• Korean Journal of Animal Reproduction
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• v.14 no.1
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• pp.36-42
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• 1990

The effect of preincubation on in vitro maturation and fertility were investigated using preovulatory oocytes with and without cumulus cells obtained from superovulated ouot-bred ICR mice. Oocytes were recovered from fully grown folicle at 10 hr after hCG administration. A part of oocytes recovered were treated with the solution of 0.1% hyaluronidase to remove cumulus cells. Both intact and treated oocytes were then incubated for 0 to 6hr in mT6 medium containing 0.3% BSA. After incubation for various times, a part of oocytes were subjected to the investigation of nuclear maturation and the remaining oocytes were used fro the induction of in vitro fertilization by adding them into medium containing capacitated mice epididymal spermatozoa. Above all, the percentage of preovulatory oocytes at the stage of metaphase II after preincubation for 0, 2, 4 and 6hr was 15.8, 36.4, 47.5 and 66.7%, respectively, suggesting the in vitro maturation of oocytes during their incubation. On the other hand, fertilizatin rate of oocytes preincubated for 0, 2, 4 and 6hr with and without cumulus cells were 41.0, 58.7, 68.7 and 75.6%, and 50.0, 45.1, 37.8 and 39.2%, respectively. No significant differences in fertilization rate between preovulatory oocytes preincubated for 6hr with cumulus cells and naturally ovulated were observed. These results suggest that cumulus cells take very important role in maturtion of oocytes in vitro. The precentage of preovulatory oocytes developed to 2-cell stage in vitro fertilization following preincubation for 0 to 6hr with and without cumulus cells ranged from 48.5 to 82.4% and 36.9 to 56.1%, respectively. Also, the rates of oocytes developed to blastocyst in vitro fertilization after preincubation for 0 to 6hr with and without cumulus cells were 28.1, 39.3, 42.5 and 44.0% and 12.5, 32.6, 24.4 and 15.5%, respectively. From these results, it could be said that fertility of preovulatory oocytes with cumulus cells could be improved to the level of that of naturally ovulated oocytes by adquate preincubation in vitro. Cumulus cells may, therefore, affect in vitro maturation, fertilization and following development of oocytes by influencing zona hardening.

• Journal of Embryo Transfer
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• v.17 no.3
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• pp.187-193
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• 2002

This study was conducted to determine the effect of extract of Cervi pantotrichum cornu on human sperm motility, Four different types of media were prepared such as plain Ham's F-10 medium(control medium), control medium containing 0.3% bovine serum albumin(BSA)(medium A), control medium containing the extract of Cervi pantotrichum cornu aqua-acupuncture medium(medium B) and medium B containing 0.3% BSA(medium C). Human semen were washed and divided into 4 fractions and sperm were cultured in those medium for up to 72 hours at 37$^{\circ}C$ in a humidified atmosphere of 5% $CO_2$ in air. A total twenty eight semen samples including 14 normozoospermia and 14 asthenospermia were used for this study. In normozoospermia group, motility of control medium and medium A, B and C were 4.1%, 1.3%, 64.5%, and 77.1%, respectively after 24 hours of incubation, and were 0.0%, 0.0%. 8.8% and 44.9%, respectively after 48 hours of incubation. In asthenospermia group, motility of control medium and medium A, B and C were 2.0%, 2.2%, 58.3% and 85.1%, respectively after 24 hours of incubation, and decreased to 0.0%, 0.2%, 5.8% and 29.6%, respectively after 48 hours of incubation. In both groups, highest sperm motility was observed in medium C group when compared with other media. Furthermore motile sperm were found in medium C after 72 hours of incubation while no motile sperm was observed in the other media. Therefore it could be concluded that the extract of Cervi pantotrichum rornu affects on the human sperm motility.

