• Clinical and Experimental Reproductive Medicine
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• v.27 no.4
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• pp.367-372
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• 2000

Objective: This study was performed to evaluate whether vitrification method could be used for the cryopreservation of human blastocysts derived from IVF program. Methods: Surplus embryos were obtained from consented IVF patients. Controlled ovarian hyperstimulation was done with midluteal GnRH agonist, gonadotropin and hCG. After oocyte retrieval and insemination, fresh embryo transfer was done at $4{\sim}8$ cell stage. The surplus embryos after ET were cultured in blastocyst medium up to 6 days after oocyte retrieval. Obtained blastocysts were cryopreserved with our vitrification method. Blastocysts were exposed to 1.5 Methylene glycol (EG) in phosphate buffered saline (PBS) for 2.5 minutes, followed by 5.5 M EG plus 1 M sucrose for 20 seconds. Then 1 to 3 blastocysts were mounted on electron microscope (EM) grid and the grid was plunged into liquid nitrogen for storage. For thawing, blastocyst-containing EM grids were sequentially transferred in 1.0 M, 0.5 M, 0.25 M, 0.125 M and 0 M sucrose solution at the intervals of2.5 minutes. And blastocysts were cultured for about 6 hours and only re-expanded blastocysts were transferred to uterus of the patients on 4 to 5 days after ovulation in natural cycle or on 18 to 19 day of artificial cycle. Results: From Oct. 1998 to Jul. 1999, 34 patients were agreed to participate in this study. The mean age and duration of infertility of the patients were 31.6 years and 4.1 years, respectively. Among 34 cycles. replacements could be done in 20 cycles (58.8%). A total 93 blastocysts were thawed and 48 (51.6%) of them survived. Thirty-eight blastocysts, mean 1.9 embryos per patient, were transferred, resulting in 5 clinical pregnancies which consisted of 1 triplet, 2 sets of twins and 2 singleton pregnancies. The pregnancy rate per transfer was 25% and implantation rate was 23.6%. Five patients delivered 7 healthy babies including 2 sets of twins at term. Conclusion: Successful pregnancies and deliveries were established after transfer of vitrified human blastocysts. Vitrification using ethylene glycol as cryoprotectant and electron microscope grid is a rapid and simple method that can be effectively applied for the cryopreservation of human blastocysts.

• Clinical and Experimental Reproductive Medicine
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• v.17 no.1
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• pp.45-56
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• 1990

Maternal serum ${\beta}$-specific human chorionic gonadotropin(${\beta}$-hCG) and pregnancy-specific ${\beta}_1$-glycoprotein($SP_1$) levels were determined more than one per week during 11-41 days post embryo transfer(ET) in 21 consecutive pregnancies after in vitro fertilization(IVF), which included 8 normal singleton pregnancies, 3 twin pregnancies, 4 clinical abortions, 1 ectopic pregnancy, and 5 preclinical abortions. The sensitivity of serum ${\beta}$-hCG and $SP_1$ radioimmunoassays was 3mIU/ml and 0.7ng/ml relatively. At the 7th to 8th week of gestation, ultrasonographic confirmation of fetal pole and fetal heartbeat was performed. Both serm ${\beta}$-hCG and $SP_1$ levels showed logarithmic increase, but log[$SP_1$] had more steep rising curve and had wider variation than log[${\beta}$-hCG] in normal singleton pregnancies. In 3 twin pregnancies and one ectopic pregnancy, both serum ${\beta}$-hCG and $SP_1$ levels located within the 95% confidence interval of the mean levels of 8 normal singleton pregnancies(normal range). In 2 clinical abortions which had a fetal pole without heartbeat, serum ${\beta}$-hCG level showed lower limit of the normal range or just below, but all $SP_1$ levels showed within the normal range. In other 2 clinical abortions which were diagnosed as blighted ovum, both serum ${\beta}$-hCG levels from 11 days post-ET and serum $SP_1$ levels from later days compared with ${\beta}$-hCG were below the normal range. In 5 preclinical abortions, serum $SP_1$ levels were within the normal range but serum ${\beta}$-hCG levels were far below the normal range. In conclusion, both serum ${\beta}$-hCG and $SP_1$ levels increased exponentially with similar pattern in normally conceived pregancy after IVF-ET. Both serum ${\beta}$-hCG and $SP_1$ levels could predict outcome of early pregnancy to a certain degree, but log[${\beta}$-hCG] levels had more significant correlation with outcome of pregnancy compared with log[$SP_1$] levels. In addition, ultrasonographic examination of fetal poles and fetal heartbeats gives very important clinical information and prognosis.

