En 1680-6433 2008-2177 jalali 1387 8 12 gregorian 2008 11 2 6 4 online 1 fulltext

En 1680-6433 2008-2177 Oocyte activation: The impact of calcium signals on fertilization Fertilization is triggered by changes in intracellular calcium concentration. In mammals, these transients in ooplasmic calcium concentration take the form of repetitive spikes, so called calcium oscillations (Ca2+-oscillations). These oscillations are important for relieve of meiotic arrest and to induce all the other events of oocyte activation. Although a surface mediated way of oocyte activation has been proposed, there is now substantial evidence to suggest that the sperm cell induces these Ca2+-oscillations by introducing a sperm specific phospholipase C, PLCζ, in the ooplasm. Ca2+-oscillations are also observed after intracytoplasmic sperm injection (ICSI), a successful technique in human assisted reproduction. In the rare cases that no fertilization is observed following ICSI, this may be due to a deficiency in PLCζ. However, artificial activating the oocytes after ICSI by increasing the calcium concentration can restore fertilization rates in these cases and support further development, as evidenced by successful pregnancies. Further evaluation of the current protocols for assisted oocyte activation is appropriate and investigation of the future application of PLCζ is warranted. Fertilization is triggered by changes in intracellular calcium concentration. In mammals, these transients in ooplasmic calcium concentration take the form of repetitive spikes, so called calcium oscillations (Ca2+-oscillations). These oscillations are important for relieve of meiotic arrest and to induce all the other events of oocyte activation. Although a surface mediated way of oocyte activation has been proposed, there is now substantial evidence to suggest that the sperm cell induces these Ca2+-oscillations by introducing a sperm specific phospholipase C, PLCζ, in the ooplasm. Ca2+-oscillations are also observed after intracytoplasmic sperm injection (ICSI), a successful technique in human assisted reproduction. In the rare cases that no fertilization is observed following ICSI, this may be due to a deficiency in PLCζ. However, artificial activating the oocytes after ICSI by increasing the calcium concentration can restore fertilization rates in these cases and support further development, as evidenced by successful pregnancies. Further evaluation of the current protocols for assisted oocyte activation is appropriate and investigation of the future application of PLCζ is warranted. Calcium, Oocyte activation, PLCζ, ICSI. Calcium, Oocyte activation, PLCζ, ICSI. 171 174 Elke Heytens Elke Heytens elke.heytens@ugent.be No Reza Soleimani Reza Soleimani No Petra De Sutter Petra De Sutter No

En 1680-6433 2008-2177 Expression of c-Jun in human granulosa cells from patients participating in in vitro fertilization programs Background: The mitogen-activated protein kinase (MAPK) pathway is one of the major signaling pathways that transmit intracellular signals initiated by extracellular stimuli to the nucleus. The stress-activated protein kinase (SAPK)/c-Jun NH2-terminal kinase is a subfamily of MAP kinases implicated in cytokine and stress responses. Objective: In this study, we have examined total and phosphorylated c-Jun in the mural and cumulus granulosa cells, and investigated also whether c-Jun can be responsible for the difference in the expression of apoptosis between mural and cumulus regions. Materials and Methods: A total of 14 consecutive couples participating in IVF program were investigated. Aspirated follicular fluid was transferred into tissue culture dishes and oocyte-cumulus cells complexes were isolated. The cells were centrifuge and fixed with Bouin’s solution and then were put on a glass slide. After fixation, the slides were stained by immunocytochemistry method. The incidence of apoptotic granulosa cells was examined by a fluorescence microscope. Results: The incidence of apoptotic granulosa cells was 1.27 ± 0.12 in the mural region and 0.38 ± 0.07 in the cumulus regions. All mural and cumulus cells expressed total c-Jun in 7 patients while phosphorylated c-Jun was also expressed in all cells of the other 7 patients. There was no difference between apoptotic and nonapoptotic cells in the expression of total and phosphorylated c-Jun. Conclusion: C-Jun may not be responsible for apoptotic effect on mural and cumulus cells. Background: The mitogen-activated protein kinase (MAPK) pathway is one of the major signaling pathways that transmit intracellular signals