• Journal of Embryo Transfer
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• v.19 no.3
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• pp.265-273
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• 2004

This study was carried out to examine the effects of fertilization media and sperm concentration on in vitro fertilization (IVF) and development (IVD) of porcine oocytes matured in vitro. Cumulus-oocyte complexes (COCs) were collected from antral follicles of porcine ovaries collected from abattoir, and were matured in vitro in modified NCSU-23 (mNCSU-23) supplemented with 10％ porcine follicular fluid (pFF). After the fertilization by experimental scheme, putative embryos were developed in vitro in NCSU-23. The results are as follows. When the oocytes were fertilized in vitro in modified TBM or modified TLP-PVA by 1 ${\times}$10$^{5}$ sperm/$m\ell$, all of the fertilization parameters were not significantly different between two media. Subsequently, as these putative embryos were developed in vitro in NCSU-23, the percentage of oocytes cleaved and of blastocysts were not different between two media, either. When the oocytes were fertilized in vitro in mTBM by 5${\times}$10$^4$, 1${\times}$10$^{5}$ or 5${\times}$10$^{5}$ sperm/$m\ell$, all of the fertilization parameters were significantly (P<0.05 or P<0.01) increased as sperm concentration was elevated. Subsequently, as these putative embryos were developed in vitro in NCSU-23, the percentage of oocytes cleaved and of blastocysts were significantly boosted (P<0.01) as sperm concentration at fertilization was elevated from 5${\times}$10$^4$ to 1${\times}$10$^{5}$ sperm/$m\ell$, but were not different between 1${\times}$10$^{5}$ and 5${\times}$10$^{5}$ sperm/$m\ell$.

• Journal of Embryo Transfer
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• v.31 no.3
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• pp.153-159
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• 2016

(-)-Epicatechin gallate (ECG) is a polyphenol compound of green tea exhibiting biological activities, such as antioxidant and anticancer effects. To examine the effect of ECG on porcine oocytes during in vitro maturation (IVM), oocytes were treated with 0-, 5-, 15-, and $25{\mu}M$ ECG. After maturation, we investigated nuclear maturation, intracellular glutathione (GSH) and reactive oxygen species (ROS) levels and subsequent embryonic development after parthenogenetic activation (PA) and in vitro fertilization (IVF). After 42 hours of IVM, the $5{\mu}M$ group exhibited significantly increased (p< 0.05) nuclear maturation (89.8%) compared with the control group (86.1%). However, the $25{\mu}M$ group observed significantly decreased (p< 0.05) nuclear maturation (83.5%). In intracellular maturation assessment the 5-, 15-, and $25{\mu}M$ groups had significantly increased (p< 0.05) GSH levels and decreased ROS levels compared with the controls. The 5- and $15{\mu}M$ group showed significantly increased (p< 0.05) embryo formation rates and total cell number of blastocysts after PA (18% and 68.9, 15% and 85.1 vs. 12% and 59.5, respectively) compared with controls. Although the $25{\mu}M$ group observed significantly lower blastocyst formation rates after PA (27.6% vs. 23.2%) than control group, the $5{\mu}M$ group showed significantly increased blastocyst formation rates after PA (37.2% vs. 23.2%) compared to the control group. Furthermore, the $5{\mu}M$ group measured significantly increased blastocyst formation rates (20.7% vs. 8.6%) and total cell number after IVF ($88.3{\pm}1.5$ vs. $58.0{\pm}3.6$) compared to the control group. The treatment of $5{\mu}M$ ECG during IVM affectively improved the porcine embryonic developmental competence by regulating intracellular oxidative stress during IVM.

• Reproductive and Developmental Biology
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• v.31 no.2
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• pp.91-96
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• 2007

