• 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.26 no.1
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• pp.75-81
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• 1999

The beneficial effect of glucose and phosphate ions in culture medium on the development of human embryos in vitro has not been fully elucidated. The purpose of this study was to evaluate the influence of fertilization and culture of embryos in glucose/phosphate-free m-TALP medium on pregnancy rates in IVF-ET program. The patients in 244 IVF-ET cycles received GnRH agonist + HMG regimens. A does of 10,000 IU HCG was administered when two or more dominent follicles reached 18mm in diameter. Thirty-six hours after HCG, oocytes were recovered transvaginally using ultrasound guidance. Aspirated oocytes were matured for 4 to 6 h in TCM-199 supplemented with 10% follicular fluid (FF). Insemination was carried out with 50,000 motile spermatozoa in TCM-199 + 10% FF or m-TALP + 5% FF + 5% fetal cord serum (FCS) according to experimental design. After 6 h, oocytes were washed 3 to 4 times and cultured in each fresh medium. After 20 h, oocytes were freed from cumulus/corona cells and examined for the presence of pronuclei. Fertilized oocytes were transferred into each co-culture drops and cultured for further incubation. On day 3, embryo transfer was performed with grade 1 and 2 embryos. Monolayers for co-culture of embryos were prepared by plating $1{\times}10^5$ cumulus cells/ml in 10ul drop of TCM-199 + 10% FF or m-TALP + 5% FF + 5% FCS media 24 h prior to the onset of co-culture. Development to 4 to 16 cell stage was observed at 70x magnification following two days of incubation. Pregnancy was confirmed by detecting increasing serum ${\beta}$-hCG concentrations for 11 days following embryo transfer. Data were analyzed by ${\chi}^2$-test. Oocytes from 244 IVF-ET cycles were randomized. The number of cycles and mean age of patients were 97 and 147, 31.3 yrs and 31.2 yrs for TCM-199 (control) and m-TALP groups, respectively. The mean number of retrieved oocytes/cycle, fertilization rates, number of embryos transferred/ET and pregnancy rates were 11.1 and 10.3, 65.1% and 67.3%, 4.1 and 4.7, 28.9% and 43.8% for TCM-199 and m-TALP groups, respectively. Differences in the pregnancy rates were found between control and m-TALP groups (p<0.05). The pregnancy rate of patients divided according to maternal age groups of ${\leq}30$, 31-35, $36{\leq}$ were 44.4% and 49.0%, 26.1% and 41.3%, 29.2% and 41.2% for control and m-TALP groups, respectively. These data indicate that culture of human embryos in glucose/phosphate-free m-TALP medium improves pregnancy rates.

• Clinical and Experimental Reproductive Medicine
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• v.39 no.2
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• pp.52-57
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• 2012

Objective: During IVF, non-transferred embryos are usually selected for cryopreservation on the basis of morphological criteria. This investigation evaluated an application for array comparative genomic hybridization (aCGH) in assessment of surplus embryos prior to cryopreservation. Methods: First-time IVF patients undergoing elective single embryo transfer and having at least one extra non-transferred embryo suitable for cryopreservation were offered enrollment in the study. Patients were randomized into two groups: Patients in group A (n=55) had embryos assessed first by morphology and then by aCGH, performed on cells obtained from trophectoderm biopsy on post-fertilization d5. Only euploid embryos were designated for cryopreservation. Patients in group B (n=48) had embryos assessed by morphology alone, with only good morphology embryos considered suitable for cryopreservation. Results: Among biopsied embryos in group A (n=425), euploidy was confirmed in 226 (53.1%). After fresh single embryo transfer, 64 (28.3%) surplus euploid embryos were cryopreserved for 51 patients (92.7%). In group B, 389 good morphology blastocysts were identified and a single top quality blastocyst was selected for fresh transfer. All group B patients (48/48) had at least one blastocyst remaining for cryopreservation. A total of 157 (40.4%) blastocysts were frozen in this group, a significantly larger proportion than was cryopreserved in group A (p=0.017, by chi-squared analysis). Conclusion: While aCGH and subsequent frozen embryo transfer are currently used to screen embryos, this is the first investigation to quantify the impact of aCGH specifically on embryo cryopreservation. Incorporation of aCGH screening significantly reduced the total number of cryopreserved blastocysts compared to when suitability for freezing was determined by morphology only. IVF patients should be counseled that the benefits of aCGH screening will likely come at the cost of sharply limiting the number of surplus embryos available for cryopreservation.

