Effect of Low-dose Aspirin on Implantation and Pregnancy Rates in Patients Undergoing Frozen-thawed Embryo Transfer / 대한불임학회지

OBJECTIVE: Low-dose aspirin have been proposed to improving endometrial receptivity and pregnancy rate in COH-IVF by increasing endometrial perfusion. However, the effect of low-dose aspirin in COH-IVF could be negligible because there have been large quantity of other important factors responsible for changing endometrial perfusion accompanied by COH procedure. In contrast, in frozen-thawed embryo transfer cycles which were not accompanied by COH procedure, the effects of low-dose aspirin in endometrial blood flow seems to be more certain than in COH-IVF cycles. In this study, we analyzed the effect of low-dose aspirin treatment on implantation and pregnancy rates in patients undergoing frozen-thawed embryo transfer METHODS: From January 2003 to December 2003, total 264 cycles from 264 patients who attended infertility clinic at Samsung Cheil Hospital were enrolled in this study. All cases included in this study, embryos were frozen and thawed at the pronuclear stage and three days after incubation, at least 2 or more good quality embryos were transferred into uterus. In study group, low dose aspirin (100 mg/day) was administrated from the first or second date of menstrual day to 9 days after embryo transfer. On the other hand, control group did not take any medicine except estradiol valerate for endometrial priming. Several variables including implantation and pregnancy rates were compared in both groups. After then, each groups were stratified by endometrial thickness checked at embryo transfer (ET) day such as (28 mm versus 0.05) After we analyzed same variables stratified by endometrial thickness checked at embryo transfer day, we could not found any significant difference between study and control groups. CONCLUSIONS: Low-dose aspirin treatment seems to have no advantage of improving implantation and pregnancy rates in patients undergoing frozen-thawed embryo transfer.

Pregnancy Outcomes after Transfer of Frozen-thawed Embryos following ICSI with Ejaculated, Fresh and Frozen-thawed Testicular Sperm / 대한산부인과학회잡지

OBJECTIVES: This study was performed to evaluate the pregnancy rate following the transfers of frozen- thawed embryos which was derived from intracytoplasmic sperm injection (ICSI) using sperm obtained by ejaculated, testicular sperm extraction (TESE), and frozen-thawed testicular sperm extraction (t-TESE). METHODS: Frozen-thawed embryos were successfully transferred to the patients in 664 cycles among 695 cycles from January 1998 to December 2002, where ICSI was done with various origins of sperm. Subjects were divided into three groups according to the origin of sperm; ejaculated sperm group as a control (n=535), TESE group (n=98) and t-TESE group (n=62). After conventional ICSI, the supernumerary PN stage or developing embryos were cryopreserved by slow freezing protocol with 1, 2-propanediol as cryoprotectant. RESULTS: The survival rate of frozen-thawed embryos was 77.7% (2515/3236) in ejaculated sperm group, 76.6% (441/576) in TESE group and 83.9% (292/348) in frozen-thawed TESE group, respectively. The difference of survival rate of between t-TESE group and other two groups was statistically significant (p<0.01). The good embryo formation rate and positive beta-hCG rate was 46.3% (1164/2515), 28.8% (148/513) in ejaculated sperm group, 49.2% (217/441), 36.6% (34/93) in TESE group and 46.2% (135/292), 34.9% (22/63) in frozen-thawed TESE group, respectively. CONCLUSION: This study demonstrates that comparable pregnancy rate and implantation rate could be achieved after the transfer of frozen-thawed embryos following ICSI using various sources of sperm. As there was no statistically significant difference in pregnacy rate between ICSI with fresh testicular sperm and with frozen-thawed testicular sperm, the sequential cryopreservation of supernumerary testicular sperm and embryos may be a useful method for increasing pregnancy outcome in infertile couples with male factor.

Efficiency of Percoll Gradient, PureSperm Gradient, and Swim-up Separation Techniques in Normal Semen Samples / 대한남성과학회지