initiated by extracellular stimuli to the nucleus. The stress-activated protein kinase (SAPK)/c-Jun NH2-terminal kinase is a subfamily of MAP kinases implicated in cytokine and stress responses. Objective: In this study, we have examined total and phosphorylated c-Jun in the mural and cumulus granulosa cells, and investigated also whether c-Jun can be responsible for the difference in the expression of apoptosis between mural and cumulus regions. Materials and Methods: A total of 14 consecutive couples participating in IVF program were investigated. Aspirated follicular fluid was transferred into tissue culture dishes and oocyte-cumulus cells complexes were isolated. The cells were centrifuge and fixed with Bouin’s solution and then were put on a glass slide. After fixation, the slides were stained by immunocytochemistry method. The incidence of apoptotic granulosa cells was examined by a fluorescence microscope. Results: The incidence of apoptotic granulosa cells was 1.27 ± 0.12 in the mural region and 0.38 ± 0.07 in the cumulus regions. All mural and cumulus cells expressed total c-Jun in 7 patients while phosphorylated c-Jun was also expressed in all cells of the other 7 patients. There was no difference between apoptotic and nonapoptotic cells in the expression of total and phosphorylated c-Jun. Conclusion: C-Jun may not be responsible for apoptotic effect on mural and cumulus cells. Apoptosis, C-Jun, Granulosa cells, In vitro fertilization, Human. Apoptosis, C-Jun, Granulosa cells, In vitro fertilization, Human. 175 179 Hassan Hassani Bafrani Hassan Hassani Bafrani hasanhasani@yahoo.com No

En 1680-6433 2008-2177 Fusion and development of 2-cell bovine embryos to tetraploid blastocyst with different voltages and durations Background: The values of embryonic stem cell and cloning are evident. Production of clone from embryonic stem cells can be achieved by introduction of stem cell into a tetraploid blastocyst. Tetraploid blastocyst can be produced in vitro by electrofusion of 2-cell embryos. Objective: The aim of this study was to assess the effect of different voltages and durations on fusion rate of bovine 2-cell embryos and their subsequent development in vitro. Material and Methods: The in vitro produced bovine 2-cell embryos were categorized into 3 groups: (1) fused group (FG); 2-cell embryos fused by exposure to different voltages (0.5, 0.75, 1, 1.25 and 1.5 kV/cm) and durations (20, 40, 60, 80 and 100 μs), (2) exposed control group (ECG); 2-cell embryos exposed to different voltages and durations but remained unfused and (3) unexposed control group (UCG); embryos cultured without exposure to any voltage. The embryos from each group were cultured and fusion, cleavage and developmental rates were compared in each group. Results: The results show that increased voltage, increases the fusion rate up to 88% for 1.5 kV/cm; however, the rate of cleavage and blastocyst formation decreases significantly to 18% and 10% respectively (p<0.05). Increased duration does not significantly increase fusion rate, however, in high voltage, increased duration decreases cleavage rate and blastocyst formation rate. Blastocyst formation rate in UCG showed a better development (32%) compared to FG (20%) or ECG (22.5%) (p<0.05). Conclusion: It can be concluded that for optimal fusion, cleavage and development, one pulse of 0.75 kV/cm for 60μs should be applied. Background: The values of embryonic stem cell and cloning are evident. Production of clone from embryonic stem cells can be achieved by introduction of stem cell into a tetraploid blastocyst. Tetraploid blastocyst can be produced in vitro by electrofusion of 2-cell embryos. Objective: The aim of this study was to assess the effect of different voltages and durations on fusion rate of bovine 2-cell embryos and their subsequent development in vitro. Material and Methods: The in vitro produced bovine 2-cell embryos were categorized into 3 groups: (1) fused group (FG); 2-cell embryos fused by exposure to different voltages (0.5, 0.75, 1, 1.25 and 1.5 kV/cm) and durations (20, 40, 60, 80 and 100 μs), (2) exposed control group (ECG); 2-cell embryos exposed to different voltages and durations but remained unfused and (3) unexposed control group (UCG); embryos cultured without exposure to any voltage. The embryos from each group were cultured and fusion, cleavage and developmental rates were compared in each group. Results: The results show that increased voltage, increases the fusion rate up to 88% for 1.5 kV/cm; however, the rate of cleavage and blastocyst formation decreases significantly to 18% and 10% respectively (p<0.05). Increased duration does not significantly increase fusion rate, however, in high voltage, increased duration decreases cleavage rate and blastocyst formation rate. Blastocyst formation rate in UCG showed a better development (32%) compared to FG (20%) or ECG (22.5%) (p<0.05). Conclusion: It can be concluded that for optimal fusion, cleavage and development, one pulse of 0.75 kV/cm for 60μs should be applied. mh.nasr-esfahani@royaninstitute.org mh.nasr-esfahani@royaninstitute.org 181 186 Mohamad Reza Darabi Mohamad Reza Darabi - No Mohamad Hosein Nasr-Esfahani Mohamad Hosein Nasr-Esfahani mh.nasr-esfahani@royaninstitute.org No

