• Clinical and Experimental Reproductive Medicine
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• v.24 no.1
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• pp.101-109
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• 1997

This study was carried to determine the possibility of finding motile spermatozoa and fertilization, pregnancy rate after testicular sperm extraction(TESE) with ICSI in obstructive and non-obstructive azoospermic patients. In 154 cases(132 patients), obstructive azoospermia was 77 cases and non-obstructive azoospermia was 77 cases. In obstructive azoospermia, patients generally showed normal spermatogenesis and included vas agenesis(n=8), multiple vas obstruction(n=7), epididymal obstruction (n=54). Total of 982 retrieved oocytes were obtained and 84.4% were injected. The fertilization rates with 2 PN and cleavage rate were 72.5% and 62.3%, respectively. 30 pregnancies(38.9%) were achieved and the ongoing pregnancies were 22 cases (28.6%). In non-obstructive azoospermia, patients showed hypospermatogenesis(n=49), maturation arrest(n=4), Sertoli cell only syndrome (n=24). The various stages of spermatogenic cell could be retrieved by TESE and could be reached normal fertilization and embryo development with ICSI. Total of 1072 retrieved oocytes obtained and 80.2% were injected. The fertilization rates with 2 PN and cleavage rate were 52.8% and 68.9%, respectively. 22 pregnancies(30.1%) were achieved and the ongoing pregnancies were 19 cases(26.0%). Conclusively, the combination of TESE with ICSI using testicular spermatozoa can achieve normal fertilization and pregnancy rate and effective method in obstructive and non-obstructive azoospermic patients.

• Clinical and Experimental Reproductive Medicine
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• v.21 no.2
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• pp.137-147
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• 1994

Controlled ovarian hyperstimulation(COH) for in vitro fertilization and embryo transfer(IVFET) often results in the production of more embryos than can be efficaciously transferred at one time. However, embryo cryopreservation provides a mechanism by which additional embryos can be stored for later thawing and transfer. From November, 1990 to October, 1992, we completed 42 transfer cycles of cryopreserved pronucleus(PN) l-cell embryos using the fixed protocol of hormonal replacement therapy in a physiological manner regardless of individual ovarian function. Artificial endometrial stimulation was performed with only exogenous estradiol and progesterone(E-P) in 36 transfer cycles (Group I) and with gonadotropin-releasing hormone agonist(GnRHa) and exogenous estradiol and progesterone(GEEP) in 6 transfer cycles(Group II ). The results were as follows. 1. The Survival rate of total cryopreserved-thawed embryos was 64.9%(198/305): 64.9% (172/265) in Group I and 65.0% (26/40) in Group II. 2. Total 168 embryos were transferred with an average of 4.7 per ET in Group I and total 26 embryos were transferred with an average of 4.3 per ET in Group II. 3. The pregnancy rate(PR) per cryopreserved-thawed ET and the implantation rate was 33.3 %(14/42) and 6.7%(13/194), respectively. The PRs per cryopreserved-thawed ET were 30.6% (11/36) in Group I and 50.0% (3/6) in Group II without significant difference. 4. The take home baby rate was 11.1%(4/36) in Group I and 33.3% (2/6) in Group II.

• Molecules and Cells
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• v.19 no.1
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• pp.46-53
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• 2005

Human embryonic stem (hES) cells, unlike most cells derived from adult or fetal human tissues, represent a potentially unlimited source of various cell types for basic clinical research. To meet the increased demand for characterized hES cell lines, we established and characterized nine new lines obtained from frozen-thawed pronucleus-stage embryos. In addition, we improved the derivation efficiency from inner cell masses (to 47.4%) and optimized culture conditions for undifferentiated hES cells. After these cell lines had been maintained for over a year in vitro, they were characterized comprehensively for expression of markers of undifferentiated hES cells, karyotype, and in vitro/in vivo differentiation capacity. All of the cell lines were pluripotent, and one cell line was trisomic for chromosome 3. Improved culture techniques for hES cells should make them a good source for diverse applications in regenerative medicine, but further investigation is needed of their basic biology.

