ObjectiveAlthough vitrification has been successfully introduced into oocyte and embryo freezing, the effect of different buffer system as a basic solution for dehydration/rehydration on the survival and developmental rates after vitrification are still unclear. And, sodium salts is main contributor to the solution effect during cooling and rewarming of embryos. Also, it was recently suggested that introducing slush-liquid nitrogen (S-LN2) into vitrification for higher cooling rate improved the integrity and subsequent development of oocytes after thawing. So, the aims of this study were to examine the effect of different basic solutions and cooling rates on the recovery of mouse blastocysts after vitrification.DesignSurvival and apoptosis of mouse blastocysts were analyzed after vitrification according to application of basic solutions with a different buffer system or choline chloride (ChCl) and/or slush-LN2.Materials and methodsMouse blastocysts derived from 2-cell embryos were collected and used for the study. In experiment I, blastocysts were loaded on electron microscopy (EM)-grids and then vitrified with NaCl-based DPBS (group 1), ChCl-based DPBS (group 2), and Mops-buffered solution (group 3), as basic solution. Additionally, we compared open pulled straws as another carrier system (group 4). In experiment II, blastocysts loaded on grids were divided into two groups and then vitrified using slush-LN2 (group 5) or not (group 6). All blastocysts were pre-equilibrated with 1.5M ethylene glycol (EG) for 2.5 min and equilibrated with 5.5M EG and 1.0M sucrose for 20 sec, and then stored in LN2. The thawed blastocysts in all groups were re-hydrated and cultured in Preimplantion-1 (P-1) medium with 10% SSS. Their survival and apoptosis after recovery were examined under microscopic observation and TUNEL assay. Statistical analysis was carried out by use of Chi-square and Student's t-test.ResultsIn experiment I, survival rate of mouse blastocysts in group 2 (78.5% (73/93)) after vitrified-thawed was higher than those in the group 1(61.4% (54/88)), 3 (63.8% (60/94)), and 4 (14.5% (8/55)), respectively (p<0.05). And in group 2, apoptosis in re-expanded blastocysts after thawing was decreased compared to those in others groups. In experiment II, survival and apoptotic rates of blastocysts in vitrified group with high cooling rate (group 5) were not different compared to those in conventional LN2 (group 6).ConclusionThe use of the high concentration of sodium in vitrification media is detrimental to survival of mouse blastocysts. Reducing or eliminating sodium in basic solution for vitrification allows mouse blastocysts to be frozen more efficiently. ObjectiveAlthough vitrification has been successfully introduced into oocyte and embryo freezing, the effect of different buffer system as a basic solution for dehydration/rehydration on the survival and developmental rates after vitrification are still unclear. And, sodium salts is main contributor to the solution effect during cooling and rewarming of embryos. Also, it was recently suggested that introducing slush-liquid nitrogen (S-LN2) into vitrification for higher cooling rate improved the integrity and subsequent development of oocytes after thawing. So, the aims of this study were to examine the effect of different basic solutions and cooling rates on the recovery of mouse blastocysts after vitrification. Although vitrification has been successfully introduced into oocyte and embryo freezing, the effect of different buffer system as a basic solution for dehydration/rehydration on the survival and developmental rates after vitrification are still unclear. And, sodium salts is main contributor to the solution effect during cooling and rewarming of embryos. Also, it was recently suggested that introducing slush-liquid nitrogen (S-LN2) into vitrification for higher cooling rate improved the integrity and subsequent development of oocytes after thawing. So, the aims of this study were to examine the