En 1680-6433 2008-2177 The effects of vitrification on spindle apparatus of in vitro matured germinal vesicle in mice Background: Routine oocytes cryopreservation remained an elusive technique in the wide ranges of available assisted reproductive technologies. The microtubules of oocytes are vulnerable to cryoprotectants and thermal change during cryopreservation. Objective: The effects of a vitrification protocol were investigated on the spindle and chromosome configurations of mice oocytes cryopreserved at the germinal vesicle stage. Materials and Methods: Germinal vesicle with cumulus cells were transferred to vitrification solution which was composed of 30% (v/v) ethylene glycol, 18% Ficoll-70 and 0.3 M Sucrose either by single step or in step-wise way. Following vitrification and in vitro maturation (MII), the matured oocytes were immonostained for meiotic spindles and chromosomes, before visualization using fluorescent microscopy. Results: A statistically significant increase was observed in the survival and maturation rate in step-wise vitrification (88.96% and 71.23% respectively) compared to single step vitrification (70.6% and 62.42% respectively) (p<0.05). Normal spindle morphology after vitrification-thawing in step-wise vitrification group (77.26%) was higher than single step vitrification group (64.24%) but lower than control group (94.75%) (p<0.05). Conclusion: The results suggest that vitrification with step-wise procedure on mice germinal vesicle oocytes has positive effects on survival and maturation rate and normal spindle configuration compare with single step vitrification procedure. Background: Routine oocytes cryopreservation remained an elusive technique in the wide ranges of available assisted reproductive technologies. The microtubules of oocytes are vulnerable to cryoprotectants and thermal change during cryopreservation. Objective: The effects of a vitrification protocol were investigated on the spindle and chromosome configurations of mice oocytes cryopreserved at the germinal vesicle stage. Materials and Methods: Germinal vesicle with cumulus cells were transferred to vitrification solution which was composed of 30% (v/v) ethylene glycol, 18% Ficoll-70 and 0.3 M Sucrose either by single step or in step-wise way. Following vitrification and in vitro maturation (MII), the matured oocytes were immonostained for meiotic spindles and chromosomes, before visualization using fluorescent microscopy. Results: A statistically significant increase was observed in the survival and maturation rate in step-wise vitrification (88.96% and 71.23% respectively) compared to single step vitrification (70.6% and 62.42% respectively) (p<0.05). Normal spindle morphology after vitrification-thawing in step-wise vitrification group (77.26%) was higher than single step vitrification group (64.24%) but lower than control group (94.75%) (p<0.05). Conclusion: The results suggest that vitrification with step-wise procedure on mice germinal vesicle oocytes has positive effects on survival and maturation rate and normal spindle configuration compare with single step vitrification procedure. Vitrification, Germinal vesicle oocyte, Mice, Microtubule. Vitrification, Germinal vesicle oocyte, Mice, Microtubule. 209 215 Reza Mahmoudi Reza Mahmoudi No iraj Amiri Iraj Amiri _ No iraj Ragardi Kashani Iraj Ragardi Kashani _ No Mehdi Abbasi Mehdi Abbasi _ No Farid Aboulhasani Farid Aboulhasani _ No