PURPOSE: The aim of this study was to compare sperm concentration, motility, viability, and morphology using Percoll gradient, PureSperm gradient, and the swim-up method in normal semen samples. MATERIALS AND METHODS: Ten normal semen samples were divided into three fractions. Motile sperm were isolated with Percoll gradient, PureSperm gradient, and swim-up method. Sperm concentration, motility, viability, and morphology were determined before and after separation. RESULTS: The sperm concentrations were not significantly different among the three METHODS: Percoll gradient(34.3+/-9.4x10(6)/ml), PureSperm gradient(37.6+/-16.6x10(6)/ml), and swim-up(27.3+/-6.4x106/ml). There was no significant difference in sperm motility among the three METHODS: Percoll gradient(93.5+/-1.6%), PureSperm gradient(92.7+/-4.4%), and swim-up(95.7+/-2.7%). When sperm motility was measured 24 hr later, the results were similar among the three METHODS: Percoll gradient(81.7+/-15.5%), PureSperm gradient(84.3+/-12.2%), and swim-up(89.4+/-5.1%). Sperm viability and morphology were slightly higher in swim-up method than the other methods, but the differences were not statistically significant. Sperm viability datas were: Percoll gradient(85.5+/-5.5%), PureSperm gradient(85.6+/-3.7%), and swim-up(88.6+/-6.6%). Morphology datas were: Percoll gradient(82.0+/-10.7%), PureSperm gradient(73.9+/-9.3%), and swim-up(83.7+/-8.5%). CONCLUSIONS: The swim-up method resulted in viability and morphology that were slightly higher than the other methods. However, all methods were useful for sperm preparation in normal semen samples.

Successful Preimplantation Genetic Diagnosis for Ornithine Transcarbamylase Deficiency, Junctional Epidermolysis Bullosa and Lactic Acidosis Using Duplex Nested PCR: Delivery of Healthy Baby by Specific Preimplantation Genetic Diagnosis for Ornithine Tran / 대한산부인과학회잡지

OBJECTIVE: Preimplantation genetic diagnosis (PGD) is reserved for couples with a risk of transmitting a serious and incurable disease, and hence avoids the undesirable therapeutic abortion. Herein, we report the result of PGD to carriers at risk of transmitting ornithine transcarbamylase (OTC) deficiency, junctional epidermolysis bullosa (EB) and lactic acidosis (LA) due to defect of pyruvate dehydrogenase alpha1 gene, respectively. METHODS: The ovarian stimulation, oocyte retrieval and ICSI procedure were undergone by conventional protocols. PGD for single gene disorders was carried out after biopsy of one or two blastomeres from the embryos on the third day. We performed the duplex nested PCR of the simultaneous amplification for the causative mutation loci as well as the SRY gene on Y chromosome in case of OTC deficiency and LA. Two different mutation loci of ITGB4 gene in EB case were amplified by the same protocol. The PCR products were analyzed by agarose gel electrophoresis, restriction fragment length polymorphism analysis or direct DNA sequencing. RESULTS: A total of 26 embryos were analyzed by duplex nested PCR. One or two blastomeres were biopsied, and successful diagnosis rate of PGD with PCR was 92.3% (24/26). There was no contamination in all PCR samples of negative controls (n=67). Five embryos (19.2%) were diagnosed as normal embryos, which were transferred to the mothers' uterus in each cases. In OTC deficiency case, singleton pregnancy was established. At 17 weeks of gestation, genetic normality of OTC gene in fetus was confirmed by amniocentesis. A healthy baby was successfully delivered at 36 weeks of gestation in OTC deficiency case. Unfortunately, pregnancies were not achievement in cases of EB and LA. CONCLUSION: This is the first report in Korea that healthy baby was born after specific PGD for OTC deficiency. Our results demonstrate that duplex nested PCR for single cell is an efficient method in identifying the gender and single gene mutation or two different mutation loci, simultaneously. This PGD procedure could provide normal healthy baby to the couple with a high risk of transmitting genetic diseases.

Improvement of Pregnancy Rate in Preimplantation Genetic Diagnosis with FISH Procedure by the Laboratory Optimization and Experiences / 대한불임학회지

OBJECTIVES: This study was performed to evaluate the laboratory system for successful PGD using fluorescence in situ hybridization (FISH) and the clinical outcome of PGD cycles in five years experiences. METHODS: A total of 181 PGD-FISH cycles of 106 couples were performed, and diagnosed chromosome normality in the preimplantation embryos. The laboratory and clinical data were classified by the following optimization steps, and statistically analyzed. Phase I: Blastomere biopsy with two kinds of pipettes, removal of cytoplasmic proteins without treatment of pepsin and culture of biopsied embryos with single medium; Phase II: Blatomere biopsy with single pipette, removal of cytoplasmic proteins with pepsin and culture of biopsied embryos with single medium; Phase III: Blastomere biopsy with single pipette, removal of cytoplasmic proteins with pepsin and culture of biopsied embryos with sequential media. RESULTS: A total of 3,209 oocytes were collected, and 83.8% (2,212/2,640) of fertilization rate was obtained by ICSI procedure. The successful blastomere biopsies were accomplished in 98.6% (2,043/2,071) of embryos, and the successful diagnosis rate of FISH was 94.7% (1,935/ 2,043) of blastomeres from overall data. Embryo transfers with normal embryos were conducted in 93.9% (170/181) of started cycles. There was no difference in the successful rate of biopsy and diagnosis among Phase I, II and III. However, the pregnancy rate per embryo transfer of Phase III (38.8%, 26/67) was significantly (p<0.05) higher than those of Phase I (13.9%, 5/36) and Phase II (14.9%, 10/67). CONCLUSIONS: The laboratory optimization and experience for the PGD with FISH procedure can increase the pregnancy rate to 38.8% in the human IVF-ET program. Our facility of PGD with FISH provides the great possibility to get a normal pregnancy for the concerned couples by chromosomal aberrations.