effect of different basic solutions and cooling rates on the recovery of mouse blastocysts after vitrification. DesignSurvival and apoptosis of mouse blastocysts were analyzed after vitrification according to application of basic solutions with a different buffer system or choline chloride (ChCl) and/or slush-LN2. Survival and apoptosis of mouse blastocysts were analyzed after vitrification according to application of basic solutions with a different buffer system or choline chloride (ChCl) and/or slush-LN2. Materials and methodsMouse blastocysts derived from 2-cell embryos were collected and used for the study. In experiment I, blastocysts were loaded on electron microscopy (EM)-grids and then vitrified with NaCl-based DPBS (group 1), ChCl-based DPBS (group 2), and Mops-buffered solution (group 3), as basic solution. Additionally, we compared open pulled straws as another carrier system (group 4). In experiment II, blastocysts loaded on grids were divided into two groups and then vitrified using slush-LN2 (group 5) or not (group 6). All blastocysts were pre-equilibrated with 1.5M ethylene glycol (EG) for 2.5 min and equilibrated with 5.5M EG and 1.0M sucrose for 20 sec, and then stored in LN2. The thawed blastocysts in all groups were re-hydrated and cultured in Preimplantion-1 (P-1) medium with 10% SSS. Their survival and apoptosis after recovery were examined under microscopic observation and TUNEL assay. Statistical analysis was carried out by use of Chi-square and Student's t-test. Mouse blastocysts derived from 2-cell embryos were collected and used for the study. In experiment I, blastocysts were loaded on electron microscopy (EM)-grids and then vitrified with NaCl-based DPBS (group 1), ChCl-based DPBS (group 2), and Mops-buffered solution (group 3), as basic solution. Additionally, we compared open pulled straws as another carrier system (group 4). In experiment II, blastocysts loaded on grids were divided into two groups and then vitrified using slush-LN2 (group 5) or not (group 6). All blastocysts were pre-equilibrated with 1.5M ethylene glycol (EG) for 2.5 min and equilibrated with 5.5M EG and 1.0M sucrose for 20 sec, and then stored in LN2. The thawed blastocysts in all groups were re-hydrated and cultured in Preimplantion-1 (P-1) medium with 10% SSS. Their survival and apoptosis after recovery were examined under microscopic observation and TUNEL assay. Statistical analysis was carried out by use of Chi-square and Student's t-test. ResultsIn experiment I, survival rate of mouse blastocysts in group 2 (78.5% (73/93)) after vitrified-thawed was higher than those in the group 1(61.4% (54/88)), 3 (63.8% (60/94)), and 4 (14.5% (8/55)), respectively (p<0.05). And in group 2, apoptosis in re-expanded blastocysts after thawing was decreased compared to those in others groups. In experiment II, survival and apoptotic rates of blastocysts in vitrified group with high cooling rate (group 5) were not different compared to those in conventional LN2 (group 6). In experiment I, survival rate of mouse blastocysts in group 2 (78.5% (73/93)) after vitrified-thawed was higher than those in the group 1(61.4% (54/88)), 3 (63.8% (60/94)), and 4 (14.5% (8/55)), respectively (p<0.05). And in group 2, apoptosis in re-expanded blastocysts after thawing was decreased compared to those in others groups. In experiment II, survival and apoptotic rates of blastocysts in vitrified group with high cooling rate (group 5) were not different compared to those in conventional LN2 (group 6). ConclusionThe use of the high concentration of sodium in vitrification media is detrimental to survival of mouse blastocysts. Reducing or eliminating sodium in basic solution for vitrification allows mouse blastocysts to be frozen more efficiently. The use of the high concentration of sodium in vitrification media is detrimental to survival of mouse blastocysts. Reducing or eliminating sodium in basic solution for vitrification allows mouse blastocysts to be frozen more efficiently.