En 1680-6433 2008-2177 Antispasmodic effect of Physalis alkekengi fruit extract Background: Studies have shown that Physalis alkekengi reduces implantation and induces antifertility in rat. In Iranian traditional medicine it is believed that this plant has abortifacient and antifertility activities. Objective: The goal of this study was to evaluate the effect of Physalis alkekengi ripe fruit hydroalcoholic extract (PFE) on uterine contractility and its possible mechanism(s). Materials and Methods: Extraction of Physalis alkekengi fruit was carried out by maceration method (70% alcohol). Uterus was dissected out from adult non-pregnant rat (Wistar) and contracted by KCl (60mM) or oxytocin (10mU/ml) in an organ bath containing De Jalon solution and the effect of PFE on the uterine contractions was investigated. Furthermore, the role of α- and β-adrenoceptors, opioid receptors, nitric oxide and cyclic guanosine monophosphate synthesis inhibitors on the extract effects were evaluated. Results: KCl- and oxytocin-induced uterine contractions were inhibited (p<0.001) by the cumulative concentrations of the extract in a concentration dependent manner. Incubation of uterus with propranolol (1μM) and L-NAME (100μM) attenuated the PFE antispasmodic effect (p<0.05). But the PFE effect was unaffected by phentolamine (1μM), naloxone (1μM) or methylene blue (10μM). In Ca2+-free with high potassium (60mM) De Jalon solution, cumulative concentrations of CaCl2 (0.1-0.5mM) induced uterine contraction concentration-dependently (p<0.001). Uterus incubation (5min) with PFE (0.25-1.75mg/ml) attenuated the CaCl2–induced contractions (p<0.05). Conclusion: It seems that the extract induced antispasmodic effect mainly via calcium influx blockade and partially through blocking β-adrenoceptors and nitric oxide (NO) synthesis. However, neither α-adrenoceptors nor opioid receptors or cGMP synthesis were involved. Background: Studies have shown that Physalis alkekengi reduces implantation and induces antifertility in rat. In Iranian traditional medicine it is believed that this plant has abortifacient and antifertility activities. Objective: The goal of this study was to evaluate the effect of Physalis alkekengi ripe fruit hydroalcoholic extract (PFE) on uterine contractility and its possible mechanism(s). Materials and Methods: Extraction of Physalis alkekengi fruit was carried out by maceration method (70% alcohol). Uterus was dissected out from adult non-pregnant rat (Wistar) and contracted by KCl (60mM) or oxytocin (10mU/ml) in an organ bath containing De Jalon solution and the effect of PFE on the uterine contractions was investigated. Furthermore, the role of α- and β-adrenoceptors, opioid receptors, nitric oxide and cyclic guanosine monophosphate synthesis inhibitors on the extract effects were evaluated. Results: KCl- and oxytocin-induced uterine contractions were inhibited (p<0.001) by the cumulative concentrations of the extract in a concentration dependent manner. Incubation of uterus with propranolol (1μM) and L-NAME (100μM) attenuated the PFE antispasmodic effect (p<0.05). But the PFE effect was unaffected by phentolamine (1μM), naloxone (1μM) or methylene blue (10μM). In Ca2+-free with high potassium (60mM) De Jalon solution, cumulative concentrations of CaCl2 (0.1-0.5mM) induced uterine contraction concentration-dependently (p<0.001). Uterus incubation (5min) with PFE (0.25-1.75mg/ml) attenuated the CaCl2–induced contractions (p<0.05). Conclusion: It seems that the extract induced antispasmodic effect mainly via calcium influx blockade and partially through blocking β-adrenoceptors and nitric oxide (NO) synthesis. However, neither α-adrenoceptors nor opioid receptors or cGMP synthesis were involved. Rat, Uterus, Physalis alkekengi, Antispasmodic. Rat, Uterus, Physalis alkekengi, Antispasmodic. 193 198 Mohammad Kazem Gharib Naseri Mohammad Kazem Gharib Naseri Gharibnaseri_m@yahoo.com No Maryam Mohammadian Maryam Mohammadian No Zahra Gharib Naseri Pharm Zahra Gharib Naseri Pharm No

En 1680-6433 2008-2177 Determination of effective dosage of FSH and hCG in Background: In vitro maturation (IVM) of oocytes reduces the costs and averts the side-effects of gonadotropin stimulation for in vitro fertilization (IVF). Reliable IVM is an intellectual, scientific and clinical challenge with a number of potential applications. Objective: The effect of hCG was evaluated on the timing and regulation of in vitro ovulation for the Syrian mice oocytes in the presence and absence of FSH. Materials and Methods: Preantral follicles, isolated from the ovaries of 6 weeks-old mice, were cultured in TCM-199 medium. The effect of 10-200 mIU/ml FSH and 1.5 IU/ml hCG was seen on the follicle maturation, as well as the changes in ovulation capacity of enclosed oocytes, after the incubation period of 6 days at 37 °C, 92% humidity and 5% CO2 in air. Results: 100 mIU/ml FSH showed increased follicle diameter, survival, germinal vesicle breakdown (GVBD) and oocyte maturation rates (p<0.0001). Significantly higher number of follicles showed cumulus attachment when ovulation