• Clinical and Experimental Reproductive Medicine
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• v.26 no.2
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• pp.193-201
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• 1999

In this study we determined fertilization processes and developmental ability of porcine oocytes following injection of round spermatid in the presence of artificial activation. Electrical stimulation at 3 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 immunocytochemistry and laser scanning confocal microscopy study revealed that micro tubules were organized from the oocyte cortex following round spermatid injection, and this seemed to move both male and female pronuclei into the oocyte center. Paternal mitochondria which are introduced with spermatid have been observed up to 4-cell. Our study indicated that either round spermatid or it's nucleus can be used to produce viable bovine embryos by injection into unfertilized oocytes.

• Development and Reproduction
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• v.2 no.2
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• pp.213-221
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• 1998

Intracytoplasmic sperm injection (ICSI) has been widely used to treat couples with infertility due to severely impaired sperm charateristics and for whom conventional in-vitro fertilization (IVF) had failed. The extent to which the morphology of the oocyte at the light microscopy level is related to the results of ICSI vis controversial. In this study, oocytes from 44 patients were reviewed. The ICSI procedure was recorded through CCD camera. The oocytes were divided into five groups according to the presence of cytoplasmic inclusions, the width of perivitelline space (PVS), the presence of cell debris in PVS, the status of first polar body and the flexibility of oolemma. The results showed that the fertilization rate and embryonic development were not associated with the morphological criteria of oocyte. The degeneraton rate of oocytes after ICSI was significantly higher (P<0.001) in the oocytes whose membranes were broken at the moment of insertion (17.7%) than the oocytes whose membranes were broken by aspiration of cytoplasm (1.6%). More oocytes with cytoplasmic inclusions (48.4% vs. 25.1%, p<0.001), wide PVS (35.2% vs. 19.0%, p<0.001), or cell debris in PVS (53.3% vs. 38.4%, p<0.05) were observed in patients with female factor infertility compared to patients with male factor infertility. These results .suggest that the fertilization rate and embryonic development after ICSI are not correlated with oocyte morphology based on the presence of cytoplasmic inclusions, size of PVS, the presence of cell debris in PVS and the status of polar body. And the degeneration rate of oocytes after ICSI was associated with the flexibility of oolemma.

• Clinical and Experimental Reproductive Medicine
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• v.24 no.1
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• pp.83-93
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• 1997

Although IVF-ET is widely applied in the treatment of couples with male factor infertility, it may fail in many infertile couples with normal semen parameters, and certain couples cannot be accepted for standard IVF-ET due to unfertilization or extremely low fertilization rate of oocytes. Recently, several procedures of microassisted fertilization (MAF) using micromanipulation have been introduced, and pregnancies and births have been obtained after partial zona dissection (PZD), subzonal insertion (SUZI), and intracytoplasmic sperm injection (ICSI). This clinical study was performed to develop and establish ICSI as an effective procedure of MAF in infertile couples who could not undergo standard IVF-ET repetitively because of failure in fertilization or extremely low fertilization rate of oocytes with the conventional fertilization technique in the previous IVF-ET cycles. From March, 1995 to May, 1996, 27 cycles of IVF-ET with ICSI in 19 infertile patients were included in study group, and the outcomes of ICSI were analyzed according to fertilization rate, cumulative embryo score (CES), and pregnancy rate. The number of oocytes retrieved after controlled ovarian hyperstimulation (COH) was $10.50{\pm}6.13$ in 30 previous cycles, and $10.57{\pm}5.53$ in 27 ICSI cycles. In ICSI cycles, the number of oocytes optimal for ICSI procedure was $7.89{\pm}4.30$, and the fertilization rate of $67.9{\pm}20.2%$ could be obtained after ICSI. The number of embryos transferred was $1.43{\pm}2.40$ in previous cycles, and $4.36{\pm}1.77$ with the mean CES of $41.8{\pm}27.4$ in ICSI cycles. In ICSI cycles, the overall pregnancy rate was 29.6% (8/27) per cycle and 42.1% (8/19) per patient with the clinical pregnancy rate of 22.2% (6/27) per cycle and 31.6% (6/19) per patient. In conclusion, MAF of human oocytes with ICSI is a promising fertilization method for IVF-ET patients, especially with the past history of failure in fertilization or low fertilization rate of oocytes in the previous IVF-ET cycles, and ICSI using micromanipulation procedures applied to human oocytes will provide a range of novel techniques which may dramatically improve the pregnancy rate in IVF-ET program and contribute much to effective management of infertile couples.