• Journal of Embryo Transfer
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• v.13 no.2
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• pp.89-96
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• 1998

Anti-phospholipid antihodies (aPL) have important roles in various pregnancy complications such as recurrent miscarrige, growth retardation, placental abruption and stillbirth. However, their biological actions on preimplantation development of oocytes are still unclear. In this study, we investigated whether either aPL containing sera or phospholipids could affect in vitro fertilization and development of mouse oocytes. Sera used in this study were collected from three patients and the presence of aPL in the sera was confirmed by enzymatic-linked immunosorbent assay. When mouse oocytes were cultured in a serum-free, Chatot, Ziomek and Bavister (CZB) medium (Experiment 1), addition of aPL-containing sera (10%) to CZB medium did not. significantly (P>0.05) influence sperm penetration of oocytes. However, development to the blastocyst stage was significantly (P<0.05) inhibited by serum addition, and formation of morulae (16-23% vs. 58%) and blastocysts (0-4% vs. 21%) was markedly reduced compared with no addition (Experiment 2). In Experiment 3, pronuclear stage embryos were cultured for 96 h in GZB medium supplemented with 1 $\mu$g /ml phosphatidyl ethanolamine, 1 $\mu$g/ml phosphatidyl inositol or 1 $\mu$g /ml phosphatidyl choline. No increase in embryo development was found after addition of the phospholipids to CZB medium. These results suggest that 1) aPL have an inhibitory role in preimplantation development of mouse embryos, and that 2) the action of aPL may be related to a specific phospholid (s) rather than the tested phospholipids in the present study.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.12 no.8
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• pp.3541-3546
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• 2011

Autophagy is an evolutionarily conserved lysosomal pathway for degrading cytoplasmic proteins, macromolecules, and organelles in addition to recycling protein and ATP synthesis. Although autophagy is very important during embryogenesis, the mechanism underlying the dynamic development during this process remains largely unknown. In order to obtain insights into autophagy in early embryo development, we analyzed gene expression levels of autophagy-related genes (ATGs) in mouse embryos developing in vitro. Using real time RT-PCR technique, ATGs including Atg2a, Atg3, Atg4b, Atg5, Atg6, Atg7, Atg9a, and Wipi3, as maternal transcripts, were only up-regulated in 1-cell embryo stage before zygotic genomic activation (ZGA), and then expression decreased from 2-cell to blastocyst embryo stage. ATGs including Dram and Atg9b were expressed abundantly in 1-cell embryo state and in blastocyst embryo stage, athough Atg8 and Ulk1 were constantly expressed during preimplantation stage. However, Atg4d were only up-expressed from 4-cell to blastocyst stage. These results suggest that autophagy is related in mouse embryo, which possibly gives an important role for early development.

• Korean Journal of Animal Reproduction
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• v.17 no.2
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• pp.141-148
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• 1993

This studies were carried out to investigate the effects of co-culture with cumulus cells, oviduct epithelial cells and uterine endometrium cells on the in-vitro fertilization and cleavage rate of bovine follicular oocytes. The ovaries were obtained from slaughtered Korean native cows. The follicular oocytes surrounded with cumulus cells were recovered by aspirating follicular fluids from the visible follicles of diameter 3~5 mm. The follicular oocytes were cultured in TCM-199 medium containing hormones and 10% FCS for 24~48 hrs in a incubator with 5% CO2 in air at 38.5$^{\circ}C$ and then matured oocytes were again cultured for 12~18 hrs with motile capacitated sperm by preincubation. The results obtained in these experiments were summarized as follows: 1. The in vitro maturatin and fertilization rate of bovine oocytes co-cultured with cumulus cells in TCM-199 medium were 64.0~74.1% and 40.0~58.6% respectively. And in-vitro fertilization rate of cumulus-enclosed oocytes(55.4%) were significantly(p<0.05) higher than cumulus-denuded oocytes(23.1%). 2. The in-vitro maturatin and fertilization rate of bovine oocytes co-cultured with 1$\times$104 cells/ml, 1$\times$106 cells/ml, 1$\times$108 cells/ml and 1$\times$1015 cells/ml oviduct epithelial cells in TCM-199 medium were 59.3% and 40.7%, 64.0% and 48.0%, 58.3% and 37.5%, 52.0% and 32.0%, respectively. 3. The in-vitro maturation and fertilization rate of bovine oocytes co-cultured with 1$\times$104 cells/ml, 1$\times$106 cells/ml, 1$\times$108 cells/ml and 1$\times$1015 cells/ml uterine endometrium cells in TCM-199 medium were 56.0% and 36.0%, 60.7% and 42.9%, 59.3% and 37.0%, 52.0% and 36.0%, respectively. 4. When the in-vitro fertilized oocytes were co-cultured with cumulus cells, oviduct epithelial cells and uterine endometrium cells, the development rate to be blastocyst was 12.2%, 15.6% and 11.7%, respectively and rates were higher than that of control, 2.1%(P<0.05).

