spare embryos is desirable. One of the improvement of cryopreservation methods is vitrification. We cryopreserved mouse day 3 embryos by vitrification using the three different vitrification solution (EFS40, VS11 and VS3a). EFS40 solution is consisted of 40% (v/v) ethylene glycol, Ficol170 30% (w/v) and 0.5M sucrose and VS11 is 6.0M ethylene glycol and 1.8M glycerol. And VS3a is 6.5M glycerol and 6% (w/v) BSA (bovine serum albumin). First we tested the toxicity of three vitrification solution by exposure to these solution during 3 min. After washing by thawing solution, the survival rates of each groups are 95.5%, 90.9% and 84.4% (EFS40, VS11 and VS3a). High percentages of them developed to expanded blastocyst and hatching embryos in culture 48hrs 94.2%, 97.7%, 100% and 97.4% (no treatment group, EFS40, VS11 and VS3a). So there is no significant differences among the each group. Second, after thawing of vitirfied embryos, the survival rates of each groups are 96.8% (slow freeze), 94.1% (EFS40), 85.5% (VS11) and 80.0% (VS3a, P vs. no freeze or EFS40 is 0.01). Vitrified embryos exhibited a high rate of development in vitro after 48hrs culture. The percentages of each group to blastocyst and hatching embryos are 88.7% (no freeze), 91.8% (slow freeze), 93.4% (EFS40), 87.7% (VS11) and 73.0% (VS3a, P vs. other group is 0.01). The results suggest that there is no significant differences in exposure of various vitrification solution and day 3 mouse embryos can be vitrified in solution EFS40 and VS11 by simple procedure.

• Reproductive and Developmental Biology
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• v.28 no.2
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• pp.83-87
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• 2004

The objective of this study was performed to establish the in vitro culture system of porcine in vitro maturation and in vitro fertilization(IVM/IVF) embryo. These studies was to determine the effects of melatonin, nitric oxide donor(SNP), and the combination effects of SNP and melatonin in porcine IVM/IVF embryos. In routine porcine IVM/IVF procedure, oocytes were cultured for 40∼44h incubation, and the zygotes were cultured for 40∼44h in NCSU 23 medium. Then 2 to 8 cell embryos were removed cumulus cell and were allotted randomly to NCSU 23 containing different concentration of melatonin, SNP and SNP plus melatonin in 5% $O_2$, 5% $CO_2$ and 90% $N_2$ at 38.5$^{\circ}C$. Cell numbers of blastocyst were also counted using double fluorescence stain method. In NCSU 23 medium treated with melatonin 0, 1, 5 and 10 nM, the developmental rate of morula plus blastocysts were 33.3%, 39.1%, 33.3% and 27.9%, respectivly. This result show that the developmental rate of morula and blascytocys treated with 1 nM melatonin was higher than in any other groups(P<0.05). The developmental rates of morula plus blastocysts were 41.9% in 0 uM SNP, 25.6% in 50 uM and 28.4% in 100 uM, respectively. The developmental rate of morula plus blastocysts were decreased treated with SNP in NCSU 23. In combined effects of SNP plus melatonin (0, SNP 50 uM, SNP 50 uM plus melatonin 1 nM, SNP 50 uM plus melatonin 5 nM and SNP 50 uM plus melatonin 10 nM), the developmental rates beyond morula stage of porcine embryos were 31.3%, 34.1%, 39.5%, 29.4% and 39.5%, respectively. The addition of SNP 50 uM plus maltonin 1 nM, developmental rates of blastocyst was higher rate than in any other groups. Cell numbers of blastocyst in NCSU 23 treated with melatonin 0, 1, 5 and 10 nM were 41.0, 42.6, 39.6 and 33.0, respectively. In combined effects of SNP plus melatonin (0, SNP 50 uM, SNP 50 uM plus melatonin 1 nM , SNP 50 uM plus melatonin 5 nM and SNP 50 uM plus melatonin 10 nM), cell numbers of developed blastocyst were 36.3, 34.6, 39.0, 39.9 and 39.0, respectively. These result show that the cell numbers of blastocyst treated with 0, 1 and 5 nM melatonin were higher than in 10 nM group(P<0.05), but cell numbers of blatocyst produced by SNP plus melatonin were not significantly difference in all experimental groups.