• Clinical and Experimental Reproductive Medicine
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• v.16 no.2
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• pp.205-210
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• 1989

To investigate the effects of ovarian cysts on the controlled ovarian hyper-stimulation cycles, 16 patients with 16 paired cycles for IVF-ET were analyzed. These patients had taken both type of cycles, i.e., with cyst(cyst group) and without cyst(control group). Mean diameter of ovarian cysts in cyst group was 18.2mm. There were no significant differences in hormone levels in early follicular phase between two groups. No significant differences were found in total dosage of hMG(IU) administered during the ovarian stimulation $843.8{\pm}123.0$ vs $891.0{\pm}129.8$, serum estradiol level (pg/ml) on the day of hCG administration($1542.8{\pm}1100.6$ vs $1567.5{\pm}1193.0$), the number of aspirated follicles $10.0{\pm}3.4$ vs $11.2{\pm}4.3$ and oocytes $5.3{\pm}3.3$ vs $6.2{\pm}3.1$, the fertilization rate(51.2 % vs 57.2 %) and the cleavage rate(40.5 % vs 52.0 %). Serum estradiol terminal patterns during COH in one group tended to be repeated in the other group. In conclusion, this study suggests that small ovarian cysts do not adversely impact on the controlled ovarian hyperstimulation parameters in IVF - ET program and the presence of small ovarian cyst without concomitant high basal serum estradiol level is not an indication of the cancellation of the controlled ovarian hyperstimulation for IVF-ET.

• Clinical and Experimental Reproductive Medicine
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• v.31 no.3
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• pp.169-176
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• 2004

Objective: This study was performed to evaluate and compare the embryonic developmental capacity and pregnancy rates in conventional in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) with ejaculated sperm or testicular sperm cycles. Materials and Methods: Fertilization was examined in the following morning after IVF (group I), ICSI (group II) or TESE-ICSI cycles (group III). Fertilized oocytes were co-cultured with Vero cells until embryo transfer (ET). On day 2 and $5{\sim}7$, grades of embryos (<4- or $\geq$4-cell) and blastocysts (BG1, 2, 3 or early) were evaluated. Clinical pregnancy rate was determined by detecting G-sac with transvaginal ultrasonogram. We analyzed the results by $X^2$ and Student's t-test and considered statistically significant when P value was less than 0.05. Results: Fertilization rate was significantly higher (p<0.05) in group I ($79.0{\pm}21.2%$) than in group II and III ($56.8{\pm}21.6%$ and $36.7{\pm}25.3%$). Cleavage and blastulation rate of group I ($95.8{\pm}13.8%$ and $59.5{\pm}25.3%$) were significantly higher (p<0.05) than those of group III ($83.4{\pm}18.6%$ and $40.4{\pm}36.5%$). Clinical pregnancy rate was significantly higher (p<0.05) in group I and II (40.7% and 41.7%) than that in group III (12.5%). No differences were found in the rates of multiple pregnancy and abortion among three groups. Embryonic implantation rate was higher in group I ($15.1{\pm}20.2%$, p<0.05) and II ($14.7{\pm}20.6%$, NS) than that in group III ($5.1{\pm}15.6%$). However, embryonic implantation rate was increased in ET with blastocyst(s) among three groups. Conclusions: Fertilized oocytes obtained from TESE-ICSI were harder to be successfully cultured to blastocyst stage for 5$\sim$7 days than that from IVF cycles. However, all blastocyst(s) ET increased the embryonic implantation rate equally in IVF, ICSI and TESE-ICSI cycles.

