• Clinical and Experimental Reproductive Medicine
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• v.18 no.1
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• pp.73-80
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• 1991

We compared fertilizing potential measurements by the zona-free hamster egg penetration assay with the in vitro fertilization and embryo transfer program was evaevulated for their ability to fertilize zona free hamster egg. Spermatozoa from 12 presumeably fertile donors and from the male partners of 56 infertile couples were evaluated for their ability to fertilizing potentials. Penertration rates of fertile donors were $36.2{\pm}27.7%$ ; Fertilization rates of infertile couples between with normal semen parameters and with abnormal semen parameters were $28.7{\pm}19.1$, $5.7{\pm}8.9%$, respectively. Sperm motility of couples with penetration rates between on 15-30% and on 30> were $54.1{\pm}4.6$, $55.5{\pm}8.3%$ respectively. Hamster penetration rates of couples participating in an in vitro fertilization and embryo transfer program was $38.9{\pm}29.9%$. But in one case, a positive fertility assessment was obtained in the absence of fertilization of the wife's eggs attributable to egg immaturity. This method may have potential value as a diagnostic tool in evaluation human sperm fertilization capacity which avoids the ethical and logistical problems associated with fertilizing of human eggs in vitro.

• Clinical and Experimental Reproductive Medicine
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• v.30 no.1
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• pp.105-109
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• 2003

Objective s: To assess the fertilizing capacity using sperm penetration assay (SPA) to predict the outcome of the in vitro fertilization-embryo transfer (IVF-ET) outcome. Materials and Methods: Semen samples were provided by 129 patients undergoing IVF. We attempted to correlate the extent of sperm penetration under enhanced SPA protocol with the results of fertilization, cleavage, preimplantation embryo development, and pregnancy. Results: Univariate analysis demonstrated a statistically significant correlation between fertilizing capacity and motility, kinetics, fertilization, cleavage and embryo development, and pregnancy rate. By logistic regression analysis, fertilizing capacity was found to be the only variable that was statistically significant with respect to pregnancy rate. Fertilizing capacity, cleavage rate and pregnant rate were significantly higher in pregnant group. However, the fertilization rates was comparable with both group. Conclusions: Lower fertilizing capacity could denote a poorer prognosis for establishing a pregnancy, even after satisfactory fertilization rate is achieved.

• Clinical and Experimental Reproductive Medicine
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• v.29 no.1
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• pp.57-66
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• 2002

Objective : This study was designed to investigate the interrelationship and clinical usefulness of sperm morphology by strict criteria (SM), acrosome reaction following ionophore challenge test (ARIC) and sperm penetration assay (SPA) using zona-free hamster ova as prognostic factors in in vitro fertilization. Materials and Methods: Semen samples were provided by 83 patients undergoing IVF. We first evaluated the differences between normal fertilization group and poor fertilization group on three andrologic tests. Secondly, we analyzed the relationship between the three andrologic tests and in vitro fertilization on IVF settings. Finally, we evaluated the effectiveness of the three andrologic tests as the prognostic indicators for fertilizing ability. Results: The fertilization rate of all men in the poor fertilization group was less than 30%; but there was no evidence that this poor fertilization was due to oocyte defects. The results of three andrologic tests were significatly higher in normal fertilization group. Fertilization rate (%) in vitro was highly correlated (p<0.001) with % normal sperm by SM, ARIC value (%), and SPA result. By using Receiver-Operator-Characteristic curve (ROC), we evaluated the effectiveness of these three tests. The sensitivity and specificity of SM, ARIC test and SPA in predicting fertilization potential in IVF setting were 76% and 75%, 84% and 90%, and 76% and 95%, respectively. Conclusion: Our data suggest that the three andrologic tests can be reliable tools as prognostic factors of sperm fertilizing ability. Among these test, ARIC test and SPA gave more accurate information on fertilizing capacity. ARIC test was shown to have a predictive value for fertilizing ability comparable to that of SPA that appears to be a simple and cost-effective addition to current andrology laboratory. Combined application of these three tests may give more information on predicting sperm fertilizing capacity.

• Clinical and Experimental Reproductive Medicine
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• v.19 no.2
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• pp.153-162
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• 1992

To solve the logistical problems of the sperm penetration assay (SPA) to provide just a sufficient number of hamster ova exactly when they are needed, a new method to cryopreserve the ova has been devised (1-step dehydration and 2-step thawing). After freezing & thawing of zona-intact (ZI) and zona-free (ZF) hamster ova according to this new method, the frozen-thawed ova were compared with fresh, control ova (FO) in terms of the degree of sperm penetration in SPA using semen samples from fertile donors, subfertile, and infertile male. Each sperm sample was capacitated for 42 hours inTEST-Yolk Buffer before insemination in SPA. In fertile doner, both the penetration rate and penetration index were lower in SPA using frozen ova (ZI; 92.4%, 6.2, ZF; 63.7%, 3.9) than those of SPA using fresh ova (99.3%, 8.4). There was a significant correlation between the penetration index of SPA using FO and ZI (p<0.001), and between those of SPA using FO and ZF and ova (p<0.001). In subfertile patient, both the penetration rate and penetration index were lowered in frozen ova (ZI; 62.3%, 1.3, ZF; 21.8%, 0.4) than those of fresh ova (74.8%, 1.8). There were significant correlation between the penetration rate and penetration index in ZI ova (p<0.05 and p<0.001, respectively). In infertile patient, both the penetration rate and penetration index were ZI; 3.1%, 0.0, ZF;0.0%, 0.0, respectively. There were significant correlation between the penetration rate and penetration index in ZI ova (p<0.05).