• Reproductive and Developmental Biology
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• v.35 no.1
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• pp.33-45
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• 2011

Heat shock protein 90 (Hsp90) is ATPase-directed molecular chaperon and affects survival of cancer cell. Inhibitory effect of Hsp90 by inducing cell cycle arrest and apoptosis in the cancer cell was reported. However, its role during oocyte maturation and early embryo development is very insufficient. In this study, we traced the effects of Hsp90 inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), on meiotic maturation and early embryonic development in pigs. We also investigated several indicators of developmental potential, including structural integrity, gene expression (Hsp90-, cell cycle-, and apoptosis-related genes), and apoptosis, which are affected by 17-AAG. Then, we examined the roles of Hsp90 inhibitor on viability of primary cells in pigs. Porcine oocytes were cultured in the NCSU-23 medium with or without 17-AAG for 44 h. The proportion of GV arrested oocytes was significantly different between the 17-AAG treated and untreated group (78.2 vs 34.8%, p<0.05). After completion of meiotic maturation, the proportion of MII oocytes was lower in the 17-AAG treated group than in the control group (27.9 vs 71.0%, p<0.05). After IVF, the percentage of penetrated oocytes was significantly lower in the 17-AAG treated group (25.2%), resulting in lower normal pronucleus formation (2PN of 14.6%). Therefore, the inhibition of meiotic progression by Hsp90 inhibitor played a critical role in fertilization status. Porcine embryo were cultured in the PZM-3 medium with or without 17-AAG for 6 days. In result, significant differences in developmental potential were detected between the embryos that were cultured with or without 17-AAG. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) showed that the number of containing fragmented DNA at the blastocyst stage increased in the 17-AAG treated group compared with control (7.5 vs 4.4, respectively). Blastocysts that developed in the 17-AAG treated group had low structural integrity and high apoptotic nuclei than those of the untreated control, resulting in decrease the embryonic qualities of preimplantation porcine blastocysts. The mRNA expressions of cell cycle-related genes were down-regulated in the 17-AAG treated group compared with control. Also, the expression of the pro-apoptotic gene Bax increased in 17-AAG treated group, whereas expression of the anti-apoptotic gene Bel-XL decreased. However, the expression of ER stress-related genes did not changed by 17-AAG. Cultured pESF cells were treated with or without 17-AAG and used for MIT assay. The results showed that viability of pESF cells were decreased by treatment of 17-AAG ($2{\mu}M$) for 24 hr. These results indicated that 17-AAG decreased cell proliferation and increased cell death. Expression patterns Hsp90 complex genes (Hsp70 and p23), cell cycle-related genes (cdc2 and cdc25c) and apoptosis-related genes (Bax and Bcl-XL) were significantly changed by using RT-PCR analysis. The spliced form of pXbp-1 product (pXbp-1s) was detected in the tunicamycin (TM) treated cells, but it is not detected in 17-AAG treated cells. In conclusion, Hsp90 appears to play a direct role in porcine early embryo developmental competence including structural integrity of blastocysts. Also, these results indicate that Hsp90 is closely associated with cell cycle- and apoptosis-related genes expression in developing porcine embryos.

• Clinical and Experimental Reproductive Medicine
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• v.35 no.4
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• pp.293-301
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• 2008

Objective: To evaluate outcomes of patients with non-obstructive azoospermia (NOA) undergoing the testicular sperm extraction (TESE) combined with intracytoplasmic sperm injection (ICSI) with different histopathologic subgroups. Method: A total of 122 embryo-transferred TESE/ICSI cycles were compared among NOA subgroups; Germ-cell aplasia (GA, 40 cycles), Maturation arrest (MA, 32 cycles) and severe hypospermatogenesis (S-HS, 50 cycles). Obstructive azoospermia (OA, 667 cycles) patients were served as a control. TESE/ICSI outcomes such as fertilization rate (FR), clinical pregnancy rate (CPR) and live birth rate (LBR) were evaluated. Results: The 2PN FR of embryo-transferred TESE/ICSI cycle was 58.1% in GA, 42.2% in MA and 48.0% in S-HS, which was significantly lower than that of OA (72.9 %, p<0.001). For ICSI-spermatozoa cycles, there were no significant differences in CPR (22.6%, 29.4% and 26.1%) and LBR (16.1%, 29.4% and 19.6%) among NOA subgroups. The CPR of ICSI-spermatid cycles was 0.0%, 9.1% and 0.0% without a live birth. For ICSI-spermatocyte cycles, no clinical pregnancies occurred in any group. Conclusion: There was no significant difference in the FR of embryo-transferred TESE/ICSI cycles among NOA subgroups. The FR among all NOA subgroups was significantly lower than that of OA. Testicular histopathology in NOA did not affect successful pregnancy if spermatozoa extraction from the testis is successful and embryo transfer is possible.