• Asian-Australasian Journal of Animal Sciences
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• v.15 no.10
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• pp.1412-1421
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• 2002

To examine the feasibility of using a sperm vector system for gene transfer, we have investigated the binding and the uptaking of foreign DNA into the sperm nucleus by PCR, in situ hybridization and LSC. We have also examined the transportation of exogenous DNA into oocytes by immunofluorescene via PCR. Sperm cells were incubated with DNA/liposome complexes (1:4 ratio) in fertilization medium with BSA or without BSA. In situ hybridization demonstrated that the transfection rate of sperm cells with and without BSA was 41 and 68% respectively, when the cells were treated with liposome/DNA complexes and 13% for DNA alone. LSC analysis showed that the binding of exogenous DNA was greatly reduced by DNase I treatment which digests DNA bound onto spermatozoa, suggesting that some of the DNA was internalized into the sperm membrane. To find out whether transfected DNA was internalized into sperm intracytomembrane, sperm DNA was amplified by inverse PCR. No PCR products were detected from sperm cells, indicating that the foreign DNA was simply bound onto the sperm membrane. To investigate transfer rates of exogenous DNA into oocytes via sperm cells, we used immunofluorescene method to follow the distribution of foreign DNA via spermatozoa: a few exogenous DNA was located in the cytoplasm of early embryos (13/60, 21.7% for DNA+/liposome+/BSA) and was not located in the pronucleus and/or nucleus. These results suggest that most of the transfected sperm cells could carry the foreign DNA into the egg by in vitro fertilization, but that the transferred DNA is degraded in the developing embryos without stable integration into the zygote genome. Therefore, we have directly injected with transfected sperm cell into oocyte cytoplasm and observed that some of the exogenous DNA was detected in preimplantation embryonic cytoplasm and expressed at preimplantation stages, suggesting that exogenous DNA in early zygote has their integrity. In this study, we have not identified a noble mechanism that interfering transportation of foreign DNA into zygote genome via spermatozoa. Our data, however, demonstrated that inverse PCR and immunofluorescene methods would be used as a new tool for follow-up of gene distribution in oocyte via sperm cells.

• Clinical and Experimental Reproductive Medicine
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• v.21 no.3
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• pp.315-323
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• 1994

Mammalian oviductal epithelial cells have been known to improve in vitro fertilization and embryonic development. Recently, co-cultured human embryos with the epithelial cells in human genital tract has been reported to improve the pregnancy rate. The purpose of the study was to investigate the effects of the epithelial cells of human genital tract on the development of mouse early embryos and human fertilized oocytes. The epithelial cells of human genital tract were collected from the fallopian tubes which were obtained during hysterectomy in fertile women and from the endometrium during endometrium biopsy. Collected human ampullary cells(HACs) and endometrial cells(HECs) were cultured for 10 days to establish primary monolayer. Second passaged HACs and HECs were obtained by trypsinization were cryopreserved in PBS with 1.5 M DMSO for later use. To investigate the effect when co-cultured with HACs and HECs, we tried to apply strict quality control on mouse embryo, from two cell to blastocyst prior to human trial. The results of quality control were as follows; In Group I (Ham's F10 with 10% FCS), Group IT (co-cultured with HACs) and Group ill (co-cultured with HECs), developmental rates to blastocyst were 63.3%(253/400), 76.0%(304/ 400),74.0%(296/400), respectively. Hatching rates were 36.8%(147/400), 41.80/0(167/400), 38.0%(152/400), respectively(p<0.05). To perform the human IVF, cryopreserved HACs were thawed at 37$^{\circ}C$ waterbath, seeded on the well dish and cultured for 48 hI'S. The pronuclear stage embryos were transferred to the seeded well dish. After 24 hRS, co-cultured embryos were examined and transferred to patient's uterus. The results of human IVF when co-cultured with HACs were that fertilization and developmental rates were 61.8% (256/414), 95.3% (244/256) as compared with 57.2% (279/488) and 94.6%(264/279) in Ham's F10 supplemented with 10% FCS(control). However, 62.9% (161/256) of co-cultured human embryos showed good embryos(no or slight fragmentation) as compared with 53.8 % (150/279) in control(p < 0.05). Pregnancy rate was 40.0% (12/30) when co-cultured with HACs whereas 30.6%(11/36) in control. In conclusions, co-culture system using HACs and HECs improved the developmental and hatching rates of mouse embryo. Also, in human IVF system when co-cultured with HACs, it improved both the quality of human embryos and the pregnancy rate.

• 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.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.36 no.6
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• pp.545-553
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• 1991

To find out the basic data for the possibility of agricultural utilization for GSA (Glutamine Synthetase Activity), the effect of nitrogen on the GSA in wheat leaf discs, the variation of GSA after light treatment and the comparative activity of GS during preservation were studied. The result of this study suggested that GSA could play an important and direct regulatory role in the nitrogen assimilation by wheat. During the growth stage of wheat its integral activity was found to closely match the organic nitrogen content. GS may therefore be the rate limiting enzyme in inorganic N assimilation. Moreover, integral GSA was closely correlated with grain yield and grain nitrogen. GSA could be suitable to utilize as a parameter for super type selection and an indicator for optimum nitrogen fertilization. Throughout the experiment, the contents of NO; were increased by N fertilization so that the NO; content was not attributable to change in the level of GSA. At investigation during dark-light transition of culture, no change in the level of GSA was observed until after 8-14 hours in the light treatment. And the level of GSA in wheat leaf discs during preservation at refrigerated storage $(-20^{\circ}C)$ was stable until 12 weeks, when its leaf discs were sampled with liquid nitrogen.