• Clinical and Experimental Reproductive Medicine
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• v.35 no.2
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• pp.131-141
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• 2008

Objective: To compare the outcomes of intracytoplasmic sperm injection (ICSI) with fresh and cryopreserved-thawed testicular spermatozoa in patients with azoospermia. Methods: One hundred and nine cycles (66 couples) where ICSI was planned with fresh or cryopreserved-thawed testicular spermatozoa were included in this study; Ninety two cycles (61 couples) with fresh testicular spermatozoa (fresh group) and seventeen cycles (13 couples) with cryopreserved-thawed testicular spermatozoa (cryopreserved-thawed group). We compared ICSI outcomes such as fertilization rate, implantation rate, pregnancy rate and miscarriage rate, which were statistically analyzed using Mann-Whitney U test or Fisher's exact test, where appropriate. Results: In 9 out of the 92 cycles where ICSI was planned with fresh testicular spermatozoa, testicular spermatozoa could not be retrieved. Fertilization rate tended to be higher in the fresh group than in the cryopreserved-thawed group ($58.0{\pm}27.8%$ vs. $45.9{\pm}25.0%$, p=0.076). The number of high quality embryos was significantly higher in the fresh group ($0.9{\pm}1.2$ vs. $0.2{\pm}0.5$, p=0.002). However, there were no significant differences in clinical pregnancy rate, implantation rate and miscarriage rate between the two groups. Conclusion: The results of this study suggest that although the use of cryopreserved-thawed testicular sperm for ICSI in patients with azoospermia may reduce fertilization capacity and embryo quality, it may not affect pregnancy rate, implantation rate and miscarriage rate. If testicular sperm can be obtained before ICSI procedure, the use of cryopreserved-thawed testicular sperm may also avoid unnecessary controlled ovarian hyperstimulation and cancellation of oocyte retrieval when spermatozoa cannot be retrieved as well as damage on testicular function by repeated TESE.

• Journal of Embryo Transfer
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• v.29 no.1
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• pp.51-57
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• 2014

The objectives of this study were to investigate the effects of trehalose as a cryoprotectant for porcine freeze-dried spermatozoa, to find the optimal freeze-drying time and storage periods of freeze-dried spermatozoa, and to find out pronuclear formation rates, cleaved rates, and embryo development through intracytoplasmic injection of freeze-dried spermatozoa on porcine oocytes. The survival rates of spermatozoa after freeze-drying with trehalose treatment were significantly higher than those of them without trehalose treatment (p<0.05). The highest survival rates were found at 75 mM trehalose treatment. The longer storage periods after freeze-drying seemed to have a lower survival rates. Development in culture of pig by ICSI with trehalose treatment were significantly higher than those of them without trehalose treatment (p<0.05). Shorter freeze-drying time of spermatozoa was resulted in the highest cleaved rates and embryo development.

• Clinical and Experimental Reproductive Medicine
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• v.36 no.4
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• pp.249-254
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• 2009

Objective: The aim of this study was to compare the fertilization and cleavage rates of human in vitro matured oocytes after fertilized by conventional in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). Methods: A total of 135 GV stage oocytes were obtained from 59 women who received ovarian stimulation and IVF during Jan 2007 to Oct 2008. Ovarian hyperstimulation was performed using hMG or recombinant FSH with GnRH antagonist and then ovulation triggered by recombinant hCG. The immature oocytes obtained from stimulation cycles were cultured in IVM medium up to 48 hrs; commercial medium supplemented with rFSH 75 mIU/mL, rhCG 0.5 IU/mL and rEGF 10 ng/mL. The in vitro matured oocytes were fertilized by conventional IVF (41 GV oocytes) or ICSI method (94 GV oocytes). Results: Maturation rate were 51.2% and 59.6% in conventional IVF group and ICSI group, respectively. There was no significant difference in fertilization rates between two groups; 71.4% and 80.4%, respectively. The cleavage rate was also similar in two groups. Conclusion: The presented data suggest that conventional IVF has comparable fertilization and cleavage potential compared with ICSI as the insemination method of immature human oocytes obtained from stimulated cycle.