Development of Effective Cryopreservation Method for Mouse Oocytes / 대한불임학회지

OBJECTIVE: The purpose of this study was to evaluate the efficacy and effect of various cryopreservation method on the survival and the cytoskeletal stability of metaphase II mouse oocyte. METHODS: Mouse ovulated oocytes were collected and cryopreserved by a modified slow-freezing method with 1.5 M 1,2-propanediol (PrOH)+0.1 M sucrose or by vitrification using cryo loop and EM grid with 40% ethylene glycol+0.6 M sucrose. Four hours after thawing, intact oocytes were fixed and stained with fluorescein isothiocyanate (FITC)-conjugated monoclonal anti-beta-tubulin antibody to visualize spindle and propidium iodide (PI) to visualize chromosome. Spindle morphology was classified as follows: normal (barrel-shaped), slightly and absolute abnormal (multipolar or absent). RESULTS: Survival rate of the frozen-thawed oocytes in vitrification group was significantly higher than that of slow-freezing group (62.7% vs. 24.4%, p<0.01). Vitrification with cryo loop showed significantly higher survival rate than that with EM grid (67.7% vs. 53.5%, p<0.05). On the other hand, proportion of normal spindle and chromosome configurations of the frozen-thawed oocytes between two vitrification group was not significantly different. CONCLUSION: For mouse ovulated oocytes, vitrification with cryo loop may be a preferable procedure compared to slow-freezing method. Further study should be needed to investigate developmental competency of frozen-thawed mouse oocytes.

Expression of Sperm-specific Cation Channel CatSper in Human Spermatozoa / 대한비뇨기과학회지

PURPOSE: We aimed to elucidate the expression and intracellular localization of sperm-specific cation channel CatSper in human spermatozoa. Moreover, the relationship between the expression of CatSper mRNA and the motility of ejaculated human spermatozoa were investigated. MATERIALS AND METHODS: Using cDNAs extracted from the ejaculated sperm of patients (n=39), the expression of CatSper mRNA was observed by RT-PCR. Semi-quantitative analysis of the CatSper mRNA expression was performed by comparing with the expression of GAPDH mRNA. To elucidate the expression and intracellular localization of CatSper protein, double fluorescent immunocytochemistry for CatSper and beta-tubulin was performed. RESULTS: The CatSper mRNA was expressed in all of the sperm samples. Using semi-quantitative analysis for the amount of CatSper mRNA expression, no significant difference was found between the normozoospermia and asthenozoospermia groups (1.5+/-0.6 vs. 1.4+/-0.6, p=0.623). Polyclonal antiserum, generated against a recombinant protein of the N-terminal 160 amino acids of human CatSper, was used. In double fluorescent immunocytochemistry, CatSper protein was found to be expressed in the flagellum of the ejaculated human spermatozoa, and localized in the connecting piece, mid-piece and principal piece, with the exception of the end piece of the flagellum. Moreover, the proportion of CatSper-positive sperm was similar in both the normozoospermia and asthenozoospermia groups. CONCLUSIONS: To the best of our knowledge, this is the first time ejaculated human spermatozoa have been shown to express the mRNA and protein of CatSper. The results of our RT-PCR and immunocytochemistry suggest that CatSper may play a role in the motility of ejaculated human spermatozoa.

