En 1680-6433 2008-2177 jalali 1388 10 1 gregorian 2009 12 22 7 4 online 1 fulltext

En 1680-6433 2008-2177 Isolation and primary culture of ES-like colonies from NMRI mouse embryos Background: Embryonic stem (ES) cells are pluripotent cells conventionally isolated from early embryos. Studies have shown that ES cells serve as a practical model for biomedical studies. Objective: The aim of the present study was to optimize culture conditions for establishment of ES-like colonies from NMRI mouse blastocysts as well as 2-cell stage embryos. Materials and Methods: Both expanded blastocysts and 2-cell stage embryos were co-cultured on mouse embryonic fibroblast (MEF). Plating capacity and formation of Inner cell mass (ICM) were examined daily. The differentiation and growth behavior of ICM cells were examined with various procedures. ICMs derived from initially cultured 2-cell or blastocyst embryos were disaggregated either mechanically or enzymatically and seeded onto MEF with or without leukemia inhibitory factor (LIF). The resulted colonies were disaggregated and reseeded onto MEF and the colonies that were morphologically similar to ES cells were evaluated for pluripotency using alkaline phosphatase (ALP) expression as a stem cell marker. Results: No morphologically good ES-like colony was isolated from 2-cell embryos after passages while 273 (79%) good-looking ICMs were isolated from 352 blastocysts. Four sets of colonies remained undifferentiated following passages. Enzymatic method of ICM disaggregation was superior to the mechanical method. Besides all ES-like colonies were obtained from the ICMs cultured in presence of MEF and LIF. Conclusion: Our results show that NMRI mouse ICMs could be isolated and cultured from blastocyst stage embryos with a suitable culture system and ES-like cell colonies remain undifferentiated when cultured with MEF and LIF. Background: Embryonic stem (ES) cells are pluripotent cells conventionally isolated from early embryos. Studies have shown that ES cells serve as a practical model for biomedical studies. Objective: The aim of the present study was to optimize culture conditions for establishment of ES-like colonies from NMRI mouse blastocysts as well as 2-cell stage embryos. Materials and Methods: Both expanded blastocysts and 2-cell stage embryos were co-cultured on mouse embryonic fibroblast (MEF). Plating capacity and formation of Inner cell mass (ICM) were examined daily. The differentiation and growth behavior of ICM cells were examined with various procedures. ICMs derived from initially cultured 2-cell or blastocyst embryos were disaggregated either mechanically or enzymatically and seeded onto MEF with or without leukemia inhibitory factor (LIF). The resulted colonies were disaggregated and reseeded onto MEF and the colonies that were morphologically similar to ES cells were evaluated for pluripotency using alkaline phosphatase (ALP) expression as a stem cell marker. Results: No morphologically good ES-like colony was isolated from 2-cell embryos after passages while 273 (79%) good-looking ICMs were isolated from 352 blastocysts. Four sets of colonies remained undifferentiated following passages. Enzymatic method of ICM disaggregation was superior to the mechanical method. Besides all ES-like colonies were obtained from the ICMs cultured in presence of MEF and LIF. Conclusion: Our results show that NMRI mouse ICMs could be isolated and cultured from blastocyst stage embryos with a suitable culture system and ES-like cell colonies remain undifferentiated when cultured with MEF and LIF. NMRI mouse Inner cell mass Embryonic stem cell. NMRI mouse Inner cell mass Embryonic stem cell. 145 152 Seyed Hassan Eftekhar Vaghefi No Nehleh Zareii Fard No Zhinoosossadat Shahidzadeh No Seyed Noureddin Nematollahi-mahani nnematollahi@kmu.ac.ir No