• Clinical and Experimental Reproductive Medicine
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• v.28 no.2
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• pp.161-167
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• 2001

Objective: Granulocyte-macrophage colony stimulating factors known to be secreted in murine and human reproductive tract. The development of human, bovine and murine embryos could be promoted by addition of GM-CSF in culture medium. However, the pregnancy and implantation rate of embryos cultured in GM-CSF have not been evaluated. The aim of this study was to assess the effect of GM-CSF in embryo development, pregnancy and implantation rate. Methods: A total of 191 IVF cycles were divided into control and GM-CSF supplement group (control=96, GM-CSF=95). The embryos were cultured for three day with or without 2 ng/ml of recombinant human GM-CSF. The quality of embryo, developmental velocity, pregnancy and implantation rates were compared. Results: There was no difference in age, number of gonadotropin ampules used, number of oocytes and fertilization. The number of ICSI cycle was higher in GM-CSF group. In GM-CSF group, G-1 grade embryos were the highest in proportion (56.4%), while G-2 grade embryos were highest (44.3%) in control group. The developmental velocity of embryos were not different between GM-CSF and control group. The pregnancy and implantation rates were significantly higher in GM-CSF group than control (47.4% vs. 33.3%, 17.0% vs. 11.1% respectively). Conclusion: By adding GM-CSF in culture medium, the quality of embryo, pregnancy and implantation rate could be improved.

• Clinical and Experimental Reproductive Medicine
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• v.25 no.3
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• pp.287-292
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• 1998

The objective of this study was to confirm whether the vitrification method using EFS35 has detrimental effect for cytoskeleton and chromosome constitution of the mouse oocytes by indirect immunocytochemistry and chromosome analysis. Mouse oocytes were vitrified using EFS35 which consisted of 35% ethylene glycol, 18% ficoll, 0.3 M sucrose and 10% FBS in M2 medium. The results obtained in this experiment were summarized as follows: When the survival rates after being exposed or vitrified in EFS35 were examined, there were not different between two groups (97.7 and 89.3%). Also, when the microtubule morphology and microfilament distribution in vitrified oocytes were examined, normal percentage of two cytoskeleton in vitrified group (95.5 and 100%) was not different from that in control (97.5 and 100%) and exposed group (92.3 and 100%). In addition, the rate of oocytes containing a normal chromosome number in vitrified group (73.5%) after IVF was not different from that in control (79.5%) and exposed group (78.7%). These results indicated that the cytoskeletal morphology and chromosome constitution of mouse oocytes were not affected by cryoprotectant (EFS35) or freezing apd that vitrification methods using EFS35 was suitable for cryopreservation of mouse oocytes.

• Clinical and Experimental Reproductive Medicine
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• v.25 no.3
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• pp.251-260
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• 1998

It is well known that the clinical test for responsibility of accurate fertilization capacity in male partners is very important to diagnose and treat the infertility. However, it has been reported that the traditional semen analysis cannot accurately predict fertilization and pregnancy potential. The present study was performed to evaluate the acrosomal reaction to ionophore challenge (ARIC) test as a prognostic indicator for fertilization of sperm and oocyte in an in vitro fertilization and embryo transfer (IVF-ET) program. From March 1996 to Februry 1997, 30 couples undergoing IVF program were allocated to this study group. All female partners in the study group were 35 years old or less and their serum level of basal follicle stimulating hormone (FSH) and estradiol $(E_2)$ were normal. All the male partners have normal parameters of semen analysis. The ARIC tests were performed on the day of ovum pick up and in vitro insemination in all the male partners. The controlled ovarian hyperstimulation (COH) using luteal long protocol of gonadotropin releasing hormone (GnRH) agonist was used in all couples for IVF-ET. The acrosomal reaction with $10{\mu}l$ of 10% DMSO was induced spontaneously in $10.1{\pm}9.8%$, and acrosomal reaction with calcium ionophore A 23187 was induced in $27.4{\pm}18.1%$, and the ARIC value was $17.4{\pm}16.2%$. There were no significant correlation between the ARIC value and the fertilization rate ($r^2$=0.044, p=0.268). There were also no significant correlation between the ARIC value and the percentage of the grade I, II embryos ($r^2$=0.046, p=0.261). On the basis of above results, it was suggested that ARIC test might not be a useful prognostic indicator for fertilization in IVF-ET in male partners with normal parameters of conventional semen analysis. We guessed that IVF-ET could be performed to the patients primarily without universal appilcation of ARIC test to all male partenrs, and if fertilization failure occurs, the micro assisted fertilization (MAF) such as intracytoplsmic sperm injection (ICSI) might be used as an alternative mode of treatment with acceptable success rate.