• Reproductive and Developmental Biology
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• v.28 no.2
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• pp.77-82
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• 2004

The purpose of this study was performed to establish the in vitro culture system of porcine in vitro maturation and in vitro fertilization(IVM/IVF) embryo. These studies was to determine the effects of antioxidants(aesculetin, taurine and melatonin) in porcine IVM/IVF embryos. In routine porcine IVM/IVF procedure, oocytes were cultured for 40∼44h incubation in NCSU 23 mediumand matured oocytes were inseminated with frozen semen. Then 2 to 8 cell embryos were removed cumulus cell and were allotted randomly to NCSU 23 containing different concentration of antioxidants in 5% $O_2$ and 5% $CO_2$ at 38.5$^{\circ}C$. Cell numbers of blastocyst were also counted using double fluorescence stain method. Aesculetin were added to NCSU 23 medium at concentration of 1 ug, 5 ug, and 10 ug, when treated with 10 ug(35.7%) of aesucletin at the rate of embryos of the morula plus blatocsyts were higher than those of any other groups (30.2%, 29.5% and 29.2%)(P<0.05). The developmental rates beyond morula stage of porcine embryos in NCSU 23 medium supplemented with taurine 0, 2.5 and 5.0 mM were 26.1%, 26.9% and 31.7%, respectively. The addition of 5.0 mM taurine was higher the developmental rate beyond morula stage than in any other groups. In NCSU 23 medium treated with melatonin 0, 1, 5 and 10 nM, the developmental rate of morula plus blastocysts were 33.3%, 39.1%, 33.3% and 27.9%, respectively. The developmental rate of morula and blascytocys treated with 1nM melatonin was higher than in any other groups(P<0.05). Cell numbers of blastocyst in NCSU 23 treated with melatonin 0, 1, 5 and 10nM were 41.0, 42.6, 39.6 and 33.0, respectively. These results indicate that aesculetin, taurine and melation can increase the developmental rate beyond the morulae and blastocysts in porcine embryos.

• Journal of Embryo Transfer
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• v.15 no.3
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• pp.219-230
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• 2000

This study was carried out to determine the factors on achieving good viability of embryos biopsied fur sexing, to investigate pregnancy rate following embryo transfer(ET) with sexed embryos, and to confirm the accuracy for the calves bort following ET with sexed embryos by polymerase chain reaction(PCR). To investigate viability of Hanwoo embryos after biopsy for sexing, fresh and frozen/thawed embryos were biopsied according to different developmental day of blastocysts, different stage of blastocysts, and different biopsy grade and the embryos themselves were incubated for 2 hours in TCM199 after microsection to be evaluated morphologically for recovery as blastocyst. The results obtained were as follows : 1. The rate of oocytes cleaved in vitro and the rate of blastocyst of the cleaved oocytes were 52.5% and 21.6%, respectively. The rate of blastocyst on day 8 was 11.2%, denoting the highest rate during whole culture period posterior to in vitro fertilization(IVF) 2. After biopsy for sexing, the viability rate of blastocyst on day 7, 8 and 9 was 75.0%, 88.4%, and 100.0%, respectively and the viability of early, mid, and expanded blastocyst after biopsy was 75.0%, 88.9%, and 91.1%, respectively The viability rate of fresh and frozen/thawed embryos was 89.9%, 71.4%, respectively. And the viability of expanded, hatching, and hatched blastocyst of frozen/thawed embryos was : 75.0%, 75.0%, and 50.0%, respectively. The viability of embryos according to biopsy grade of 10∼20%, 21∼30%, and 31∼40% was 85.7%, 91.5%, and 71.4%, respectively. 3. Pregnancy rate after transfer with biopsied embryo between flesh and frozen/thawed embryos was 22.6% and 20.0%, respectively. 4. In comparison between sex by PCR method and sex of calves born after embryo transfer, the accuracy of sex deterimination was 92.3% (12/13).