En 1680-6433 2008-2177 Comparison of maternal serum Tumor Necrosis Factor-alpha (TNF-α) in severe and mild preeclampsia versus normal pregnancy Background: Preeclampsia is a disorder unique to pregnancy and has long been recognized as an important contributor of maternal and fetal morbidity and mortality. It is suggested that cytokines such as Tumor Necrosis Factor-alpha (TNF-α) have an important role in the pathogenesis of preeclampsia and may cause generalized endothelial dysfunction. Objective: The aim of this study was comparison of maternal serum TNF-α in severe and mild preeclampsia versus normal pregnancy. Materials and Methods: This study was performed on 37 women with preeclampsia (17 mild and 20 severe preeclampsia) and 41 normotensive pregnant women with similar gestational age at third trimester of pregnancy. All the preeclamptic cases had blood pressure ≥ 140/90 mmHg and proteinuria ≥ 300 mg in a 24-h urine sample. Maternal serum TNF-α concentration was compared in all of them. Results: The level of TNF-α concentration was not statistically different between the studied groups. No significant correlation was found between preeclampsia and control group as they were compared in the view of maternal serum TNF-α concentration. Conclusion: These findings suggest that serum TNF-α is not significantly associated with preeclampsia. Background: Preeclampsia is a disorder unique to pregnancy and has long been recognized as an important contributor of maternal and fetal morbidity and mortality. It is suggested that cytokines such as Tumor Necrosis Factor-alpha (TNF-α) have an important role in the pathogenesis of preeclampsia and may cause generalized endothelial dysfunction. Objective: The aim of this study was comparison of maternal serum TNF-α in severe and mild preeclampsia versus normal pregnancy. Materials and Methods: This study was performed on 37 women with preeclampsia (17 mild and 20 severe preeclampsia) and 41 normotensive pregnant women with similar gestational age at third trimester of pregnancy. All the preeclamptic cases had blood pressure ≥ 140/90 mmHg and proteinuria ≥ 300 mg in a 24-h urine sample. Maternal serum TNF-α concentration was compared in all of them. Results: The level of TNF-α concentration was not statistically different between the studied groups. No significant correlation was found between preeclampsia and control group as they were compared in the view of maternal serum TNF-α concentration. Conclusion: These findings suggest that serum TNF-α is not significantly associated with preeclampsia. Tumor Necrosis Factor-alpha (TNF-α) Preeclampsia Normal pregnancy. Tumor Necrosis Factor-alpha (TNF-α) Preeclampsia Normal pregnancy. 153 158 Fatemeh Vahid Roudsari No Sedigheh Ayati ayatis@mums.ac.ir No Hossein Ayatollahi No Habibollah Esmaeily No Maliheh Hasanzadeh No Masoud Shahabian No Leila Pour Ali No

En 1680-6433 2008-2177 The ovarian stimulation effects on Muc1 expression of the mouse endometrium before implantation Background: Acceptance of uterus and reaction between endometrium and embryo has an important role for implantation. Muc1 an integral membrane mucin is expressed on the apical surface of uterine epithelial cells and could have effects on its receptivity. Objective: The aim of this study was to evaluate the changes in Muc1 expression of gravid mouse endometrium with and without hyperstimulation before implantation. Materials and Methods: Adult female NMRI mice were divided into control and experimental groups. Experimental group superovulated using an intraperitoneal injection of Pregnant Mare’s Serum Gonadotrophin (PMSG) followed 48 hours later by another injection of Human Chorionic Gonadotropic hormone (HCG). The female mice have mated with normal male mice. All control and hyperstimulated groups subdivided into six groups. After mating female mice were examined by vaginal plaque as day of zero and in 0-5 days after copulation they were sacrificed by cervical dislocation. Then the middle 1/3 parts of their uterine horns were obtained and stained by immunohistochemicaly technique for Muc-1 detection. Results: Our results showed that in the control and hyperstimulated groups the Muc1 expression is markedly reduced in the luminal uterus epithelium at the time of implantation. Furthermore luminal and glandular uterus epithelium did not exhibit the same decrease in Muc1 expression during the receptive phase. Conclusion: Ovarian hyperstimulation didn’t alter the Muc1 expression markedly in surface and glandular epithelium of endometrium which could affect on its receptivity. Background: Acceptance of uterus and reaction between endometrium and embryo has an important role for implantation. Muc1 an integral membrane mucin is expressed on the apical surface of uterine epithelial cells and could have effects on its receptivity. Objective: The aim of this study was to evaluate the changes in Muc1 expression of gravid mouse