• Clinical and Experimental Reproductive Medicine
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• v.25 no.1
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• pp.1-8
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• 1998

To investigate the influences on semen parameters and fertilizing capacity of immunoglobulin (Ig) isotypes and regional distribution of antisperm antibody (ASA) on the human sperm surface. Sixty-seven ASA-positive patients were compared with 96 ASA-negative donors. ASAs in semen showed significant negative effects on both semen parameters and fertilizing capacity; in those with ASAs in the sperm head and/or tail, the reductions were significant. In the head as well as the tail, there was close correlation between fertilizing capacity and both IgG and IgA. Both semen parameters and fertilizing capacity are significantly affected by the presence of ASA in semen. In particular, antibodies IgG to sperm head and/or tail, and antibodies IgA to sperm tail appeared to have a highly detrimental effect on fertilizing capacity.

• Clinical and Experimental Reproductive Medicine
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• v.21 no.1
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• pp.1-11
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• 1994

The occurrence and time course of capacitation, acrosomal loss, and hyperactivated motility require quantitative definition in order to characterize fertile human sperm. Recently the method has been developed to estimate the quality of spermatozoa by using kinematic parameters such as curvilinear velocity(VCL), average path velocity(VAP), linearity(LIN), straightness(STR), amplitude of lateral head displacement(ALH), and beat cross frequence(BCF) from Computer Assisted Sperm Analysis (CASA). In this study, using the Hamilton Thorn motility analyzer HTM 2030(Hamilton Thorn Research, Beverly, MA), we attempted to identify the spermatozoa with hyperactivated motility (HA) objectively and to monitor hyperactivation of human spermatozoa during incubation in capacitating media and after treatment of calcium ionophore as compared with acrosome status. And we examined whether HA are related to the result of SPA. Semen samples obtained from 16 healthy men were prepared by swim up technique and preincubated in a capacitating media(modified BWW medium) for 5 hours and treated with calcium ionophore solution. The acrosome reaction was detected with PSA-FITC labelling of the acrosome and in vitro sperm ferilizing capacity was assessed by the zona free hamster ovum penetration assay (SPA). The incidence of hyperactivated sperm was 2.6% in fresh semen, 14.3% of the swim up population, 13.7% after 5h of incubation. Significant increase of percentage of hyperactivated sperm was observed after the incubation (p<0.05) but after treatment, no significant changes of percentage of hyperactivated sperm(l1.8%) in contrast to significant rise in the percentage of acrosome reacted cells. Correlation analysis failed to show any significant relationship between the percentage of sperm with HA and SPA score. In conclusion, although no direct correlations were found between the results of SPA and HA, hyperactivation of sperm is associated with capacitation and monitoring hyperactivated sperm will be expected as a method of evaluating the functional quality of sperm such as SPA.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.13-13
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• 2001

This study was undertaken to evaluate the effects of catalase using xanthine (X) - xanthine oxidase (XO) system on in vitro maturation and fertilization in pig. When follicular oocytes were cultured in maturation medium with X and/or XO, the maturation rates were not significantly different between in medium with and without catalase despite of different culture periods. However, significantly (P<0.05) higher maturation rates were obrained in culture with X-XO system. The rates of degenerated oocytes were increased with culture periods prolonged, and were significantly (P<0.05) higher in medium without than with catalase at 120 h of culture. On the other hand, the parthenogenetic oocytes were observed with high proportions at 72 h of culture, hut were not different in medium with and without catalase at various times of culture. In another experiment, the frozen-thawed boar spermatozoa treated with X-XO system for in vitro fertilization. The penetration rates were higher in medium with that than without catalase during the in vitro fertilization with, none (P<0.05), XO and X＋XO. On the other hand, when sperm were treated with none, X, XO and X＋XO, lipid peroxidation were higher in medium without that than with catalase. However, the changes in sperm penetration and lipid peroxidation showed opposite patterns. The sperm suspensions were also treated with X and/or XO for assay of sulfhydryl (-SH) group content. Under the above all conditions, sperm-SH group were higher detected In medium with that than without catalase. The activity of sperm binding to zona pellucida was also evaluated through binding to salt-stored porcine oocytes. In control group, sperm binding to zona pellucida were higher than in medium with X, XO and X＋XO groups. No significant differences, however, were observed between medium with and without catalase. In conclusion, the exposure of follicular oocytes and spermatozoa to X-XO system may be caused stimulating in vitro maturation and fertilization in pig. This work was supported by grant No. 2000-1-22200-001-3 from the Basic Research Program of the Korea Science & Engineering Foundation.