started within 16-24 hours post hCG (97% and 80% respectively; p<0.0001) as compared to the cultures without hCG or when the ovulation time increased from 24 hours post hCG. Combination of FSH and hCG showed 97% (p<0.0001) ovulation as compared to that seen for FSH-containing medium only (81%) or control (10%). Conclusion: The combined administration of 1.5 IU/ml hCG and 100 mIU/ml FSH induces the in vitro follicle maturation, ovulation capacity and proper timing of mice oocytes. Background: In vitro maturation (IVM) of oocytes reduces the costs and averts the side-effects of gonadotropin stimulation for in vitro fertilization (IVF). Reliable IVM is an intellectual, scientific and clinical challenge with a number of potential applications. Objective: The effect of hCG was evaluated on the timing and regulation of in vitro ovulation for the Syrian mice oocytes in the presence and absence of FSH. Materials and Methods: Preantral follicles, isolated from the ovaries of 6 weeks-old mice, were cultured in TCM-199 medium. The effect of 10-200 mIU/ml FSH and 1.5 IU/ml hCG was seen on the follicle maturation, as well as the changes in ovulation capacity of enclosed oocytes, after the incubation period of 6 days at 37 °C, 92% humidity and 5% CO2 in air. Results: 100 mIU/ml FSH showed increased follicle diameter, survival, germinal vesicle breakdown (GVBD) and oocyte maturation rates (p<0.0001). Significantly higher number of follicles showed cumulus attachment when ovulation started within 16-24 hours post hCG (97% and 80% respectively; p<0.0001) as compared to the cultures without hCG or when the ovulation time increased from 24 hours post hCG. Combination of FSH and hCG showed 97% (p<0.0001) ovulation as compared to that seen for FSH-containing medium only (81%) or control (10%). Conclusion: The combined administration of 1.5 IU/ml hCG and 100 mIU/ml FSH induces the in vitro follicle maturation, ovulation capacity and proper timing of mice oocytes. Follicle stimulating hormone, HCG, Preantral follicles, Oocyte maturation, GVBD. Follicle stimulating hormone, HCG, Preantral follicles, Oocyte maturation, GVBD. 199 204 Aisha Javed1 Aisha Javed1 _ No Saeed Rezaei-Zarchi Saeed Rezaei-Zarchi _ No Morteza Anvari Morteza Anvari _ No Madiha Javeed Madiha Javeed _ No Fatemeh Barzegari Firouzabadi Fatemeh Barzegari Firouzabadi _ No Amer Jamil| _ No Seyed Mehdi Kalantar Seyed Mehdi Kalantar - No Habibollah Nazem Habibollah Nazem - No

En 1680-6433 2008-2177 Neonatal mortality of low birth weight infants in Yazd, Iran Background: Low birth weight (LBW) is one of the major determinants of neonatal survival as well as postnatal morbidity. Objective: The main objective of the present study was to determine neonatal mortality rate (NMR) in LBW infants in Yazd, Iran. Materials and Methods: In a prospective-cohort study, all births in the maternity hospitals of Yazd, Iran in 2004 were evaluated and mortality rate in LBW population over the course of the first month of extra uterine life was determined. Results: In total, 8.4% (507 of 6016 births) of all newborns were LBW and 18.7% (95/507) of all LBW neonates died. Neonatal mortality rate in Yazd was 24/1000 live births .Two- third (95 /143) of all neonatal deaths occurred in LBW. Neonatal mortality rate (NMR) in LBW, Moderately low birth weight (MLBW), Very low birth weight (VLBW) and Extremely low birth weight (ELBW) were 23, 11.5 , 62.5 and 117 times more than that of normal weight newborns, respectively. Nearly 65% of all LBW neonatal deaths occurred in first 24 hours after birth. Overall NMR, Early Neonatal mortality rate (ENMR) and Late Neonatal mortality rate (LNMR) in LBW were 187, 118 and 9.8 in 1000 live births, respectively. The main causes of mortality among LBW in order of prevalence were respiratory distress syndrome (RDS) (59%), asphyxia (20%), septicemia (12%) and congenital malformation (9%). Conclusion: Neonatal mortality rate in Yazd is high and LBW accounted for two-third of neonatal deaths. Therefore, effort should be intensified to implement effective strategies for the reduction of LBW births and improving the care of these vulnerable neonates. Background: Low birth weight (LBW) is one of the major determinants of neonatal survival as well as postnatal morbidity. Objective: The main objective of the present study was to determine neonatal mortality rate (NMR) in LBW infants in Yazd, Iran. Materials and Methods: In a prospective-cohort study, all births in the maternity hospitals of Yazd, Iran in 2004 were evaluated and mortality rate in LBW population over the course of the first