endometrium with and without hyperstimulation before implantation. Materials and Methods: Adult female NMRI mice were divided into control and experimental groups. Experimental group superovulated using an intraperitoneal injection of Pregnant Mare’s Serum Gonadotrophin (PMSG) followed 48 hours later by another injection of Human Chorionic Gonadotropic hormone (HCG). The female mice have mated with normal male mice. All control and hyperstimulated groups subdivided into six groups. After mating female mice were examined by vaginal plaque as day of zero and in 0-5 days after copulation they were sacrificed by cervical dislocation. Then the middle 1/3 parts of their uterine horns were obtained and stained by immunohistochemicaly technique for Muc-1 detection. Results: Our results showed that in the control and hyperstimulated groups the Muc1 expression is markedly reduced in the luminal uterus epithelium at the time of implantation. Furthermore luminal and glandular uterus epithelium did not exhibit the same decrease in Muc1 expression during the receptive phase. Conclusion: Ovarian hyperstimulation didn’t alter the Muc1 expression markedly in surface and glandular epithelium of endometrium which could affect on its receptivity. Endometrium Muc1 expression Ovarian stimulation. Endometrium Muc1 expression Ovarian stimulation. 157 162 Marzieh Panahi marzpanah@yahoo.com No Azam Soleimani No Mahmoud No Abdolrahman Abdolrahman Dezfoolian No

En 1680-6433 2008-2177 Effect of withania somnifera on levels of sex hormones in the diabetic male rats Background: There are evidences regarding the prevalence of dysfunction in sexual function and behavior in diabetic people. Experimental studies revealed a positive effect of withania somnifera on sexual function and behaviors. Objective: In this research the effect of withania somnifera on sexual function in diabetic male Wistar rats was assessed by measuring the serum levels of testosterone progesterone estrogen FSH and LH. Materials and Methods: Experimental diabetes mellitus type I was induced by intraperitoneal injection of a single dose (60 mg/kg) of streptozotocin (STZ) in Wistar male rats. Oral withania somnifera root was given in pelleted food at ratio of 6.25% for 4 weeks. The levels of gonadadotropic hormones (LH FSH) progesterone estrogen and testosterone in animals’ serum were determined after 4 weeks in all groups. Results: Withania somnifera root was effective in lowering FSH serum level in somnifera-treated animals compared to controls (p Background: There are evidences regarding the prevalence of dysfunction in sexual function and behavior in diabetic people. Experimental studies revealed a positive effect of withania somnifera on sexual function and behaviors. Objective: In this research the effect of withania somnifera on sexual function in diabetic male Wistar rats was assessed by measuring the serum levels of testosterone progesterone estrogen FSH and LH. Materials and Methods: Experimental diabetes mellitus type I was induced by intraperitoneal injection of a single dose (60 mg/kg) of streptozotocin (STZ) in Wistar male rats. Oral withania somnifera root was given in pelleted food at ratio of 6.25% for 4 weeks. The levels of gonadadotropic hormones (LH FSH) progesterone estrogen and testosterone in animals’ serum were determined after 4 weeks in all groups. Results: Withania somnifera root was effective in lowering FSH serum level in somnifera-treated animals compared to controls (p Withania somnifera Diabetes Sex hormones Rat. Withania somnifera Diabetes Sex hormones Rat. 163 168 Zahra Kiasalari No Mohsen Khalili najafabady@yahoo.com No Mahbobeh Aghaei No

En 1680-6433 2008-2177 The effect of administrating indomethacin or hyoscine before embryo transfer on ART outcome (a pilot study) Background: In spite of the great progress in assisted reproductive techniques ( ART) and although good quality embryos are transferred pregnancy rates have remained around 30%-35% due to low implantation rates. Objective: The aim of this study was to assess and compare the effects of administrating indomethacin or hyoscine suppositories prior to embryo transfer on the pregnancy rate in ART cycles. Materials and Methods: This double-blind clinical trial was performed in Vali-e-Asr Hospital as a pilot study from August 2005 through December 2006 on 66 infertile women in ART