Implantation Rate and Clinical Pregnancy Rate According to Dosage and Timing of Progesterone Administration for Secretory Endometrial Preparation in Frozen-Thawed Embryo Transfer Cycles / 대한불임학회지

OBJECTIVE: To evaluate the difference of implantation rate (IR) and clinical pregnancy rate (CPR) between two protocols of endometrial preperation in women undergoing frozen-thawed embryo transfer (FET) cycles. METHODS: This study was performed during the different time periods: A retrospective study from January 2000 to June 2001 (phase I) and a prospective study from July 2001 to March 2002 (phase II). All the patients received estradiol valerate (6 mg p.o. daily) starting from day 1 or 2 of the menstrual cycle without pituitary down regulation. Progesterone was administered around day 14 after sonographic confirmation of endometrial thickness > or = 7 mm and no growing follicle. In Group A (n=88, 99 cycles) of phase I, progesterone was administered i.m. at a dose of 50 mg daily from one day prior to thawing of pronuclear (PN) stage frozen embryo or three days prior to thawing of 6-8 cell stage frozen embryo and then each stage embryos were trasnsferred 2 days or 1 day later after thawing. In Group B (n=246, 299 cycles) of phase I, patients recieved progesterone 100 mg i.m. from one day earlier than group A; two days prior to PN embryo thawing, four days prior to of 6-8 cell embryo thawing. During the phase II, to exclude any differences in embryo transfer procedures, in Group 1 (n=23, 28 cycles) of phase II embryo was transfered by one who have used the progesterone protocol since the phase I. In Group 2 (n=122, 139 cycles) of phase II embryo was transfered by one who use the progesterone protocol from the phase II. RESULTS: When compared across the phase and group, there were no significant differences in the characteristics. During the phase I, there were significant increase in IR (14.4% vs 5.9%, p=0.001) and CPR (28.3% vs 14.5%, p=0.000) in group A. During the phases II, IR (11.8% vs 10.6%) and CPR (27.6% vs 27.3%) show no differences between two groups. CONCLUSIONS: In FET cycles, IR and CPR are increased significantly by the change of dosage and timing of progesterone administraton. And the timing is considered to be more important factor because the dosage of progesterone did not affect implantation window in previous studies. Therefore, we suggest that progesterone administration in FET cycle should begin from one day prior to PN stage embryo thawing and three days prior to 6-8 cell stage embryo thawing.

Recombinant Human Follicle Stimulating Hormone (rFSH) versus Highly Purified Urinary FSH (uFSH): Oocyte and Embryo Quality / 대한산부인과학회잡지

OBJECTIVE: To estimate the efficacy of recombinant human follicle stimulating hormone (rFSH) versus highly purified urinary human FSH (uFSH) in women undergoing controlled ovarian hyperstimulation (COH) for in vitro fertilization and embryo transfer (IVF-ET). METHODS: From 1 January 2001 to 31 August 2001, A total of 254 cycles from 241 patients who attended infertility clinic at Samsung cheil hospital was enrolled in this study. With pituitary down regulation using GnRH agonist by short protocol, rFSH (Puregon(R), Organon, Netherlands) was administered in 131 cycles and uFSH (Metrodin-HP(R), Serono, Switzerland) was administered in 123 cycles. We analyzed ovarian response, pregnancy rate, live birth rate, oocyte quality and embryo quality. RESULTS: The clinical characteristics of two groups were not different. Total FSH dosages (1322.3+/-526.2 IU versus 2124.4+/-881.9 IU, p<0.001) and dosages per retrieved oocyte (90.6+/-36.0 IU versus 138.0+/-57.2 IU, p<0.001) were significantly lower in rFSH group than uFSH group. Clinical pregnancy rate and live birth rate of two groups were not significantly different. The rate of good quality oocyte (Grade I and II) from retrieved oocytes was higher in rFSH group (68.2% versus 64.8%, p=0.024), but after preincubating oocytes for 4 to 6 hours and removing cumulus cells in intracytoplasmic sperm injection (ICSI) cycles, nuclear maturity of oocytes were not significantly different. The quality of transferred embryos were not significantly different too. CONCLUSION: rFSH offered more effective ovarian response in COH and better quality of retrieved oocytes, compared with uFSH.

The Efficacy of Recombinant Human Follicle Stimulating Hormone (rhFSH) in Human IVF-ET Program / 대한불임학회지