With increasing use of blastocyst culture as a routine protocol in human clinical IVF, cryopreservation of excessive human blastocysts has become an important part of infertility treatment. Vitrification technique for cryopreservation of human blastocysts has been developed and applied in clinical settings. In our previous study, the vitrification protocol showed promising results for both oocytes and blastocysts. In this study we have modified our original method to improve efficiency of vitrification for blastocysts. We report the survival, pregnancy and implantation rats of vitrified blastocysts. Clinical trial of vitrification for human blastocysts After embryo transfers in IVF cycles, surplus embryos that developed to blastocyst stage were first equilibrated in cryoprotectant solution containing 1.5 mol/L ethylene glycol (EG) for 5min at 37°C followed by introducing vitrification solution containing 5.5 mol/L EG + 1.0 mol/L sucrose for 20sec at room temperature. Blastocysts were then placed on the electron microscope (EM) grids and immediately plunged to the liquid nitrogen (LN2) directly. A total of 407 blastocysts from 127 egg retrieval cycles were cryopreserved using vitrification technique. For thawing, the EM grid holding the embryos was taken out of the liquid nitrogen container, then immediately transferred to organ culture dish sequentially containing 1.0ml of thawing solutions which is 1.0, 0.5, 0.25, 0.125, 0 mol/L of sucrose at intervals of 2.5 min at 37°C. Vitrified blastocysts were thawed around 4PM the day before embryo transfer, and cultured in G2.2 or G2.3 medium overnight. Re-expanded blastocysts were judged for survival following morning, then transferred to the patients in the afternoon. A total of 89 blastocysts obtained from 19 cycles of oocyte collection were vitrified. Of which, 77 blastocysts were thawed, and 53 embryos (69%) survived. A total of 47 blastocysts were transferred to 19 patients in 20 embryo transfer cycles. The mean number of blastocysts transferred per cycle was 2.4 ± 0.9. Of the 47 embryos transferred, 24(51%) were determined to be implanted about 4 weeks after embryo transfer by ultrasound exam. Of 20 transfers, 16 resulted in clinical pregnancy; the pregnancy rate was 80% per warming cycle and 84% per vitrification cycle. There were no cancelled embryo transfers after thawing. Ten patients delivered 13 babies (3 sets of twins and 7 of singleton deliveries) between November 2003 and March 2005. Two patients are on going. Two patients ended miscarriage and two patients performed therapeutic abortion after amniocentesis. Significantly high pregnancy and implantation rates were observed with vitrified blastocysts. This indicates that the vitrification technique would be a highly efficient method in clinical application of cryopreservation of supernumerary blastocysts in ART program.

Oocytes meiotic cell cycle progression is thought to be orchestrated by protein phosphorylation-dephosphorylation cascades. MPF (maturation- or metaphase-promoting factor) and MAP (microtubule-associated or mitogen-activated protein) kinase are two key molecules involved in this process. MPF plays a pivotal role in driving the oocyte meiotic cell cycle progression and MAP kinase plays an important role in controlling chromatin and microtubule behavior. However, the role of MAP kinase phosphorylation during maturation, fertilization, and early embryonic development has not been studied in humans. We investigated change of MAP kinase phosphorylation pattern of oocytes during in vitro maturation and early embryonic development in humans. Experimental study utilizing human oocytes and embryos. Human oocytes and embryos were collected from consented stimulated IVF patients and divided into 4 groups. Group 1 : immature oocytes (GV stage oocytes), Group 2 : unfertilized mature oocyte (MII stage oocytes), Group 3 : fertilized embryos (tripronuclei embryos, 3PN), Group 4 : cleavaged embryos (2-cell and 8-cell stage embryos). Immature oocytes were retrieved 34–36 hours after hCG administration and collected for analysis. Twenty hours after insemination, the oocytes were examined for the presence of pronuclei and then collected the unfertilized oocytes. The tripronucleated embryos were obtained 18–24 hours after retrieval. Two-cell and 8-cell embryos were cultured in P-1 medium and collected after 2 and 3 days. Four groups used for detection of MAP kinase phosphorylation assay. MAP kinase phosphorylation was revealed by western immunoblotting. Proteins extracted from oocytes and embryos, electrophoresised, transferred to the nitrocellulose membrane, the membrane was incubated with primary anti-phosphorylated MAP kinase antibody followed by biotynylated second antibody. After incubation for 1 min in the ECL detection solution, we exposed the membrane to autoradiography film for detection. This experiment was repeated three times. MAP kinase was not phosphorylated at the GV stage oocytes but mature oocytes contained two forms of MAP kinase, ERK 1, ERK 2 that become phosphorylated. After insemination and culture, MAP kinase phosphorylation of 3PN embryos, 2-cell embryos was still present in a phosphorylated form. However, the MAP kinase was dephosphorylated at the 8-cell stage embryos. This study suggests that phosphorylation/dephosphorylation event of MAP kinase occur stage-specific during oocyte maturation and early embryonic development in humans. Further studies of the mechanism of MAP kinase role are necessary for ART program.