• Journal of Animal Science and Technology
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• v.48 no.6
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• pp.785-796
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• 2006

This study was designed to evaluate the changes in sperm motility, viability, HOST(hypo-osmotic swelling test), IVP(in vitro penetration), SCSA(sperm chromatin structure assay) during storage of liquid semen collected from boars with different farrowing rates using AI, and to find the relationship between boar fertility through AI and sperm diagnostic parameters during semen storage. The results of HOST were significantly decreased according to the increasing of in semen storage days and the results of IVP were significantly decreased at 3 days of semen storage (P<0.05). The %Red was significantly different among the >80%, 70󰠏80% and <70% farrowing rate group at semen storage day 6(P<0.05). The correlation coefficients between the %Red and farrowing rate were increased according to the semen storage. In conclusion, these results suggest that the sperm parameters evaluated in these studies may be useful indicators to predict the fertility of AI and evaluate the semen quality in boars.

• Clinical and Experimental Reproductive Medicine
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• v.19 no.2
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• pp.143-152
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• 1992

The present experiments were focussed to modify a short slow-cooling protocol used for freezing of early stage embryo(Testart et al., 1986) and also to apply the modified method for the cryopreservation of hamster oocytes with Zona or without. The protocol was modified by changing the 4-step equilibration into 1-step and the 1-step thawing into 2-step. The oocytes were added in 1.5M PROH and 0.1M Sucrose, seeded at $-7^{\circ}C$, slow cooled($0.3^{\circ}C$/min) to $-30^{\circ}C$ before plunging to $-196^{\circ}C$. The oocytes were thawed at $23-25^{\circ}C$ air(20sec/150sec) and/or 33-35 water(10sec). The survival of the frozen-thawed oocytes was determined by morphologic criteria and their fertilizing ability was also estimated by Sperm Penetration Assay(SPA) system(Chang et al, 1990) using fertile men semen sample. One-step equilibration showed slightly higher survival rate(83.9% vs. 71.0%) and fertilization rate(83.9% vs. 71.0%) compared with four-step(p>0.05). And two-step thawing(air & water exposing) of oocytes frozen after 1-step equilibration showed significantly higher survival rate(96.3%) than one-step thawing at air(85.2%) or water(65.0%) only(p<0.05). Therefore, by the modified method(l-step equilibration & 2-step thawing), Zona-intact(ZI) and Zona-free(ZF) oocytes were frozen and thawed. ZI-oocytes showed significantly higher survival rate(95.4%, 308/323 vs. 67.6%, 240/355) than ZF-oocytes(P<0.01). But the survival of ZF-oocytes was as high as ZI-oocytes in fourteen of twenty-four replicates. ZI-oocytes was also significantly higher fertilization rate($92.4{\pm}8.9%$ vs. $63.7{\pm}18.5%$) and higher mean number of penetrated sperm($6.2{\pm}4.2$ vs. $3.9{\\pm}3.3$) than ZF-oocytes, but not higher than control(fresh oocytes;$99.3{\pm}2.4%$, $8.4{\pm}4.2$)(P<0.001). Conclusively, this modified method will contribute to freeze effectively the hamster oocytes for simplifing of the logical consideration of performing SPA and also to freeze the human and other animal oocytes.

• Journal of Embryo Transfer
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• v.13 no.2
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• pp.89-96
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• 1998

Anti-phospholipid antihodies (aPL) have important roles in various pregnancy complications such as recurrent miscarrige, growth retardation, placental abruption and stillbirth. However, their biological actions on preimplantation development of oocytes are still unclear. In this study, we investigated whether either aPL containing sera or phospholipids could affect in vitro fertilization and development of mouse oocytes. Sera used in this study were collected from three patients and the presence of aPL in the sera was confirmed by enzymatic-linked immunosorbent assay. When mouse oocytes were cultured in a serum-free, Chatot, Ziomek and Bavister (CZB) medium (Experiment 1), addition of aPL-containing sera (10%) to CZB medium did not. significantly (P>0.05) influence sperm penetration of oocytes. However, development to the blastocyst stage was significantly (P<0.05) inhibited by serum addition, and formation of morulae (16-23% vs. 58%) and blastocysts (0-4% vs. 21%) was markedly reduced compared with no addition (Experiment 2). In Experiment 3, pronuclear stage embryos were cultured for 96 h in GZB medium supplemented with 1 $\mu$g /ml phosphatidyl ethanolamine, 1 $\mu$g/ml phosphatidyl inositol or 1 $\mu$g /ml phosphatidyl choline. No increase in embryo development was found after addition of the phospholipids to CZB medium. These results suggest that 1) aPL have an inhibitory role in preimplantation development of mouse embryos, and that 2) the action of aPL may be related to a specific phospholid (s) rather than the tested phospholipids in the present study.