• Journal of Embryo Transfer
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• v.19 no.1
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• pp.19-25
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• 2004

The objective of this study was to optimize the selection of sperm, optimal culture system of in vitro derived porcine embryos. The results obtained were summarized as follows: 1. When oocytes were inseminated with liquid sperm and frozen-thaw sperm, the cleavaged rate of liquid sperm (46.2%) was higher than that of frozen-thaw sperm (39.7%), however there were not show significant different each other. The blastocyst rates of liquid sperm (15.8%) was significantly higher than that of frozen-thawed sperm (9.3%)(P< 0.05). 2. When oocytes were inseminated with epididymal sperm after 1, 2 and 3 day storage, the cleavaged rate of epididymal sperm after 1, 2 and 3 day storage was 60.5, 61.0 and 56.8% respectively. The morulae (17.4, 19.9 or 17.3%) and blastocyst (8.7, 15.4, 11.3%) rate of epididymal sperm after 1, 2 and 3 day storage was no significantly respectively(P< 0.05). 3. In vitro developed to cleavaged rate of G1.3/G2.3 media used for culture was significantly(P< 0.05) higher as 62.1% compared with the results using the media NCSU23(52.8), however in vitro developed to blastocyst rate of NCSU23(11.6%) media was significantly(P< 0.05) higher than that'of G1.3/G2.3(4.7%). 4. When the fertilized oocytes were cultured with NCSU23 in addition to 1 mM glutathione(GSH), the cleavaged rate of treated groups of GSH(62.3%) was significantly higher than that of control(53.5%) respectively(P< 0.05). And in vitro developed to blastocyst rates of treated groups of GSH(15.6%) was higher than that of control(12.6%) however, there was no significant difference(P< 0.05).

• Korean Journal of Animal Reproduction
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• v.22 no.4
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• pp.395-403
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• 1998

In this study we determined fertilization processes and developmental ability of porcine oocytes following injection of round spermatid in the presence and absence of artificial activation. Electrical stimulation at 2 h before spermatid injection significantly increased the incidence of normal fertilization as compared to those following injection without stimulation or with stimulation immediately after injection. The incidences of two pronuclear formation and apposition were not different in oocytes between following intracytoplasmic spermatid and spermatid nucleus injection. Indirect immunocyto chemistry and laser scanning confocal microscopy study revealed that microtubuels were organized from the oocyte cortex following round spermatid injection, and this seemed to move both male and female pronuclei into the oocyte center. At 6 days following injection blastocoele formation was seen in the eggs following round spermatid (25%) and round spermatid nucleus injection (27%). However, none of oocytes developed to the blastocyst stage at 6 days following sham injection. The average cell numbers of blastocysts at 8 days following injection of spermatid and spermatid nucleus were 87 to 99. These results suggested that either round spermatid or it's nudeus can be used to produce viable embryos by injection into unfertilized oocytes in the pig.

• Clinical and Experimental Reproductive Medicine
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• v.20 no.2
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• pp.107-115
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• 1993

Procedures to separate motile. normal & motile and acrosome-reacted sperm with high efficiency have clinical application in Assisted Reproductive Technology in terms of increasing the probability of fertilization by a normal sperm and subsequent normal embryonic development. This study evaluated the effects of 10 sperm preparation techniques [Swim-up from a washed pellet (SU). Swim-up from semen (SO). Continuous Percoll Gradients I (PIC). Discontinuous Percoll Gradients I (PID). Continuous Percoll Gradients II(P II C). Discontinuous Percoll Gradients II(P II D), SpermPrep (SFC). Wang's tube (WT). Albumin Gradients (AG), Low temperature capacitation (LTC)] on motility (%), normal morphology (%), motile sperm recovery rate(%). morphologically normal & motile sperm recovery rate (%), true acrosome reaction (%) and fertilizing ability. A P II D proved to be an effective means of separating morphologically normal & motile sperm. Our results indicated the P II D has advantages as compared with other methods in terms of recovery rate. enhancement of motility and normal morphology. And a LTC seems to be an effective means of enhancing the true acrosome reaction and fertilizing ability. These results suggest that the combined method of LTC and P II D for separation of morphologically normal & motile sperm and acrosome reacted sperm may be a useful procedure for intrauterine insemination and in vitro fertilization in the management of male factor infertility as well as for isolation of subpopulation of sperm for basic research.