Objective: Most of embryologists on human infertility clinics have faced the demand of cryopreservation for zona pellucia (ZP)-opened embryos obtained from microsurgical manipulation according to rapid developing human ART program. And now, vitrification has been widely introduced to cryopreservation of mammalian oocytes and embryos. The aim of this study was to evaluate the effect of vitrification procedure on development of the ZP-opened or a blastomere-biopsied embryo, and to analyze the effect of the microsurgical removal of damaged blastomere before or after vitrification. Design: Intact survival and development of mouse embryos were analyzed after vitrification following microsurgical manipulation. Materials and Methods: Late 2-cell embryos were collected from superovulated ICR mouse and cultured up to 4-cell in vitro. First of all, embryos with intact ZP were pre-equilibrated in DPBS with 1.5M ethylene glycol (EG) for 2.5 min. Embryos were then vitrified with DPBS with 5.5M EG and 1.0M sucrose for 20 sec. Those were loaded onto electron microscope copper grids or into pulled straws to store in liquid nitrogen. Stored embryos were thawed by a five-step method, and their survival and development were analyzed. Second, embryos with ZP-opened or a blastomere-biopsied were loaded into pulled straws, and then vitrified and thawed. Third, one of blastomeres was destroyed after opening of ZP, and then removed or remained before or after vitrification using pulled straws. Their survival and development were examined after thawing. Results: After vitrified-thawed, survival and development up to blastocyst of embryos with ZP-intact loaded in pulled straws were not different when compared to one on grids (87.5 vs. 85.5% and 83.4 vs. 80.3%). Intact survival and blastocyst formation rate of embryos with ZP-opened or a blastomere-biopsied were 84.6% vs. 84% and 74.4% vs. 77.4%, respectively. And, intact recovery rate of embryos removed the damaged blastomere (DB) before vitrification was not different compared to that of embryos remained the one. But, blastocyst formation rate was higher in the DB-removed group than in DB-remained group (70.7% (46/65) vs. 55.1% (38/69), p<0.05). Also, micromanipulated embryos after vitrification showed similar pattern of development to those before vitrification. Conclusion: Vitrification is very simple and effective method for cryopreservation of normal and micromanipulated embryos. Also, removing of DB before or after vitrification should improve embryonic development. These results would provide a useful tool for cryopreservation of biopsied embryos obtained from preimplantation genetic diagnosis program or reconstructed embryos derived from nuclear transfer. Supported by: a grant from the INTERDISCIPLINARY RESEARCH PROGRAM of the KOSEF (1999-2-205-002-5).