OBJECTIVES: Recently, recombinant FSH (rFSH) has been manufactured using a Chinese hamster ovary cell line transfected with the gene encoding human FSH. Both rFSH and urinary gonadotropin (uFSH) could be used for controlled ovarian hyperstimulation (COH). However, uFSH implies a number of disadvantages, such as batch-to-batch inconsistency, no absolute source control, dependence on large amounts of urine, low specific activity, and low purity. The purpose of this study was to evaluate the efficacy of rFSH in human IVF-ET program. MATERIALS AND METHODS: A total of 508 infertile women was enrolled in this study. They are classified into rFSH group (n=177) or uFSH group (n=331), and all of them were matched by age and cause of infertility in same period. The Puregon(R) (Organon, Holland) was used as rFSH, and the Metrodin-HP(R) (Serono, Switzeland) and Humegon(R) (Organon, Holland) was used as uFSH. We subdivided the patients into three age groups. The outcomes of IVF-ET program were analyzed using the statistical package for social sciences (SPSS). RESULTS: There was no significant differences in the level of estradiol on hCG injection day, the numbers of retrieved oocytes, matured oocytes, fertilized oocytes, transferred embryos, frozen embryos between the two groups. The total dose (IU) of gonadotropin for COH was significantly lower in the rFSH group compared to uFSH group (1339+/-5491.1 vs 2527.8+/-1075.2 IU, p<0.001). Clinical pregnancy rate per embryo transfer in the rFSH group showed increasing tendency, compared to the uFSH group, but there was no statistical significance (35.2% vs 29.3%). Our results demonstrated that the relative efficiency of rFSH compared with uFSH is higher in older patients. CONCLUSIONS: The ovarian stimulatory effect and clinical outcome of recombinant FSH was similar to that of the urinary gonadotropin. The IVF-ET cycles with significantly lower dose of gonadotropin in rFSH group showed comparable results. Therefore, we suggest that recombinant FSH is more potent and effective than urinary gonadotropin.

Influence on the Pregnancy Rate of Motility of Fresh and Frozen Testicular Spermatozoa in Obstructive Azoospermic Patients / 대한불임학회지

OBJECTIVE: ICSI with testicular sperm could achieve optimal fertilization and pregnancy. This study was performed to observe the influence on fertilization and pregnancy of motility of fresh testicular sperm and sperm extracted from frozen-thawed seminiferous tubules in obstructive azoospermia. MATERIALS ANDMETHODS: We analysed clinical outcome of ICSI using fresh testicular sperm and sperm extracted from thawed seminiferous tubules. The presence of motility were compared to determine the factor for optimal fertilization and pregnancy rates. RESULTS: In 316 cases of TESE-ICSI in obstructive azoospermia, ICSI with fresh testicular sperm (fresh sperm group) were 163 cases and ICSI with sperm testicular sperm extracted from frozen-thawed seminiferous tubule (thawed sperm group) were 153 cases. The fertilization rates were 71.3% and pregnancy rates were 32.5% in fresh sperm group, in thawed sperm group, 65.1% and 33.3% respectively. The fertilization and pregnancy rates of motile and non-motile testicular sperm were 72.9% and 33.6%, 50.0% and 18.2%, respectively (p<0.05). The fertilization and pregnancy rates of motile and non-motile sperm extracted from the thawed seminiferous tubule were 67.8% and 34.7%, 55.1% and 28.1%, respectively (p<0.05). The comparative of the results of ICSI using motile fresh testicular sperm and motile sperm extracted from thawed seminiferous tubule, fertilization and pregnancy rates were not significantly different (72.9% and 33.6%, 67.8% and 34.7%, respectively). CONCLUSION: These results suggest that successful pregnancy in TESE-ICSI treatment is influenced by the motility of fresh testicular sperm and sperm extracted from thawed seminiferous tubule in obstructive azoospermic patients.

The Effect of Exocelomic Fluid in the First Trimester Pregnancy on Trophoblast Cell Proliferation in Vitro / 대한산부인과학회잡지

OBJECTIVE: To investigate the effect of exocelomic fluid in first trimester pregnancy on trophoblast cell proliferation in vitro. METHODS: The coelomic fluid was obtained from women with apparently normal pregnancies (n=9) and women presenting with missed abortion (n=22). The concentrations of cytokines in coelomic fluid were determined by two steps sandwich ELISA. The detection limits were the 4 pg/ml for IFN-gamma, 1 pg/ml for TNF-alpha, 2 pg/ml for IL-6 and 5 pg/ml for IL-10, respectively. The data are presented as mean+/-SEM. Statistical analysis was performed by Mann-Whitney U test. Trophoblast cell (Jeg-3 choriocarcinoma cell line) proliferation in vitro was determined using colorimetric immunoassay, based on the measurement of BrdU incorporation using DNA synthesis. The optical absorbance of the samples at 450 nm was measured using an ELISA reader. The data are presented as absorbance in the samples (mean+/-SEM). Statistical analysis was performed using regression analysis and t-test. RESULS: Th-2 type cytokines are present to some extent and IL-6, one of Th-1 type cytokines, also exists in the coelomic fluid from the missed abortion. Coelomic fluids from the majority of normal pregnancies inhibited trophoblast proliferation in vitro significantly higher than fluids from the missed abortion. CONCLUSION: These data showed that exocoelomic fluids may have a unique immune privilege surrounding developing embryo in the early pregnancy. Further studies are required to determine the goowth factors in coelomic fluids from normal pregnancies and missed abortion, and to evaluate the influence on the development of early pregnancy complications.