In vitro development of porcine embryo is affected by culture condition. One possible factor is osmolarity of culture medium. 1his study examined whether high osmolarity of culture medium at the early culture stage improves development of preimplantation porcine in vitro fertilization (IVF) and nuclear transfer (NT) embryos. NT and IVF embryos were divided into three groups and the basic medium was PZM-3 ($250{\sim}270$ mOsmol, control group). The control group of embryos was cultured in PZM-3 for whole culture period. Other two groups of embryos were cultured in a modified PZM-3 with 0.05 M sorbitol or 0.05 M sucrose ($300{\sim}320$ mOsmol, sorbitol or sucrose group) for the first 2 days, and then cultured in PZM-3 for further culture. NT embryos cultured in sucrose group showed a significantly higher developmental rate to the blastocyst stage with a decreased apoptosis rate compared to the sorbitol (p<0.05). For IVF, sucrose group showed a significantly increased the blastocyst formation rate with a decreased apoptosis rate compared to the control (p<0.05). This study represents that the high osmolarity in the early embryo culture stage can enhance the in vitro development of porcine NT and IVF embryos to the blastocyst stage with reduced apoptosis of cells.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.79-79
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• 2002

본 연구에서는 in vitro에서 성숙된 난자의 핵성숙(Polar Body extrusion)에 소요되는 시간과 배반포 단계로의 발달능력 사이의 관계를 비교하여 조기에 발달능력을 가진 embryo를 선발할 수 있는 IVP 체계를 개발하고자 하였으며 in vitro maturation(IVM)에 따른 first polar body(PB) 형성, IVM과 IVF 시간이 oocyte의 발달에 미치는 영향과 생산된 배반포의 세포수를 평가하였다. IVM은 TCM199 배양액을 사용하였고 in vitro fertilization(IVF)은 Fer -TALP용액을 사용하였으며 in vitro culture(IVC)는 CRlaa 배양액을 사용하여 2일까지는 0.3% BSA를 3일 부터는 10%FBS와 bovine oviduct epithelial cell을 첨가하여 배양하였다. IVM 시간에 따른 PB의 출현율은 0hr(0%), 6hr(0%), 12hr(0%), 14hr(8.7%), 16hr(40.5%), 18hr(48.0%), 20hr(65%), 22(68%) 그리고 24hr(74.5%)을 보였으며 IVM 시간에 따른 cleavage 및 8cell 발달율 사이에는 유의적인 차이가 없었으나 배반포(BL) 및 8cell에서 배반포로 발달률은 18시간(BL 31$\pm$6, BL/8cell 82 $\pm$5%)에서 가장 높게 나타났으며 24시간(BL 17$\pm$2, BL/8cell 60$\pm$8%)과 유의적인 차이를 보였다(P<0.05). IVC 7일째 배반포의 총세포수와 trophoblast(TE) 세포수는 IVM 18시간(mean$\pm$S.E.; total: 131.1$\pm$34.0, TE: 97.6$\pm$29.6)에서 24시간(total: 112.2$\pm$17.5, TE: 80.1$\pm$15.6)보다 유의하게 많은 것으로 나왔으나(P<0.05) 7일째의 inner cell mass(ICM) 숫자(18hr 33.5$\pm$12.8 vs 24hr 32.1$\pm$12.0)와 8일째 ICM, TE 그리고 총 세포수에는 유의성 있는 차이가 없었다. IVM 18시간에서 PB 형성과 8cell 발달률 사이에 높은 상관성을 보였고 배반포 및 8cell에서 배반포 단계로 높은 발달률을 보였으며 생산된 배반포의 TE 숫자와 총 세포수가 유의하게 많은 것으로 나타났다. 따라서 IVM 18시간 실시하였을 경우 보다 많은 세포수를 가진 배반포 발달 가능성이 높은 embryo를 조기에 선발 가능할 것으로 사료된다.

• Clinical and Experimental Reproductive Medicine
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• v.47 no.3
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• pp.213-220
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• 2020