Objective: There were controversial reports on result of cryopreservation using the EG or PROH as a cryoprotectant. Recently, EG has been used in mammals as a cryoprotectant for the cryopreservation of oocytes and embryos because of low toxicity and rapid permeation into the cells. Therefore, this study was conducted to compare the effects of EG and PROH at the cleavage stage embryos in mouse and human.Design: In experiment 1, mouse intact 6, 8-cell embryos were collected from ICR mouse and divided into two groups: group1; EG, n = 347, group 2; PROH, n = 319. In experiment 2, human cleavage stage embryos derived from tripronucleate embryos were obtained from patients undergoing IVF and divided into two groups: group 1; cleavage embryos with EG, n =39; group 2; cleavage embryos with PROH, n = 36. The survival rates included embryos with at least intact 1 blastomere as a survival rate. The developmental rates of frozen-thawed embryos with EG and PROH in mouse and human were examined after culture.Materials/Methods: Mouse and human embryos were cryopreserved with 1.5 mol/l EG + 0.2mol/l sucrose and 1.5 mol/l PROH + 0.2mol/l sucrose using the slow freezing method. The embryos survived after thawing in all groups were cultured in Preimplantion-1 (P-1) medium with 10% SSS. The rate of survival and the number of embryos developed to blastocyst stages were examined under a stereomicroscope after culture. Statistical analysis was carried out by use of a X2 test.Results: In experiment1, the rates of survival were 80.4% (279) in group 1 and 79.3% (253) in group 2. The developmental rates to blastocyst were 44.1%(123) in group 1 and 45.1%(114) in group 2, respectively. In experiment 2, rates of survival were 94.9%(37) in group1 and 97.2(35) in group2.Conclusions: Cryopreservation of mouse and human embryos at cleavage stage by using EG or PROH had no statistical difference in the survival rate and developmental rate to blastocyst. Further study is requested for correlation between the penetration time and the concentration of cryoprotectant. Objective: There were controversial reports on result of cryopreservation using the EG or PROH as a cryoprotectant. Recently, EG has been used in mammals as a cryoprotectant for the cryopreservation of oocytes and embryos because of low toxicity and rapid permeation into the cells. Therefore, this study was conducted to compare the effects of EG and PROH at the cleavage stage embryos in mouse and human. Design: In experiment 1, mouse intact 6, 8-cell embryos were collected from ICR mouse and divided into two groups: group1; EG, n = 347, group 2; PROH, n = 319. In experiment 2, human cleavage stage embryos derived from tripronucleate embryos were obtained from patients undergoing IVF and divided into two groups: group 1; cleavage embryos with EG, n =39; group 2; cleavage embryos with PROH, n = 36. The survival rates included embryos with at least intact 1 blastomere as a survival rate. The developmental rates of frozen-thawed embryos with EG and PROH in mouse and human were examined after culture. Materials/Methods: Mouse and human embryos were cryopreserved with 1.5 mol/l EG + 0.2mol/l sucrose and 1.5 mol/l PROH + 0.2mol/l sucrose using the slow freezing method. The embryos survived after thawing in all groups were cultured in Preimplantion-1 (P-1) medium with 10% SSS. The rate of survival and the number of embryos developed to blastocyst stages were examined under a stereomicroscope after culture. Statistical analysis was carried out by use of a X2 test. Results: In experiment1, the rates of survival were 80.4% (279) in group 1 and 79.3% (253) in group 2. The developmental rates to blastocyst were 44.1%(123) in group 1 and 45.1%(114) in group 2, respectively. In experiment 2, rates of survival were 94.9%(37) in group1 and 97.2(35) in group2. Conclusions: Cryopreservation of mouse and human embryos at cleavage stage by using EG or PROH had no statistical difference in the survival rate and developmental rate to blastocyst. Further study is requested for correlation between the penetration time and the concentration of cryoprotectant.