• Korean Journal of Animal Reproduction
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• v.26 no.1
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• pp.41-51
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• 2002

The present study was carried out to examine the effect of cysteamine in vitro maturation (IVM) of porcine oocytes and development of porcine IVM/IVF Embryos. The results were summarized as follows : 1. The rates of nuclear maturation, penetrated oocytes, pronuclear formation, polyspermic oocytes and mean numbers of the penetrated sperm were not different in NCSU23 maturation medium with 0, 25, 50 and 100 $\mu$M cysteamine (P〉0.05). 2. The rates of blastocyst formation at day 7 after in vitro fertilization in 0, 25, 50 and 100 $\mu$M cysteamine were 17.9$\pm$6.1, 17.4$\pm$6.3, 24.2$\pm$1.9 and 16.9$\pm$2.0%, respectively. And the total cells were 30.7$\pm$2.4, 34.9$\pm$2.8, 39.6$\pm$2.3 and 36.8$\pm$3.6, respectively. Fifty $\mu$M cystealnine group was significantly higher than those of any other treatment groups (P＜0.05). 3. The ratios of ICM/total cells in 20~40% category were 20.5, 41.6, 19.5 and 31.5%, respectively. Twenty five $\mu$M cysteamine group was higher than those of other groups. 4. The rates of blastocyst formation at day 7 in the NCSU-23 culture medium of porcine IVF-produced embryos with 0, 25, 50, and 100 $\mu$M cysteamine were 16.0$\pm$0.2, 13.6$\pm$1.7, 25.0$\pm$0.8 and 15.7$\pm$4.5%, respectively. And the total cells were 27.0$\pm$3.7, 36.1$\pm$4.8, 34.0$\pm$3.8 and 25.2$\pm$4.4, respectively. Fifty $\mu$M cysteamine group was significantly higher than those of any other treatment groups (P＜0.05). 5. The ratios of ICM/total cells in 20~40% category were 53.8, 30.0, 16.6 and 11.1%, respectively. The addition groups of cysteamine were lower than those of control group. In conclusion, these results suggested that the addition of 50 $\mu$M cysteamine in the IVM medium and 25~50 $\mu$M cysteamine in IVC medium were effective on the blastocyst formation and total cells of blastocysts.

• Clinical and Experimental Reproductive Medicine
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• v.21 no.3
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• pp.233-240
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• 1994

This study was conducted to compare the endocrine milieu, and pregnancy rates in In Vitro Fertilization and Embryo Transfer(IVF-ET) program employing combined with gonadotropin releasing hormone agonist(GnRH-a) and pergonal(LH 75lU+FSH 75lU) when either human chorionic gonadotropin(HCG) or progesterone were used for luteal phase support. A total number of 40 IVF-ET treatment cycles were prospectively studied. Ovarian hyperstimulation method was modified ultrashort protocol using GnRH-a. All patients started Decapeptyl at menstrual cycle day # 2, and HMG was started at # 3 days. When leading follicle was ${\geqq}$18mm or at least two follicles were ${\geqq}$14mm in diameter, HCG 10000lU intramuscularly was injected. After 36 hours HCG administration, oocytes were retrieved as usual guided by transvaginal ultrasound. Embryo were transfered 36-48 hours later. The patient's cycles were prospectively randomized to receive HCG(20cycles) or Progesterone (20cycles) for luteal support. The progesterone group received 25mg 1M starting from the day of ET. The HCG group received 1500IU 1M. on days 0, +2, +5 after ET. Estadiol($E_2$) and Progesterone($P_4$) were measured on the day of oocyte aspiration, ET day, and every 6 days thereafter. Results were follows as; 1. Estradiol, progesterone and LH levels on the day of HCG trigger, retrieved oocytes and number of transfered embryo were not significantly different in both groups. 2. On the day of aspiration and embryo transfered day, $E_2$, $P_4$ level were significantly higher in progesterone group than HCG group(p<0.01). 3. $E_2$, $P_4$ level on 6 days after ET were significantly higher in progesterone group than HCG group(p<0.01). But, $P_4/E_2$ ratio was not different in both groups. 4. $E_2$, $P_4$ level 12 days after ET were decreased abruptly in both groups and higher hormonal level appeared in HCG group(P<0.01). 5. The total pregnancy rate in the HCG group was 40% (8/20) and in the progesterone group 15%(3/20). 6. Comparing the pregnant and nonpregnant cases progesterone group was not different the hormonal status. In HCG group, pregnant cases appeared in higher $P_4$, $P_4/E_2$ ratio than nonpregnanct cases(P<0.01).