month of extra uterine life was determined. Results: In total, 8.4% (507 of 6016 births) of all newborns were LBW and 18.7% (95/507) of all LBW neonates died. Neonatal mortality rate in Yazd was 24/1000 live births .Two- third (95 /143) of all neonatal deaths occurred in LBW. Neonatal mortality rate (NMR) in LBW, Moderately low birth weight (MLBW), Very low birth weight (VLBW) and Extremely low birth weight (ELBW) were 23, 11.5 , 62.5 and 117 times more than that of normal weight newborns, respectively. Nearly 65% of all LBW neonatal deaths occurred in first 24 hours after birth. Overall NMR, Early Neonatal mortality rate (ENMR) and Late Neonatal mortality rate (LNMR) in LBW were 187, 118 and 9.8 in 1000 live births, respectively. The main causes of mortality among LBW in order of prevalence were respiratory distress syndrome (RDS) (59%), asphyxia (20%), septicemia (12%) and congenital malformation (9%). Conclusion: Neonatal mortality rate in Yazd is high and LBW accounted for two-third of neonatal deaths. Therefore, effort should be intensified to implement effective strategies for the reduction of LBW births and improving the care of these vulnerable neonates. Low Birth Weight, Neonatal Mortality Rate, Neonate. Low Birth Weight, Neonatal Mortality Rate, Neonate. 205 208 Motahhareh Golestan Motahhareh Golestan mogolestan@yahoo.com No

En 1680-6433 2008-2177 The effects of vitrification on spindle apparatus of in Background: Routine oocytes cryopreservation remained an elusive technique in the wide ranges of available assisted reproductive technologies. The microtubules of oocytes are vulnerable to cryoprotectants and thermal change during cryopreservation. Objective: The effects of a vitrification protocol were investigated on the spindle and chromosome configurations of mice oocytes cryopreserved at the germinal vesicle stage. Materials and Methods: Germinal vesicle with cumulus cells were transferred to vitrification solution which was composed of 30% (v/v) ethylene glycol, 18% Ficoll-70 and 0.3 M Sucrose either by single step or in step-wise way. Following vitrification and in vitro maturation (MII), the matured oocytes were immonostained for meiotic spindles and chromosomes, before visualization using fluorescent microscopy. Results: A statistically significant increase was observed in the survival and maturation rate in step-wise vitrification (88.96% and 71.23% respectively) compared to single step vitrification (70.6% and 62.42% respectively) (p<0.05). Normal spindle morphology after vitrification-thawing in step-wise vitrification group (77.26%) was higher than single step vitrification group (64.24%) but lower than control group (94.75%) (p<0.05). Conclusion: The results suggest that vitrification with step-wise procedure on mice germinal vesicle oocytes has positive effects on survival and maturation rate and normal spindle configuration compare with single step vitrification procedure. Background: Routine oocytes cryopreservation remained an elusive technique in the wide ranges of available assisted reproductive technologies. The microtubules of oocytes are vulnerable to cryoprotectants and thermal change during cryopreservation. Objective: The effects of a vitrification protocol were investigated on the spindle and chromosome configurations of mice oocytes cryopreserved at the germinal vesicle stage. Materials and Methods: Germinal vesicle with cumulus cells were transferred to vitrification solution which was composed of 30% (v/v) ethylene glycol, 18% Ficoll-70 and 0.3 M Sucrose either by single step or in step-wise way. Following vitrification and in vitro maturation (MII), the matured oocytes were immonostained for meiotic spindles and chromosomes, before visualization using fluorescent microscopy. Results: A statistically significant increase was observed in the survival and maturation rate in step-wise vitrification (88.96% and 71.23% respectively) compared to single step vitrification (70.6% and 62.42% respectively) (p<0.05). Normal spindle morphology after vitrification-thawing in step-wise vitrification group (77.26%) was higher than single step vitrification group (64.24%) but lower than control group (94.75%) (p<0.05). Conclusion: The results suggest that vitrification with step-wise procedure on mice germinal vesicle oocytes has positive effects on survival and maturation rate and normal spindle configuration compare with single step vitrification procedure. Vitrification, Germinal vesicle oocyte, Mice, Microtubule Vitrification, Germinal vesicle oocyte, Mice, Microtubule 209 215 Reza Mahmoudi Reza Mahmoudi No