Objective: The in vitro maturation of immature oocytes retrieved from unstimulated ovaries has been interested in assisted reproductive technology. Oocyte maturation, characterized by germinal vesicle (GV) break-down, formation of first meiotic spindle, extrusion of the first polar body, and arrest in metaphase of the second meiotic division. However, numerous anomalies in the nuclear and cytoplasmic maturation were induced during in vitro maturation. This study was conducted to analyze spindle and chromosome configurations of in vitro maturation failure metaphase I (MI) arrested oocytes collected from PCO patients.Design: Arrested oocytes at the MI stage were collected after 48h culture. The chromosome and spindle configuration were observed by immunostaining.Materials/Methods: Immature oocytes were aspirated using ultrasound guided aspiration method with specially designed injection needle from unstimulated PCO patients. Oocytes with a germinal vesicle were cultured in maturation medium for 48h. Oocytes were removed cumulus cells with hyaluronidase and mechanical pipetting after culture. Oocytes without an intact GV and 1st polar body were defined as metaphase I. Oocytes were used for immunostaining by using monoclonal anti-tubulin antibody and TRITC-conjugated second antibody.Results: Absent (85%) or disorganized (15%) spindle and dispersed chromosomes (100%) were observed in all analyzed oocytes.Conclusions: This result suggested that the MI arrested oocytes was related to the MI spindle, with absence of microtubules and dispersion of the chromosomes. Objective: The in vitro maturation of immature oocytes retrieved from unstimulated ovaries has been interested in assisted reproductive technology. Oocyte maturation, characterized by germinal vesicle (GV) break-down, formation of first meiotic spindle, extrusion of the first polar body, and arrest in metaphase of the second meiotic division. However, numerous anomalies in the nuclear and cytoplasmic maturation were induced during in vitro maturation. This study was conducted to analyze spindle and chromosome configurations of in vitro maturation failure metaphase I (MI) arrested oocytes collected from PCO patients. Design: Arrested oocytes at the MI stage were collected after 48h culture. The chromosome and spindle configuration were observed by immunostaining. Materials/Methods: Immature oocytes were aspirated using ultrasound guided aspiration method with specially designed injection needle from unstimulated PCO patients. Oocytes with a germinal vesicle were cultured in maturation medium for 48h. Oocytes were removed cumulus cells with hyaluronidase and mechanical pipetting after culture. Oocytes without an intact GV and 1st polar body were defined as metaphase I. Oocytes were used for immunostaining by using monoclonal anti-tubulin antibody and TRITC-conjugated second antibody. Results: Absent (85%) or disorganized (15%) spindle and dispersed chromosomes (100%) were observed in all analyzed oocytes. Conclusions: This result suggested that the MI arrested oocytes was related to the MI spindle, with absence of microtubules and dispersion of the chromosomes.

Objective: Insulin like growth factors (IGFs) in the intraovarian autocrine control mechanism may serve as a central signal, and the granulose cell is their site of production and action. The role of intraovarian IGF, especially in correlation to oocyte maturity has not been fully understood in human. This study was conducted to examine the correlation between the ovarian IGF I and II and IR (type 1 receptor) and the maturity of oocytes from various type of follicle and patient.Design: The comparison of the pattern of IGFI, IGFII and IGFIR mRNA expression: 1. according to the oocyte maturity in individual follicle (stimulated IVF cycle: cumulus cell(CC) and granulose cell(GC)) 2. according to the patients (stimulated IVF cycle, natural cycle and nonstimulated polycystic ovarian syndrome (PCOS) cycle) 3. according to oocyte maturity (MII and GV oocytes).Materials/Methods: Expression of mRNA of IGFI, IGF II and IGF IR in oocytes, purified cumulus cells and granulose cells was explored by a semiquantitative reverse transcription polymerase chain reaction (RT-PCR). The band intensity of PCR products was measured by using the imaging Densitometer.Results: 1. The expression pattern of IGF I, IGF II and IGF IR according to maturity presents different intensity in individual follicle from the same patients 2. The expression patterns of IGF I, IGF II and IGF IR in GC and CC obtained from the same follcle show different intensity, respectively 3. PCOS patients from the nonstimulated cycle presents different intensity and patterns of IGF I, IGF II and IGF IR compared to stimulated cycle. 