cycles. Controlled ovarian hyperstimulation was done using recombinant FSH (Gonal-F) with a long GnRH analogue protocol. After obtaining written consent the subjects were randomly allocated into three equal groups (n=22). Groups A and B received indomethacin and hyoscine rectal suppositories respectively 30 minutes before embryo transfer and group C was the control group. Data were analyzed by χ2 t-test ANOVA and Kruskall Wallis tests. Results: Overall pregnancy rate was 31% (n=21) with 13.6% (n=3) in group A 45.5% (n=10) and 36% (n=8) in groups B and C respectively which shows that pregnancy rate is significantly higher in the group using hyoscine compared to the other two groups (p=0.04). Uterine muscle cramps were experienced by 3 women (13.6%) in group C while none were reported by women in groups A or B which shows a significant difference (p Background: In spite of the great progress in assisted reproductive techniques ( ART) and although good quality embryos are transferred pregnancy rates have remained around 30%-35% due to low implantation rates. Objective: The aim of this study was to assess and compare the effects of administrating indomethacin or hyoscine suppositories prior to embryo transfer on the pregnancy rate in ART cycles. Materials and Methods: This double-blind clinical trial was performed in Vali-e-Asr Hospital as a pilot study from August 2005 through December 2006 on 66 infertile women in ART cycles. Controlled ovarian hyperstimulation was done using recombinant FSH (Gonal-F) with a long GnRH analogue protocol. After obtaining written consent the subjects were randomly allocated into three equal groups (n=22). Groups A and B received indomethacin and hyoscine rectal suppositories respectively 30 minutes before embryo transfer and group C was the control group. Data were analyzed by χ2 t-test ANOVA and Kruskall Wallis tests. Results: Overall pregnancy rate was 31% (n=21) with 13.6% (n=3) in group A 45.5% (n=10) and 36% (n=8) in groups B and C respectively which shows that pregnancy rate is significantly higher in the group using hyoscine compared to the other two groups (p=0.04). Uterine muscle cramps were experienced by 3 women (13.6%) in group C while none were reported by women in groups A or B which shows a significant difference (p Embryo transfer Hyoscine Indomethacin Pregnancy rate ART. Embryo transfer Hyoscine Indomethacin Pregnancy rate ART. 169 173 Farnaz Sohrabvand fsohrabvand@yahoo.com No Fedyeh Haghollahi No Masoomeh Maasomi No Leila Asgarpoor No Mamak Shariat No Mahtab Hamedani No

En 1680-6433 2008-2177 Effects of pentoxifylline and vitamin E on pregnancy rate in infertile women treated by ZIFT: a randomized clinical trial Background: Reviewing the literature reveals that pentoxifylline (PTX) plus tocopherol (vitamin E) are used mainly to promote sperm quality. However trials focusing on the effects of these drugs in female partner are limited. Combination of pentoxifylline and vitamin E appeared to improve the pregnancy rate in patients with a thin endometrium by increasing the endometrial thickness and improving ovarian function. Objective: To determine whether combined PTX and tocopherol treatment can improve clinical pregnancy rate. Materials and Methods: One hundred twelve infertile women undergoing standardized controlled ovarian hyperstimulation for ICSI- ZIFT entered this randomized clinical trial. Patients were randomized to equal groups of combined PTX and tocopherol therapy or none (not receiving PTX and tocopherol). These drugs were administered to the intervention group for two cycles before starting ICSI-ZIFT cycle. Main outcome measure was clinical pregnancy rate. SPSS.11 software (SPSS Inc. Chicago IL.) was used for data collection and analysis. Results: The clinical pregnancy was higher in the intervention (combined PTX and tocopherol) group in comparison to the other group (57.14% vs 39.29% p=0.01). However there was no difference in the mean endometrial thickness number of retrieved oocytes the number of metaphase II oocytes and grade of them in both groups. Conclusion: This study showed that PTX plus tocopherol could improve the ZIFT outcome in infertile couples. Local effects and anti oxidative characteristics of these drugs may be the cause of better results. Background: Reviewing the literature reveals that pentoxifylline (PTX) plus tocopherol (vitamin E) are