• Korean Journal of Animal Reproduction
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• v.24 no.2
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• pp.143-154
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• 2000

Most eggs initiated the fertilization processes but arrested at specific stages. The stages included failure of the oocyte to exit from the meiotic metaphase-II with or without sperm penetration, failure of appropriate sperm aster formation, inability to form proper male and female pronuclei, failure of suitable pronuclear apposition, and failure to form proper number of either male or female pronuclei. Various images of defective microtubule organization and chromatin configuration during IVF and ICSI procedures were observed. We discussed the data with previous research results during normal fertilization in humans and other mammals. In conclusion, various aberrant patterns in microtubule assembly and chromatin configuration, which were assessed in the present study, could be used as criteria to improve assisted reproductive technology in clinics. However, further cellular and molecular characterization is needed to clarify these aberrant patterns of cytoskeletal assembly.

• Clinical and Experimental Reproductive Medicine
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• v.24 no.1
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• pp.111-118
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• 1997

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. A 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 (${\geqq}$ 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.

• Clinical and Experimental Reproductive Medicine
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• v.42 no.4
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• pp.143-148
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• 2015

Objective: The aim of the current study was to determine the predictive value of anti-$M{\ddot{u}}llerian$ hormone (AMH) levels for pregnancy outcomes in patients over 40 years of age who underwent in vitro fertilization or intracytoplasmic sperm injection-embryo transfer (IVF/ICSI-ET) cycles. Methods: We retrospectively analyzed the medical records of 188 women aged 40 to 44 years who underwent IVF/ICSI-fresh ET cycles due to unexplained infertility in the fertility center of CHA Gangnam Medical Center. Patients were divided into group A, with AMH levels <1.0 ng/mL (n=97), and group B, with AMH levels ${\geq}1.0ng/mL$ (n=91). We compared the clinical pregnancy rate (CPR) in the two groups and performed logistic regression analysis to identify factors that had a significant effect on the CPR. Results: The CPR was significantly lower in group A than group B (7.2% vs. 24.2%, p<0.001). In multivariate logistic regression analysis, AMH levels were the only factor that had a significant impact on the CPR (odds ratio, 1.510; 95% confidence interval, 1.172-1.947). The area under the receiver operating characteristic curve for AMH levels as a predictor of the CPR was 0.721. When the cut-off level of AMH was set at 1.90 ng/ mL, the CPR was 6.731-fold higher in the group with AMH levels ${\geq}1.90ng/mL$ than in the group with AMH levels <1.90 ng/mL (p<0.001). Conclusion: Our study showed that AMH levels were predictive of clinical pregnancy in infertility patients over 40 years of age. Further prospective studies should be conducted to validate the predictive capability of AMH levels for the outcome of clinical pregnancy.

• Clinical and Experimental Reproductive Medicine
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• v.44 no.1
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• pp.33-39
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• 2017

Objective: The aim of this study was to assess the changes of follicular fluid (FF) and serum levels of cerebellin precursor protein 1 (cbln1) and betatrophin in patients with polycystic ovary syndrome (PCOS) undergoing in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) with a gonadotropin-releasing hormone (GnRH) antagonist protocol. Methods: Twenty infertile women with PCOS and 20 control women diagnosed as poor responders undergoing ovarian stimulation with a GnRH antagonist were included. Blood samples were obtained during ovum pick-up. Follicular fluid from a dominant follicle was collected from the subjects. Using enzyme-linked immunosorbent assays, FF and serum levels of cbln1 and betatrophin were measured in both groups of participants. Metabolic and hormonal parameters were also determined and correlated with each other. Results: Both groups of women had similar serum and FF betatrophin levels ($55.0{\pm}8.9ng/mL$ vs. $53.1{\pm}10.3ng/mL$, p=0.11). The serum and FF betatrophin levels of poor responders were found to be similar ($49.9{\pm}5.9ng/mL$ vs. $48.9{\pm}10.7ng/mL$, p=0.22). Conversely, the FF cbln1 levels of PCOS women were found to be significantly higher than the serum cbln1 levels ($589.1{\pm}147.6ng/L$ vs. $531.7{\pm}74.3ng/L$, p<0.02). The FF cbln1 levels of control participants without PCOS were significantly higher than their serum cbln1 levels ($599.3{\pm}211.5ng/L$ vs. $525.3{\pm}87.0ng/L$, p=0.01). Positive correlations were detected among body mass index, insulin resistance, serum insulin, total testosterone, and betatrophin levels in the PCOS group. Conclusion: Follicular fluid betatrophin and cbln1 concentrations may play a pivotal role on follicular growth in PCOS subjects undergoing IVF/ICSI with an antagonist protocol.