4. MII (NF oocyes) and GV oocytes presented different density in all type. 5. There are various alternatively spliced form and different band intensity in IGF I and IGF II according to their sources.Conclusions: The results presented that human ovarian IGF and their receptor was produced differently according to maturity. And these pattern may have different role in autocrine and paracrine fashion in the human follicle. Various spliced form and intensity according to the source of patients should be confirmed and considered for clinical application. And, further studies are needed to understand the physiological role of IGF system correlated with our results. Objective: Insulin like growth factors (IGFs) in the intraovarian autocrine control mechanism may serve as a central signal, and the granulose cell is their site of production and action. The role of intraovarian IGF, especially in correlation to oocyte maturity has not been fully understood in human. This study was conducted to examine the correlation between the ovarian IGF I and II and IR (type 1 receptor) and the maturity of oocytes from various type of follicle and patient. Design: The comparison of the pattern of IGFI, IGFII and IGFIR mRNA expression: 1. according to the oocyte maturity in individual follicle (stimulated IVF cycle: cumulus cell(CC) and granulose cell(GC)) 2. according to the patients (stimulated IVF cycle, natural cycle and nonstimulated polycystic ovarian syndrome (PCOS) cycle) 3. according to oocyte maturity (MII and GV oocytes). Materials/Methods: Expression of mRNA of IGFI, IGF II and IGF IR in oocytes, purified cumulus cells and granulose cells was explored by a semiquantitative reverse transcription polymerase chain reaction (RT-PCR). The band intensity of PCR products was measured by using the imaging Densitometer. Results: 1. The expression pattern of IGF I, IGF II and IGF IR according to maturity presents different intensity in individual follicle from the same patients 2. The expression patterns of IGF I, IGF II and IGF IR in GC and CC obtained from the same follcle show different intensity, respectively 3. PCOS patients from the nonstimulated cycle presents different intensity and patterns of IGF I, IGF II and IGF IR compared to stimulated cycle. 4. MII (NF oocyes) and GV oocytes presented different density in all type. 5. There are various alternatively spliced form and different band intensity in IGF I and IGF II according to their sources. Conclusions: The results presented that human ovarian IGF and their receptor was produced differently according to maturity. And these pattern may have different role in autocrine and paracrine fashion in the human follicle. Various spliced form and intensity according to the source of patients should be confirmed and considered for clinical application. And, further studies are needed to understand the physiological role of IGF system correlated with our results.

Objective: The stimulatory effects of EGF and FSH on oocyte maturation have been reported in various mammalian species. And some reports presented FSH enhanced the effect of EGF on oocyte maturation. However, the mechanism of the interaction between EGF and FSH on nuclear maturation of mammalian oocytes is not fully understood. Therefore, we observed the effect of EGF and FSH on nuclear maturation during in vitro maturation of mouse oocytes. Also, we examined the interaction between EGF and FSH on nuclear maturation of mouse oocytes using the EGFR inhibitor. Design: The maturation rates of cumulus oocyte complex (COC) were examined in medium supplemented with or without EGF, FSH or EGFR inhibitor. Materials/Methods: Germinal vesicle (GV) stage oocytes were obtained from 3–4weeks PMSG primed BCF1 hybrid mice and cultured in TCM-199 medium with 0.4%PVP supplemented with/without EGF (1ng/ml), FSH (1ng/ml) or EGFR specific tyrosine kinase inhibitors: Tyrphostin AG 1478 (500nM) for 14–15hr. Rapid staining method were used for the assessment of nuclear maturation. Results: The nuclear maturation rates (MII %) of EGF, FSH and FSH/EGF combination group (83.3% and 88.1% and 96.4%) were significantly higher than that of control group (50.9%; p <0.05).) The effect on nuclear maturation of EGF was significantly blocked by EGFR inhibitor (tyrphostin AG 1478), however the effect of FSH was not blocked by tyrphostin AG1478. The results showed the effects of EGF, FSH and EGFR inhibitor on nuclear maturation of cumulus-intact mouse oocytes. Conclusions: The results confirmed that EGF had a physiological role in the regulation of mouse oocyte maturation. And further study is needed to search the signaling pathway between FSH and EGF associated with maturation. Table TableMaturation rate of mouse oocytes with EGF, FSH, and EGFR inhibitorEGFEGFR InhibitorFSHNo. of oocytes (Examined)No. (%) of oocytes at the stage of GVNo. (%) of oocytes at the stage of GVBDNo. (%) of oocytes at the stage of MII−−−11216 (14.3)39 (34.8)57 (50.9)+−−1262 (1.6)19 (15.1)105 (83.3)∗Values with different superscripts within each column are significantly different (P < 0.05)−−+126-15 (11.9)111 (88.1)∗Values with different superscripts within each column are significantly different (P < 0.05)+−+112-4 (3.6)108 (96.4)∗Values with different superscripts within each column are significantly different (P < 0.05)++−989 (9.2)45 (45.9)44 (44.9)−++105-15 (14.3)90 (85.7)∗Values with different superscripts within each column are significantly different (P < 0.05)+++110-18 (16.4)92 (83.6)∗Values with different superscripts within each column are significantly different (P < 0.05)∗ Values with different superscripts within each column are significantly different (P < 0.05) Open table in a new tab