Analysis of Factors Affecting Survival and Pregnancy Rate in Frozen-thawed Embryo Transfers / 대한불임학회지

OBJECTIVE: The purpose of this study was to determine the important factors affecting survival and pregnancy rate in frozen-thawed embryo transfer cycles. METHODS: we performed reprospective analysis in 738 cycles of frozen-thawed embryo transfers, in relation to the insemination methods, the freezing stage of embryo, patient's age, infertility factors and the origin of injected sperm in ICSI cycles. After conventional IVF or ICSI, the supernumerary PN stage zygotes or multicellular embryos were cryopreserved by slow freezing protocol with 1,2-propandiol (PROH) as a cryoprotectant. RESULTS: The survival rates of thawed embryos were 69.3% (1585/2287) in conventional IVF group and 71.7&% (1645/2295) in ICSI group. After frozen-thawed embryo transfers, 27.0% (92/341) and 32.0% (109/341) of pregnancy rates were achieved in conventional IVF and ICSI group, respectively. There were no significant difference in the survival and pregnancy rates according to the insemination methods, the freezing stage and patient's age. However, the pregnancy rate (36.2%) of male factor infertility (22.9%). In ICSI group, the origin of injected sperm did not affect the outcome of frozen-thawed embryo transfer cycles. CONCLUSION: The present study demonstrates that acceptable clinical outcomes can be achieved after the transfer of frozen-thawed embryos regardless of the stage of embryos for freezing, the patient's age and the origin of injected sperm.

Th1 Cytokine ( IFN-gamma ) Secretion Pattern of Peripheral Blood Mononuclear Cells Response to Trophoblast Antigen in Women with Unexplained Recurrent Spontaneous Abortion and Normal Fertile Controls / 대한산부인과학회잡지

OBJECTIVE: A dichotomous Thl and Th2 cytokine profile has been associated with reproductive failure and success, respectively. The purpose of our study was to determine the levels of Thl cytokine (IFN- y ) secreted by peripheral blood mononuclear cells (PBMCs) form women with unexplained recurrentabortion (URA) and fertile controls in response to trophoblast antigen. METHODS: PBMCs were isolated from 30 nonpregnant women with URA and from 10 nonpregnant fertile controls. Following 4 days of culture (1 * 10(6) cells/mL) with and without a protein extract derived from a trophoblast cell line (30 ug/mL, protein). None of the women had allergies, atopy or recent infection. Cytokines were measured in supernatants with enzyme-linked immunosorbent assay (ELISA) kits. IFN- r kit was obtained from BOISOURCE (lower limit of sensitivity, 15.6 pg/mL for IFN- r ). All values below the lowest limit of sensitivity as determined by test kit standards were considered negative. The cytokine stimulation test is considered positive if the IFN- r concentration increases by 200% or more with the trophoblast antigen stimulation. Datas are presented as mean+ SEM. Nonparametric testing (Mann-Whitney U) was used for analysis with P<0.05 considered statistically significant. RESULTS: The Thl-type cytokine (IFN- r ) was detected in 20(67%) of 30 supernatants from women with URA. In contrast, 2 (20%) of trophoblast-activated PBMC culture supernatants from the 10 parus women with normal reproductive histories was detected IFN- r and but were significantly lower than levels in women with URA who had secreted IFN- r upon trophoblast stimulation (99.80+ 18.17 pg/mL versus 166.47 + 36.96 pg/mL, p<0.05). Spontaneous secretion of IFN- r was significantly higher in culture supernatants from women with URA than in supernatants from women with successful reproductive histories (41.36.09+6.99 pg/mL versus 25.89+9.34 pg/mL, p<0.05). CONCLUSION: These data indicate that there are significant differences between women with URA and women with normal reproductive histories in their regulation of the Thl-cytokine (IFN- r) in response to trophoblast. Thl-type immunity to trophoblast is associated with URA and may play a role in reproductive failure.