used mainly to promote sperm quality. However trials focusing on the effects of these drugs in female partner are limited. Combination of pentoxifylline and vitamin E appeared to improve the pregnancy rate in patients with a thin endometrium by increasing the endometrial thickness and improving ovarian function. Objective: To determine whether combined PTX and tocopherol treatment can improve clinical pregnancy rate. Materials and Methods: One hundred twelve infertile women undergoing standardized controlled ovarian hyperstimulation for ICSI- ZIFT entered this randomized clinical trial. Patients were randomized to equal groups of combined PTX and tocopherol therapy or none (not receiving PTX and tocopherol). These drugs were administered to the intervention group for two cycles before starting ICSI-ZIFT cycle. Main outcome measure was clinical pregnancy rate. SPSS.11 software (SPSS Inc. Chicago IL.) was used for data collection and analysis. Results: The clinical pregnancy was higher in the intervention (combined PTX and tocopherol) group in comparison to the other group (57.14% vs 39.29% p=0.01). However there was no difference in the mean endometrial thickness number of retrieved oocytes the number of metaphase II oocytes and grade of them in both groups. Conclusion: This study showed that PTX plus tocopherol could improve the ZIFT outcome in infertile couples. Local effects and anti oxidative characteristics of these drugs may be the cause of better results. Endometrium Pentoxifylline ZIFT Vitamin E Pregnancy outcome. Endometrium Pentoxifylline ZIFT Vitamin E Pregnancy outcome. 175 179 Ashraf Aleyasin No Marzieh Aghahosseini No Mahshid Mohseni No Atossa Mahdavi mahdavi_a@farabi.tums.ac.iratossa_mahdavi @yahoo.com No

En 1680-6433 2008-2177 Developmental consequences of mouse cryotop-vitrified oocyte and embryo using low concentrated cryoprotectants Background: The risk of multiple pregnancies often present in programs of In Vitro Fertilization (IVF) is an important force for embryo cryopreservation. On the other hand ethical restriction and assurance of potential fertility following chemo/radio therapy has led scientists to focus on female gamete preservation. Objective: Optimizing vitrification protocol by using less concentrated cryoprotectants (CPAs) in order to decrease CPAs toxicity. Materials and Methods: Mouse Metaphase-II (M-II) oocytes and four cell-stage embryos were collected. Oocytes Survival Fertilization and Developmental Rates (SRs FRs DRs) were recorded after cryotop-vitrification/warming. As well as comparing fresh oocytes and embryos the data obtained from experimental groups (exp.) applying 1.25 1.0 0.75 molar (M) CPAs were analyzed in comparison to those of adopting 1.5 M CPAs [largely-used concentration of Ethylen Glycol (EG) and Dimethyl-sulphoxide (DMSO)]. Results: The data of oocytes exposed to 1.25 M concentrated CPAs were in consistency with those exposed to 1.5 M and control group in terms of SR FR and DR. As less concentration was applied the more decreased SRs FRs and DRs were obtained from other experimental groups. The results of embryos which were exposed to 1.25 M and 1.0 M were close to those vitrified with 1.5 M and fresh embryos. The results of 0.75 M concentrated CPAs solutions were significantly lower than those of control 1.5 M and 1.0 M treated groups. Conclusion: CPAs limited reduction to 1.25 M and 1.0 M instead of using 1.5 M for oocyte and embryo cryotop-vitrification procedure may be a slight adjustment. Background: The risk of multiple pregnancies often present in programs of In Vitro Fertilization (IVF) is an important force for embryo cryopreservation. On the other hand ethical restriction and assurance of potential fertility following chemo/radio therapy has led scientists to focus on female gamete preservation. Objective: Optimizing vitrification protocol by using less concentrated cryoprotectants (CPAs) in order to decrease CPAs toxicity. Materials and Methods: Mouse Metaphase-II (M-II) oocytes and four cell-stage embryos were collected. Oocytes Survival Fertilization and Developmental Rates (SRs FRs DRs) were recorded after cryotop-vitrification/warming. As well as comparing fresh oocytes and embryos the data obtained from experimental groups (exp.) applying 1.25 1.0 0.75 molar (M) CPAs were analyzed in comparison to those of adopting 1.5 M CPAs [largely-used