Objective: Intracytoplasmic sperm injection (ICSI) is now widely used for male factor treatment in human IVF/ET program. Although poor sperm parameters including count, sperm motility and morphology has been reported not important in ICSI program, sperm motility is a prerequisite indicator to find viable spermatozoa in the treatment of severe asthenozoospermia. Pentoxifylline (PF) and other methylxanthines are known to influence sperm metabolism and motility. Because of its enhancing effect on sperm motility and its role in facilitating the acrosome reaction, the agent is now commonly used in human ICSI program to easily find motile spermatozoa. It has been postulated that PF, being a phosphodiesterase inhibitor, increase sperm kinetic parameters and the number of spermatozoa exhibiting hyperactivated motility by raising the intracellular content of cAMP, a molecule involved in the generation of sperm energy. The aim of study was to evaluate the effect of PF on the conventional ICSI program undergone in severe asthenozoospermia.Design: A study was undertaken at the Infertility Medical Center, CHA General Hospital comparing the clinical results of ICSI between PF treated and PF untreated group.Materials/Methods: Total 348 cycles of ICSI program were undertaken at CHA General Hospital from January, 1996 to September, 2000, were divided into two groups: injected with PF treated sperm (PFT, 204 cycles) or non PF treated sperm (NPFT, 144 cycles) and the clinical results of PFT group were compared with those of NPFT.Results: PF-treatment on sperm increased their motility of normozoospermia and severe asthenozoospermia. Fertilization rate of PFT group was higher than those of ICSI programs undertaken using sperm of NPFT (69.7% vs. 62.9%, p <0.01). And, embryo transfer and pregnancy rates of PFT were slightly higher than those of NPFT (93.1%, 44.2% vs. 90.3%, 36.2%).Conclusions: These results showed that treatment of PF has a beneficial role on selection of viable spermatozoa in severe asthenozoospermia. Objective: Intracytoplasmic sperm injection (ICSI) is now widely used for male factor treatment in human IVF/ET program. Although poor sperm parameters including count, sperm motility and morphology has been reported not important in ICSI program, sperm motility is a prerequisite indicator to find viable spermatozoa in the treatment of severe asthenozoospermia. Pentoxifylline (PF) and other methylxanthines are known to influence sperm metabolism and motility. Because of its enhancing effect on sperm motility and its role in facilitating the acrosome reaction, the agent is now commonly used in human ICSI program to easily find motile spermatozoa. It has been postulated that PF, being a phosphodiesterase inhibitor, increase sperm kinetic parameters and the number of spermatozoa exhibiting hyperactivated motility by raising the intracellular content of cAMP, a molecule involved in the generation of sperm energy. The aim of study was to evaluate the effect of PF on the conventional ICSI program undergone in severe asthenozoospermia. Design: A study was undertaken at the Infertility Medical Center, CHA General Hospital comparing the clinical results of ICSI between PF treated and PF untreated group. Materials/Methods: Total 348 cycles of ICSI program were undertaken at CHA General Hospital from January, 1996 to September, 2000, were divided into two groups: injected with PF treated sperm (PFT, 204 cycles) or non PF treated sperm (NPFT, 144 cycles) and the clinical results of PFT group were compared with those of NPFT. Results: PF-treatment on sperm increased their motility of normozoospermia and severe asthenozoospermia. Fertilization rate of PFT group was higher than those of ICSI programs undertaken using sperm of NPFT (69.7% vs. 62.9%, p <0.01). And, embryo transfer and pregnancy rates of PFT were slightly higher than those of NPFT (93.1%, 44.2% vs. 90.3%, 36.2%). Conclusions: These results showed that treatment of PF has a beneficial role on selection of viable spermatozoa in severe asthenozoospermia.