Objective: The aim of this study was to explore the potential adverse effect of spontaneously decreasing serum estradiol (SE) levels on in vitro fertilization (IVF) outcomes. Methods: This retrospective single-subject study analyzed IVF cycles conducted at a hospital IVF unit between 2010 and 2017. Overall, 2,417 cycles were analyzed. Only cycles with spontaneously decreasing SE before human chorionic gonadotropin (hCG) triggering were included. Each patient served as her own control, and subsequent cycles were analyzed for recurrent SE decreases. The main outcome was the number of oocytes retrieved. Results: Cycle characteristics were similar between the study (SE decrease) and control groups, with the exception of the median SE on the day of hCG triggering (899.7 pg/mL; interquartile range [IQR], 193-2,116 pg/mL vs. 1,566.8 pg/mL; IQR, 249-2,970 pg/mL; p< 0.001). The study group, relative to the control group, had significantly fewer total oocytes (5 [IQR, 2-9] vs. 7 [IQR, 3-11]; p= 0.002) and significantly fewer metaphase II (MII) oocytes (3 [IQR, 1-6] vs. 4 [IQR, 2-8]; p= 0.001) retrieved. The study group had fewer cleavage-stage embryos than the control cycles (3 [IQR, 1-6] vs. 4 [IQR, 2-7]; p= 0.012). Compared to cycles with a ≤ 20% SE decrease, cycles with a > 20% decrease had significantly fewer total and MII oocytes retrieved. SE decrease recurred in 12% of patients. Conclusion: A spontaneous decrease in SE levels adversely affected IVF outcomes, with a linear correlation between the percentage decrease and the number of oocytes retrieved. SE decrease can repeat in later cycles.

• Journal of Embryo Transfer
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• v.9 no.1
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• pp.15-21
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• 1994

This experiment was investigated the effect of cell stage of embryos at 48 hours post-insemination On in vitro development of IVF embryos. The ovaries of Korean native cows or heifers were obtained from an abattoir and kept on 25 to 28$^{\circ}C$ and transported to laboratorty within 2 hrs. The oocytes were matured in vitro(IVM) for 24 hrs. in TGM-199 supplemented with 35 $\mu$g/$m\ell$ FSH, 10 $\mu$g/$m\ell$ LH, 1 $\mu$g/$m\ell$ estradiol-17$\beta$ and granulosa cells at 39$^{\circ}C$ under 5% $CO_2$ in air. They were fertilized in vitro(IVF) by epididymal spermatozoa treated with heparin for 24 hrs. , and then the zygotes were co-cultured in vitro(IVC) with bovine oviductal epithelial cells for 7 to 9 days. At 48 hrs. post-insemination, the embryos were classfied into 5 to 8-cell, 3 to 4-cell or 2-cell stage and then were co-cultured in vitro(IVC) with bovine oviductal epithelial cells until the embyos reached blastocyst stage. Embryos developed to blastocyst stage were stained with Hoechst 33342 for cell counting. The embryos of 5 to 8-cell stage at 48 hrs. post-insemination with grade I oocytes were significantly (P<0.05) better developed to blastocysts(63.0%) than 3 to 4-cell(42.0%) and 2-cell stage(2.7%) embryos which delayed in the early cleavage, and those embryos cleaved faster in the very early stage seemed to develop to blastocysts earlier. These results indicate that the embryos cleaved faster at 48 hrs. post-insemination seemed to develop to blastocysts earlier.

• Journal of Embryo Transfer
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• v.14 no.2
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• pp.113-119
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• 1999

This study was performed to improve the development of the in vitro fertilized bovine embryos by the condition of in vitro maturation. COCs were matured in TCM 199 supplemented with 0.1% PVA, 10ng/ml EGF, Hormones (5$\mu\textrm{g}$/ml FSH, 10 IU hCG, 1 $\mu\textrm{g}$/ml estradiol 17-$\beta$) or granulsa cell+Hormones atmosphere 39$^{\circ}C$, 5% CO2, 95% air for 24hrs. Matured oocytes were fertilized with frozen-thawed semen capacitated with 5mM caffein in BO medium for 20 hrs. IVF embryos were cultured in TCM 199 containing with hormones(same as matured medium), 10% FBS and co-culture with bovine oviduct epitherial cells. Maturation rates of COCs were showed 73.8%, 78.5%, 83.2% and 87.6% respectively, and were significant differences between PVA, EGF, and Hormones, GC+Hormones(p<0.05). The cleavage rates of IVF embryos were revealed 72.5%, 78.4%, 82.3% and 84.2% and showed same tendency as maturation rates(p<0.05). The blastocysts matured by above maturation condition and cultured for 7~10 days after fertilization had 34.4, 43.6, 52.3 and 59.3 cells had no differences among the treatments. These results suggest that high molecules as a substitutes of serum and growth factor may induce nuclear resumption of COCs but we need more study to produce transferable IVF blastocysts by use of that agents.