concentration of Ethylen Glycol (EG) and Dimethyl-sulphoxide (DMSO)]. Results: The data of oocytes exposed to 1.25 M concentrated CPAs were in consistency with those exposed to 1.5 M and control group in terms of SR FR and DR. As less concentration was applied the more decreased SRs FRs and DRs were obtained from other experimental groups. The results of embryos which were exposed to 1.25 M and 1.0 M were close to those vitrified with 1.5 M and fresh embryos. The results of 0.75 M concentrated CPAs solutions were significantly lower than those of control 1.5 M and 1.0 M treated groups. Conclusion: CPAs limited reduction to 1.25 M and 1.0 M instead of using 1.5 M for oocyte and embryo cryotop-vitrification procedure may be a slight adjustment. Cryotop Cryoprotectant Embryo Mouse Oocyte Vitrification. Cryotop Cryoprotectant Embryo Mouse Oocyte Vitrification. 181 188 gf ghgfhgf No

En 1680-6433 2008-2177 The effects of LIF and EGF on mouse oocyte maturation fertilization and development in vitro Background: Mammalian oocytes are exposed to a mixture of many different growth factors and cytokines which provides an optimized microenvironment for oocyte maturation. In the lack of this natural microenvironment in vitro the quality of oocyte and embryos appears to be suboptimal. Objective: This study was undertaken to investigate the effects of EGF and LIF on in vitro maturation fertilization and cleavage rates in mouse oocytes. Materials and Methods: The GV oocytes were collected from female NMRI mice and randomly divided into control and 3 treatment groups. Oocytes in treatment groups were cultured in the maturation medium supplemented with 50 ng/ml rhLIF (Treatment 1) 10ng/ml EGF (Treatment 2) and 50 ng/ml LIF+ 10ng/ml EGF (Treatment 3) for 24 hours at 37°C in humidified 5% CO2 in air. The matured oocytes were fertilized in vitro and cultured for 96 hours. Finally the developmental rates were assessed and embryos were stained using Hoechst 33258. Results: There was a higher maturation rate in treatment groups compared to the control group. There was not any significant difference in the rate of fertilization among the groups. The rates of cleavage (79.1%) and blastocyst formation (62.2%) were significantly higher in LIF + EGF group comparing to the other groups. The rates of hatching in groups treatment 1 (35.2%) and 3(41%) was significantly higher comparing to the other groups. Also the mean of total cell number in treatment groups significantly was higher than control (p< 0.05). Conclusion: The findings of this study suggest a beneficial effect of LIF and EGF on mouse oocyte maturation and cleavage rates. Background: Mammalian oocytes are exposed to a mixture of many different growth factors and cytokines which provides an optimized microenvironment for oocyte maturation. In the lack of this natural microenvironment in vitro the quality of oocyte and embryos appears to be suboptimal. Objective: This study was undertaken to investigate the effects of EGF and LIF on in vitro maturation fertilization and cleavage rates in mouse oocytes. Materials and Methods: The GV oocytes were collected from female NMRI mice and randomly divided into control and 3 treatment groups. Oocytes in treatment groups were cultured in the maturation medium supplemented with 50 ng/ml rhLIF (Treatment 1) 10ng/ml EGF (Treatment 2) and 50 ng/ml LIF+ 10ng/ml EGF (Treatment 3) for 24 hours at 37°C in humidified 5% CO2 in air. The matured oocytes were fertilized in vitro and cultured for 96 hours. Finally the developmental rates were assessed and embryos were stained using Hoechst 33258. Results: There was a higher maturation rate in treatment groups compared to the control group. There was not any significant difference in the rate of fertilization among the groups. The rates of cleavage (79.1%) and blastocyst formation (62.2%) were significantly higher in LIF + EGF group comparing to the other groups. The rates of hatching in groups treatment 1 (35.2%) and 3(41%) was significantly higher comparing to the other groups. Also the mean of total cell number in treatment groups significantly was higher than control (p< 0.05). Conclusion: The findings of this study suggest a beneficial effect of LIF and EGF on mouse oocyte maturation and cleavage rates. LIF EGF IVM IVF Embryo development Mouse. LIF EGF IVM IVF Embryo development Mouse. 189 194 fgfdgdf gffdgfdg No