• Clinical and Experimental Reproductive Medicine
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• v.22 no.2
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• pp.171-181
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• 1995

Ovarian hyperstimulation syndrome(OHSS) is one of the well-known complication of controlled ovarian hyperstimulation(COH). Though there have been numerous measures to prevent the occurrence of OHS, it has not been completely preventable until now. The fluid shift from the intravascular space to the third space is due to decreased oncotic pressure of the serum. The objective of this study was to evaluate if IV administration of 20% albumin in those patients with OHSS risk can make prevention of severe OHSS. We retrospectively analysed 70 patients undergoing IVF-ET who had serum peak estradiol($E_2$) level of >2,500 pg/ml and/or the number of oocytes retrieved over 20. The treatment group(n=39) received albumin while the control group(n=31) did not. After 40 grams of human albumin diluted in 1,000 ml of 0.9% sodium chloride solution, the treatment group received half of the fluid during oocyte retrieval, the remainder in the recovery suite. The results were as follows; There were significant differences in the levels of serum peak $E_2$ and number of oocytes retrieved between the two groups(p<0.05). However, there were no significant differences in the incidence of OHSS and pregnancy rate or multifetal pregnancy rate. In conclusion, administration of albumin to OHSS risk patients did not reduce the rate of OHSS in IVF-ET. However, if we consider the fact that there were differences in the level of peak serum $E_2$ and oocyte numbers, further prospective study may be needed.

• Clinical and Experimental Reproductive Medicine
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• v.38 no.1
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• pp.37-41
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• 2011

Objective: To compare the effectiveness and convenience of a pen device for the self-administration of follitropin ${\beta}$ with a conventional syringe delivering follitropin ${\beta}$ solution in patients undergoing IVF-ET. Methods: GnRH agonist long protocol was used for controlled ovarian stimulation (COS) in all subjects. A total of 100 patients were randomized into the pen device group or the conventional syringe group on the first day of COS. Local tolerance reactions were assessed within 5 minutes, at 1 hour and at 3 hours after each injection. On the day of hCG injection, patients were asked to rate their overall pain and convenience experienced with self-injection on a visual anlaogue scale (VAS). Results: There were no differences in patients' characteristics between the two groups. The duration of COS was significantly shorter in the pen device group than in the conventional syringe group. Patients included in the pen device group needed a significantly smaller amount of follitropin ${\beta}$. However, no differences between the two groups were found in IVF results and pregnancy outcome. The incidence of local pain within 5 minutes, at 1 hour and at 3 hours after the injection was significantly lower in the pen device group. VAS scores indicated that injections using the pen device were significantly less painful and more convenient. Conclusion: The pen device for self-administration of follitropin ${\beta}$ is less painful, safer and more convenient for the patients, and can be more effective because of the shorter duration and smaller dose of follitropin ${\beta}$ when compared with the conventional syringe.

• Journal of Embryo Transfer
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• v.28 no.3
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• pp.185-191
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• 2013

The purpose of this study is to improve production efficiency of vitrified-thawed transgenic bovine embryos. Transgenic bovine embryos were produced by injection of FIV-GFP lentiviral vector into perivitelline space of in vitro matured MII stage oocytes, and then in vitro fertilization. EGFP-expressing transgenic bovine blastocysts were cultured in serum-containing and serum-free medium. These blsatocysts were vitrified by pull and cut (PNC) container made with 0.25 cm plastic straw. Results indicate that total developmental rates of normal IVF embryo cultured in serum-containing and-free medium into blastocyst were not significantly different (22.3 vs 21.5%) and those of GFP-expressing transgenic bovine embryo into blastocyst showed no significant difference between serum-containing (13.9%) and-free medium (13.1%). However, developmental rate of GFP transgenic embryo was significantly (P<0.05) lower than its of normal IVF embryos. In additional study, we vitrified GFP transgenic normal bovine blastocysts using PNC vitrification method. Survival rate of vitrified-thawed GFP transgenic blastocyst (23.1%) was significantly (P<0.05) lower than its of normal blastocysts (68.9%). Although, survival rate of vitrified-thawed GFP transgenic blastocyst was lower than its of normal blastocyst, our result may suggested that PNC vitrification method is feasible to cryopreserve transgenic embryos. Our next plan will be the production of GFP express transgenic bovine derived from vitrified-thawed embryos using PNC method.