Differential Expression of Glucose Transporter Gene in Mouse Early Embryos / 대한불임학회지

The uptake of glucose for metabolism and growth is essential to most animal cells and is mediated by glucose-transporter (GLUT) proteins. The aim of this study was to determine which class of glucose transporter molecules was responsible for uptake of glucose in the mouse early embryo and at which stage the corresponding genes were expressed. In addition, co-culture system with vero cell was used to investigate the effect of the system on GLUT expression. Two-cell stage embryos were collected from the superovulated ICR female and divided into 3 groups. As a control, embryos were cultured in 0.4% BSA-T6 medium which includes glucose. For the experimental groups, embryos were cultured in either co-culture system with vero cells or glucose-free 76 medium supplemented with 0.4% BSA and pyruvate as an energy substrate. 2-cell to blastocyst stage embryos in those groups were respectively collected into microtubes (50 embryos/tube). Total RNA was extracted and RT-PCR was performed. The products were analysed after staining ethidium bromide by 2% agarose gel electrophoresis. Blastocysts were collected from each group at 120hr after hCG injection. They were fixed in 2.5% glutaraldehyde, stained with hoechst, and mounted for observation. In control, GLUT1 was expressed from 4-cell to blastocyst. GLUT2 and GLUT3 were expressed in morula and blastocyst. GLUT4 was expressed in all stages. When embryos were cultured in glucose-free medium, no significant difference was shown in the expression of GLUTI1, 2 and 3, compared to control. However GLUT4 was not expressed until morular stage. When embryos were co-cultured with vero cell, there was no significant difference in the expression of GLUT1, 2, 3 and 4 compared to control. To determine cell growth of embryos, the average cell number of blastocyst was counted. The cell number of co-culture (93.8+/-3.1, n=35) is significantly higher than that of control and glucose-free group (76.6 +/- 3.8, n=35 and 68.2+/-4.3, n=30). This study shows that the GLUT genes are expressed differently according to embryo stage. GLUTs were detectable throughout mouse preimplantation development in control and co-culture groups. However, GLUT4 was not detected from 2- to 8-cell stage but detected from morula stage in glucose-free medium, suggested that GLUT genes are expressed autocrinally in the embryo regardless of the presence of glucose as an energy substrate. In addition, co-culture system can increase the cell count of blastocyst but not improve the expression of GLUT. In conclusion, expression of GLUT is dependent on embryo stage in preimplantation embryo development.

Analysis of the Azoospermia Factor (AZF) Gene on Y Chromosome and Expression Pattern of DAZ Gene in Korean Infertile Men / 대한불임학회지

Cytogenetic observations of loss of the distal portion of the Y chromosome long arm were found to be associated with disrupted spermatogenesis. The existence of a gene involved in the regulation of spermatogenesis, the azoospermia factor (AZF), was postulated. In this study, we screened the AZF region including DAZ and DAZH genes and observed the expression pattern of DAZ and DAZH transcript in infertile men with azoospermia and oligospermia by using a sequence-tagged site (STS)-based PCR method. PCR primers were synthesized for 11 STSs that span Yq interval 6, SRY, DAZ, and DAZH, functional DAZ homologue on chromosome 3. Microdeletions were detected in 4/32 (12.5%) azoospermic men and 1/11 (9%) severe oligospermic men. Only 2 of 5 patients had microdeletions of Yq that contained the 342 gene, whereas the other 3 patients had deletions extending from intervals 5L-6F proximal to the DAZ gene on Yq. Testis biopsies of the azoospermic patients revealed a variety from Sertoli cell-only syndrome to testicular maturation arrest. Of 4 men with clinical data available, average testis size was R: 13.8 co, L: 13.8 co, serum T was 4.0+/-1.25 ng/ml, LH was 3.63+/-1.90 mIU/ml, and FSH was 8.85 +/- 5.13 mIU/ml. These values did not differ significantly from the remainder of the patients tested. We could not observed the DAZ transcript in 2 patients, who have no mature spermatozoa. In 11,6% of patients microdeletions of the AZF could be detected. These deletions in the AZF region seem to be involved causing spermatogenic failure. But the frequency of microdeletions proximal to DAZ suggests that DAZ is not the only gene associated with spermatogenic failure.

Fertilization and Pregnancy Rate of Testicular Sperm after Testicular Sperm Extraction (TESE) with Intracytoplasmic Sperm Injection(ICSI) / 대한불임학회지

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.

Results of Transfer of Cryopreserved Supernumerary Embryos Obtained after Conventional in vitro Fertilization and Intracytoplasmic Sperm Injection (ICSI) / 대한불임학회지