• Clinical and Experimental Reproductive Medicine
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• v.25 no.3
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• pp.239-244
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• 1998

The purpose of this study is compare IVF cycle outcome in poor responders between clomiphene citrate (CC) stimulated and controlled ovarian hyperstimulation (COH) protocol. A total of 94 patients responding poorly in previous IVF cycles (estradiol<600 pg/ml or less than 3 oocytes retrieved) subsequently underwent either COH (COH group: 122 cycles, 68 patients) or CC-stimulated cycles (CC group: 43 cycles, 26 patients). CC was administered for five consecutive days starting on cycle day 3 at a dose of 100 mg daily. Serial transvaginal ultrasound examination was done from cycle day 8. Urine was collected $3\sim4$ times before hCG injection for the detection of LH surge. The hCG was administered when serum estradiol reached greater than 150 pg/ml and mean follicle diameter>16 mm. In COH group, ovarian stimulation was done using short protocol (GnRH-a/FSH/HMG/hCG). No difference in age or number of transferred embryos was found between CC group and COH group. COH group had significantly (p<0.05) higher mean peak level of $E_2$ ($810{\pm}112$ vs $412{\pm}55$ pg/ml) and greater number of retrieved oocytes ($3.0{\pm}0.2$ vs $2.0{\pm}0.2$) than CC group. CC group had transferred embryos $(1.8{\pm}0.2)$ compared with $(2.1{\pm}0.2)$ in COH group. However, CC group had higher pregnancy rate than COH group per retrieval [26.9% (7/26) vs 6.2% (6/97)], or per transfer [31.8% (7/22) vs 7% (6/86)]. Although cycle cancellation rate in CC group (48.8%) was higher than that of COH group (21.3%), the pregnancy rate per cycle in CC group was still higher (16.3%) than COH group (4.9%). In addition, implantation rate in CC group was 17.5% (7/40), which was significantly (p<0.01) higher than 3.9% (7/180) in COH group. These data suggest that oocyte and embryo quality are lower in COH cycles of poor responders than CC cycles. We suggest that clomiphene citrate stimulated IVF cycle may be more efficient than COH IVF cycle in poor responders in terms of lower costs and higher pregnancy performance.

• Clinical and Experimental Reproductive Medicine
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• v.30 no.2
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• pp.119-126
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• 2003

Objective: This study was carried out to compare the clinical outcomes of intracytoplasmic sperm injection (ICSI) in patients with obstructive azoospermia according to sperm retrieval site and technique; microsurgical epididymal sperm aspiration (MESA), percutaneous epididymal sperm aspiration (PESA), testicular sperm extraction by open biopsy (TESE). Methods: The outcomes of ICSI and IVF-ET were evaluated and compared among 3 groups. Seventy three men suffering from infertility due to obstructive azoospermia had 107 ICSI cycles using MESA (21 cycles in 15 patients), PESA (26 cycles in 17 patients) and TESE (60 cycles in 41 patients). Results: In the clinical outcomes in patients undergoing ICSI with epididymal or testicular sperm, there were no significant differences in fertilization rate (66.1% vs. 60.5%), cleavage rate (94.9% vs. 97.6%), cumulative embryo score (CES) (51.3 vs. 58.8), implantation rate (7.9% vs. 6.1), and clinical pregnancy rate per ET (30.4% (14/46) vs. 25.4% (15/59)) between both groups. Also, in the clinical outcomes in ICSI patients using MESA, PESA, TESE, there were no significant differences in fertilization rate (61.8%, 69.4%, 60.5%), cleavage rate (92.1%, 97.3%, 97.6%), CES (38.1, 52.0, 58.8), implantation rate (9.5%, 6.6%, 6.1%), and clinical pregnancy rate per ET (35% (7/20), 26.9% (7/26), 25.4% (15/59)) among 3 groups. Conclusion: When compared with MESA or TESE, PESA, the clinical outcomes were similar in ICSI patients with obstructive azoospermia whatever the origin or the technique of sperm retrieval. However, we considered PESA is more time-saving and cost effective for ICSI in patients with obstructive azoospermia.