• Clinical and Experimental Reproductive Medicine
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• v.31 no.1
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• pp.9-17
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• 2004

Objective: The aim of this study were to compare the effects of EG and PROH on cryopreservation of mouse and human embryos, and to find the optimal protocol for embryo freezing. Methods: Human embryos derived from fertilized eggs showing 3 pronuclei (PN) and mouse embryos were divided into two groups respectively: dehydrated with 1.5 M EG + 0.2 M sucrose or 1.5 M PROH + 0.2 M sucrose using the slow freezing method. Moreover mouse embryos were controlled the exposure time of cryoprotectant during dehydration or rehydration steps. Results: The survival rates of human embryos were 79.2% (84/106) in EG group and 77.9% (88/113) in PROH group. In mouse embryos, the survival and development rates up to blastocyst were 70.6% (245/347), 44.1% (123/279) in EG group and 62.1% (198/319), 45.1% (123/279) in PROH group, respectively. However, in EG group, partially damaged embryos after thawing were decreased compared to PROH group. In combination group, when the exposure time during dehydration and rehydration were reduced, the survival and embryonic developments were increased slightly, but not significant. Conclusion: Cryopreservation of mouse and human embryos at cleavage stage by using EG or PROH exhibited no statistical difference in the survival rate and/or developmental rate to blastocyst. However, the use of EG for cryopreservation of embryos might reduce the exposure time of the cryoprotectant because of a high permeation of EG and result in lessen its toxic effects.

• Clinical and Experimental Reproductive Medicine
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• v.24 no.3
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• pp.281-292
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• 1997

The objective of this study was to compare retrospectively the survival and pregnancy rates(PR) of cryopresered-thawed embryos obtained from intracytoplasmic sperm injection (ICSI) or conventional in vitro fertilization (IVF). Ninety-six cycles of cryopresered-thawed embryo transfer (ET) were performed in 79 patients from June, 1996 to September, 1997 and grouped as followings: 20 cycles (16 patients) inseminated by ICSI (ICSI Group) and 76 cycles (63 patients) by conventional IVF (IVF Group). Slow-freezing and rapid-thawing protocol was used with 1.5M propanediol (PROH) and 0.1M sucrose as cryoprotectant. All embryos were frozen-thawed at the two pronuclear (2 PN) stage excluding four cycles in which the early cleavage stage embryos were frozen, and allowed to cleave in vitro for one day before ET. The duration from freezing to thawing was comparable in both groups ($mean{\pm}SD$, $112.1{\pm}80.0$ vs. $124.8{\pm}140.1$ days). The age of female ($31.2{\pm}3.4$ vs. $32.6{\pm}3.3$ years) and the endometrial thickness prior to progesterone injection ($9.4{\pm}2.0$ vs. $9.3{\pm}1.8$ mm) were also comparable in both groups. There was no significant difference in the outcomes of cryopreserved-thawed ET between two groups: survival rate ($85.2{\pm}16.1%$ vs. $82.2{\pm}19.7%$), cleavage rate ($96.9{\pm}6.7%$ vs. $94.7{\pm}13.0%$), cumulative embryo score (CES, $54.5{\pm}31.1$ vs. $49.0{\pm}20.0$), preclinical loss rate (5.0% vs. 5.3%), clinical miscarriage rate (0% vs 29.4%), clinical PR per transfer (35.0% vs. 22.4%), implantation rate (9.9% vs. 5.6%), and multifetal PR (42.9% vs. 17.6%). In conclusion, human embryos resulting from ICSI can be cryopreserved-thawed and transferred successfully, and the survival rate and PR are comparable to conventional IVF.

• Clinical and Experimental Reproductive Medicine
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• v.34 no.4
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• pp.285-291
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• 2007

Objective: To evaluate the outcomes of in vitro fertilization and embryo transfer (IVF-ET) in women over 40 years of age. Methods: A total of 170 patients (271 cycles) over 40 years of age who underwent IVF-ET at Seoul Women's Hospital (Incheon, Korea) were analyzed in this study retrospectively. The patients were grouped into the women <44 years old group and the women $\geq$44 years old group. Statistical analysis was performed using Student's t-test and Fisher's exact test as appropriate. Results: An overall clinical pregnancy rate per retrieval was 11% (30/271). Of these, clinical miscarriage rate were 33% (10/30) and the overall delivery rate was 7.4% (20/271) per retrieval, respectively. The women $\geq$44 years old group had significantly higher cancellation rate (13% vs. 25%), lower number of retrieved oocytes (6.17$\times$4.62 vs. 4.13$\times$4.07), decreased number of 2PN (4.83$\times$3.61 vs. 3.46$\times$3.12), and reduced embryos for transfer (3.52$\times$1.72 vs. 2.81$\times$1.83) than the women <44 years old group. We found significantly lower clinical pregnancy rate (13.0% vs. 2.1%) and live birth rate (9.0% vs. 0.0%) in the women $\geq$44 years old group than the women <44 years old group. Conclusion: The present study has shown that IVF outcome is seriously impaired in the women $\geq$44 years old.