Intracytoplasmic sperm injection (ICSI) recently has been utilized widely as the most successful technique to overcome the unfertilization problem in cases of severe male infertility in couples who could not be treated by conventional IVF. Recently, indications of ICSI have been extended further and more fertilized oocytes become available. Thus, it is necessary to examine the efficiency of freezing the surplus embryos obtained from ICSI. We compared the survival rate and the future outcome of cryopreserved embryos obtained either after conventional IVF or ICSI during the same period. After ICSI or IVF, five best-quality embryos from each patient were transferred in the stimulation cycle and the surplus pronuclear (PN) stage oocytes or multicellular embryos were cryopreserved by slow freezing protocol with 1,2-propanediol (PROH) as a cryoprotectant.4 total of 792 embryos from ICSI trial were thawed and 65.2% (516/792) survived. The survival rates of PN stage oocyte, multicellular embryo and PN + multicellular embryo were 63.5%, 68.2%, 64.0%, respectively. After 111 transfers, 34 pregnancies were achieved, corresponding to a clinical pregnancy rate of 30.6% per transfers. We thawed 1033 embryos from IVF trials and 57.5% (594/1033) survived. In IVF cycle, the survival rates of PN stage oocyte, multicellular embryo and PN + multicellular embryo were 58.2%, 65.2%, 40.2%, respectively. Thirty eight clinical pregnancies were established after 134 transfers, corresponding to a pregnancy rate of 28.4% per transfer The cleavage rate of thawed PN stage oocytes from ICSI trial (61.3%) was significantly higher than those from conventional IVF (53.4%). The developmental rates of good embryo (> or = grade II) in thawed PN stage oocytes obtained from conventional IVF and ICSI were 63% and 65%, respectively. We concluded that PN stage oocytes, multicellular embryos resulting from ICSI procedure can be successfully frozen/thawed with reasonable clinical pregnancy rates comparable to those of IVF.

Efficacy of Coculture System in the Patients with Poor Prognoses on Human IVF-ET Program / 대한불임학회지

SUMMARY: The present study was carried out to evaluate whether the coculture system of human embryos with Vero cells can improve the quality of embryo or overcome the repetitive implantation failures in order to obtain pregnancy. From January to December 1996, a total 202 cases which patients with the problems of repetitive implantation failures (group I) or those with the poor embryonic quality in their previous cycles (group II) was analysed. The quality of cocultured embryo, pregnancy, on-going and implantation rates between coculture and control groups were compared. Of 93 cases in group I, coculture was performed in 34 cases and conventional IVF for the rest. Of 109 cases in group II, 36 for coculture and 73 for conventional IVF. In group I, pregnancy, on-going and implantation rates in coculture group (14/34 (41.2%), 9/34 (26.5%), 16/81 (19.8%), respectively) were higher than those of control (11/59 (18.6%), 8/59 (13.6%), 12/152 (7.9%), respectively). There is significance in the pregnancy and implantation rates (p=0.028 and p=0.015). In group II, pregnancy, on-going and implantation rates in coculture group (8/36 (22.2%), 5/36 (13.9%), 8/87 (9.2%), respectively) were higher than those of control (5/73 (6.8%), 3/73 (4.1%), 3/158 (1.9%), respectively). Like the result of group 1, there is significance in the pregnancy and implantation rates (p=0.028 and p=0.022). Coculture system with Vero cells works well in the groups of the two indications. Although the case of 3 day-coculture was small as 15 cases in group II, 3 day-coculture improved pregnancy rate (4/15 (26.7%)). Therefore, 3 day-coculture with assisted hatching is recommended to the patients with poor embryonic quality. In conclusion, coculture system with Vero cells can be suggested as an effective method which improves pregnancy rate in those who have repetitive implantation failures or whose embryonic quality was poor in their previous cycles.

A study on the patterns of expression of the DAZ and HSP genes in the testicular tissue of men with azospermia

In order to examine whether microdeletions on the Y chromosome exist or not and observe the aspects of expression of DAZ which is suggested to be essential in spermatogenesis in testicular tissue, tissues of 21 patients with azospermia were analyzed by using PCR methods and reverse transcription-PCR. The primers used for the analysis of the microdeletions on the Y chromosome were gene-specific. According to the results of the PCR with genomic DNA of the peripheral blood extracted from each patient, of the 21 men with azospermia 2 displayed microdeletions of the DAZ gene in the Y chromosome but none of HSP70A and HSP70B. And the reverse transcription-PCR of the RNA extracted from the testicular tissue of the patients gave results which found no amplified products of the mRNA of DAZ in the patients with microdeletions of that gene as expected, and confirmed patterns of expression of the mRNA of HSP70B in 5 whereas none could be seen with regard to HSP70A. Furthermore, the sperm of these 5 men were discovered t In accordance with the results previously described, one can see that the microdeletions of DAZ are associated with spermatogenesis and contemplate that HSP70B plays an important part in the maturation process of sperm. But it is considered that there is no correlation between the genes DAZ and HSP and since factors associated with the process of spermatogenesis are being continously discovered more studies on this should be advanced.