• Clinical and Experimental Reproductive Medicine
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• v.34 no.3
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• pp.173-178
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• 2007

Objective: To compare the outcome of twin pregnancies after in-vitro fertilization (IVF) with that of spontaneously conceived twins. Methods: We analyzed 146 twins retrospectively, who had been delivered between January 2000 and July 2005. After reviewing their obstetric medical records, 72 twins conceived with IVF (IVF group) and 51 spontaneously conceived twins (spontaneous group), whose gestational age was over 24 weeks, were recruited for this study. Results: There were no significant statistical differences of demographic features such as maternal age, gestational age and fetal weight between the two groups. However, the risk of pregnancy-induced hypertension (PIH) and gestational diabetes (G-DM) had significantly increased in the group of IVF (OR 2.59; 95% CI 1.01$\sim$6.66). The risk of fetal weight discordancy rate (>20%) and fetal-sex homology rate in IVF group were decreased (OR 0.37; 95% CI 0.14$\sim$0.96, OR 0.45; 95% CI 0.21$\sim$0.99). Conclusion: In this comparative study, maternal PIH and G-DM risks of IVF group are higher than that of spontaneous group. But, fetal-weight discordancy risk and fetal-sex homology rate were lower in IVF group.

• Reproductive and Developmental Biology
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• v.35 no.4
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• pp.491-498
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• 2011

During mammalian fertilization, germ cell-specific hyaluronidases, such as sperm adhesion molecule 1 (SPAM1) and hyaluronoglucosaminidase 5 (Hyal5), are important for the dispersal of the cumulus mass. In this study, we demonstrated that bull Hyal5 is a single copy gene on chromosome 4 that is expressed specifically in the testis. In addition, we expressed recombinant bull SPAM1 and Hyal5 in human embryonic kidney 293T cells and showed that these enzymes possessed hyaluronidase activity. We also demonstrated that a polyclonal antibody against bull sperm hyaluronidase inhibits sperm-egg interactions in an in vitro fertilization (IVF) assay. Our results suggested that bull Hyal5 may have a critical role in bull fertilization.

• Clinical and Experimental Reproductive Medicine
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• v.22 no.2
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• pp.211-220
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• 1995

Though the endometriosis is not always related with infertility, endometriosis causes infertility in some patients. There are many treatment modalities of infertile patients who have endometriosis. In recent years, Assisted Reproductive Technology(ART) have been widely accepted as being a useful tool for the treatment of infertile endometriotic patients. The objective of this study was to evaluate the outcome of ART in infertile endometriotic patients who have been carried out IVF-ET from Jan, 1992 to Dec, 1994 and to compare the results between COH/IUI and IVF-ET in the patients with endometriosis stage I. Tubal disease only patients were grouped(308 patient, 956 cycles) as a control. Endometriosis group was subdivided into 4 groups according to American Fertility Society classification; endometriosis stage I (45 patients, 61 cycles), stage II (26 patients, 39 cycles), stage III (26 pateitns, 37 cycles), stage IV (33 patients, 50 cycles). The outcomes of IVF-ET in endometriosis patients were as follows; The oocyte recovery rates were significantly lower in stage III, IV endometriosis. In case of stage III endometriosis, the fertilization rate was significantly lower than other stages of endometriosis. Clinical pregnancy rates per cycle were not different between the tubal group(22%) and the endometriosis group(25%). According to endometriosis stage, the implantation rate and clinical pregnancy rate were significantly lower in stage IV (5.6%, 16%) compared with other stages (I; 10.0%, 26%, II;9.8%, 31%, III;12.6%, 32%). It suggests that some factor like autoantibodies may inhibit implantation of embryos in stage IV endometriosis. To evaluate the possibility that simply increasing the number of gametes at the site of fertilization might account for pregnancies attributed to IVF-ET, the authors retrospectively analyzed the outcome of couples undergoing IUI during hMG cycles and CC cycles between 1992 and 1994 in the women with endometriosis stage 1. In case of stage I endometriosis, though the COH/IUI group showed lower FSH level and lesser age profile than IVF-ET group, IUI group has resulted in lower pregnancy rates(19.2%) compared with the IVF-ET group(26.2%). In conclusion, endometriotic infertile patients can get comparable pregnancy rates with the tubal factor infertility patients during IVF-ET program. Moreover even in stage I endometriosis, IVF-ET may be an more effective treatment modality than COH/IUI.