• Journal of Embryo Transfer
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• v.19 no.2
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• pp.155-163
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• 2004

The aims of this study are 1) to test oocytes and embryos collected from in-vitro to achieving the valuable protocol by culturing, vitrifying and thawing of oocytes/embryos, and 2) to transfer them to recipient, and finally have resulted in pregnancies from recipient females after surgical or nonsurgical transfer. In vitro maturation and fertilization were performed according to Funahashi et al (1994). Glucose-free NCSU 23 supplemented with 5 mM sodium pyruvate, 0.5 mM sodium lactate and 4 mg/ml bovine serum albumin for 2 days at $39^{\circ}C$, and 10% fetal bovine serum albumin was added to the culture medium thereafter. Embryos were treated with 7.5 ${\mu}g/ml$ cytochalasin-B for 30 min, centrifuged at 13,000 rpm for 13 min and then exposed sequentially to an ethylene glycol(EG) vitrification solution, aspirated into OPS, and plunged/thawed into/from liquid nitrogen. In vivo embryos were surgically collected from three dornors after AI for control group. Forty-nine embryos were washed 3 times in mPBS + 10% FBS, followed treatments : cultured, centrifuged, vitrified, recovered and transferred to recipients as in vitro prepared embryos. Three recipients were transferred individually with 100, 100 frozen embryos derived from abattoir and 34 fresh embryos by surgically, and another three recipients were transferred individually with 150, 150 frozen embryos and 100 fresh embryos by nonsurgically, respectively. all recipient sows exhibited delayed returns to estrus. To our knowledge, theses results suggest that required an improved techniques, more vigorous embryos preparation and substitute to gilt with cleaner uterous condition.

• Clinical and Experimental Reproductive Medicine
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• v.23 no.1
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• pp.109-114
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• 1996

General PCR technique alone has a limitation for preimplantation genetic diagnosis(PGD) using single blastomere. Recntly developed primer extension preamplification(PEP) technology amplifies the whole genome and thus, simultaneous multiple locus analysis became possible. In this study, we report the efficacy of PEP-PCR for PGD in three muscular dystrophy carriers undergoing IVF-ET. A total of 37 blastomeres were obtained from 40 embryos at six to eight cell stage in three IVF cycles in two DMD and one BMD carriers. Whole genome from single blastomeres were amplified using I5-base oligonucleotide random primers. PCR amplified products of exon 45 in the dystrophin gene and alphoid X/Y loci for gender determination were analysed by 2% metaphor gel electrophoresis. A total of 37 PEP-PCR replicates from 37 single blastomeres from 40 embryos and 37 blanks were performed. We obtained the reliable results for exon 45 and alphoid X/Y. Transfer of female embryos and unaffected male embryo was attempted in three couples. Unfortunately, pregnancy was not achieved in these cases. PEP-PCR is a reliable and efficient PGD method in multiple locus analysis using single blastomere.

• Journal of Plant Biotechnology
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• v.29 no.4
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• pp.253-257
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• 2002

To establish the optimum condition for plant regeneration from somatic embryos of Acanthopanax sessiliflorus Rupr. et Maxim, a medicinal plant, somatic embryos were induced from zygotic embryo-derived embryogenic callus in hormoen-free MS medium. To induce plantlet conversion, cotyledonary somatic embryos were cultured on MS solid medium with GA$_3$at various concentrations (0~10 mg/L) for three weeks. Plantlets were transferred to 1/3 MS solid medium with 0.5% charcoal for 7 weeks. Stem length was increased proportionally to the concentration and treatment period of GA$_3$. Also, the highest leaf width (8.9 mm) and leaf number (2.84) of plantlet were obtained when plantlets were converted on 5,10 mg/L GA$_3$pretreatments, respectively. The highest plant conversion frequency (66.7%) was obtained when the somatic embryos were cultured on medium containing 5 mg/L GA$_3$ for 3 weeks and then were transferred to 1/3 MS medium with 0.5% charcoal. The highest survival rate of soil transfer was 90% when plantlets were regenerated on medium with 5 mg/L GA$_3$ for 3 weeks and then transferred to plastic pots containing vermiculite and sand mixture for 4 weeks.

• Clinical and Experimental Reproductive Medicine
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• v.17 no.2
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• pp.115-121
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• 1990

Ultrasonically guided oocyte collection gradually replaces laparoscope in many IVF center. In present study, we compare the efficacy of both methods in our IVF program. Totally 377 cycles which were undertaken in vitro fertilization treatment were divided into 2 groups. Ultrasonically guided transvaginal follicular aspiration was performed in 188 cycles and laparoscopic follicular aspiration was performed in 189 cycles under local anesthesisa. The mean age for both groups was similar. Follicular recruitment was achieved with human menopausal gonadotropin (hMG) or a com bination of clomiphene citrate and hMG or a combination of FSH and hMG. In the ultrasonically guided aspiration group, 1821 follicles were aspirated with 61.8% of recovery rate (1125 oocytes), 81.5% of embryo transfer rate (145 cycles) and (17%), 26 cases intrauterine pregnancies were estabilished. In the laparoscopic group, 604 follicles were aspirated with 68.7% recovery rate (445 oocytes) and a 79.9% ET rate (127 cycles), 11 cases (8.7%) intrauterine pregnancies were estabilished. A valid comparison of these data is not possible because the 2 groups are dissimilar for factors known to influence oocyte development and recovery. No statistically significant differences could be demonstrated between 2 groups in all but the recovery rate and clinical pregnancy rate, In ultrasound group, the clinical pregnancy rate was significantly higher than that of laparoscope group. The potentially detrimental effect of CO2 pnemoperitonium present during laparoscope but not in ultrasound guided recovery on ova quality may underlie the observed difference in the clinical pregnancy rate between the 2 groups. Ultrasound guided aspiration seems to be as effective as laparoscopy in terms of oocyte retrieval and conception rate. Furthermore, the procedure is simple and inexpensive, it may replace laparoscopy as a method for oocyte collection in most patients who undergo IVF.

• Clinical and Experimental Reproductive Medicine
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• v.13 no.1
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• pp.67-75
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• 1986

Follicle monitoring in the normal and clomiphene·stimulated cycles were analyzed in the Seoul IVF and ET (In vitro fertilization and embryo transfer) program. Ovarian follicular diameters were measured by the real·time sector scanner and plasma estradiol levels were assayed by radioimmunoassay methods during periovulatory period. The maximum follicular sizes of the clomiphene-stimulated and normal cycles were 21.1+-3.4mm and 19.2+-0.8mm, respectively. The peak levels of plasma estradiol in the clomiphene-stimulated and normal cycles were 10538+-553.6ng/ml and 298.3+-39.6pg/ml, respectively. Daily growth rate of the follicular diameters of the clomiphene-stimulated and normal cycles were 2.1mm and 1.9mm, respectively. Mean follicular number of the clomiphene-simulated and normal cycles were 2.28+-1.12 and 1.12+-0.21, respectively. There was a good statistical correlation between the mean follicular diameters and the plasma estradiol levels in the normal ovulatory and c1omiphene-stimulated ovulatory menstrual cycles (p<0.05). Our data revealad that the mean follicular diameter and the plasma estradiol level prior to HCG administration in IVF and program should reach at the level of 17.8+-3.0mm and 949.4+-487.1 pg/ml, respectively.

• Clinical and Experimental Reproductive Medicine
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• v.38 no.3
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• pp.148-152
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• 2011

Objective: Laser-assisted intracytoplasmic sperm injection (LA-ICSI), also known as micro-opening or thinning of the zona pellucida (ZP) prior to ICSI, may help to reduce mechanical damage to the oocyte during the procedure. The aim of the present study was to evaluate and analyze the efficacy of our institutional LA-ICSI program, which features laser-assisted ZP thinning prior to ICSI, in comparison with conventional ICSI (C-ICSI), performed on patients with different clinical characteristics. Methods: Patients undergoing a total of 212 ICSI cycles were randomly divided into an LA-ICSI group (106 cycles) and a conventional ICSI group (106 cycles). To reduce tissue damage, we thinned the ZP by approximately 70%, using a laser, before ICSI. Patients thus treated formed the LAICSI group. Comparisons included the morphological quality of transferred embryos, blastocyst development of the remaining embryos, and clinical pregnancy, in terms of ICSI method and patient characteristics. Results: Fertilization, development of remaining embryos, and pregnancy rate were significantly higher in the LA-ICSI group compared with the C-ICSI group. Fertilization, embryonic development, and the pregnancy rate were all improved in younger patients (<38 years of age) and in those who underwent a low number of IVF-ET attempts (<3 trials). In addition, the pregnancy rate was increased in older patients. Conclusion: LA-ICSI may be useful in improving the chance of pregnancy in all ICSI patients.