- Email: [email protected]
Expression of lipid oxidative stress-related gene ALDH3A2 (aldehyde dehydrogenase 3 family, member A2) in human granulosa-lutein (GL) cells correlates with FSH response and pregnancy
achievement of a pregnancy within a minimum follow-up of 18 months. Analysis was by intention to treat. Information about couples’ demographics and fertility outcome was collected. The pregnancy rate (PR) and statistical evaluation measures were compared. RESULTS: Overall PR was higher in couples with BBT than in those without BBT (P<0.001, 51.9% vs. 33.9%; relative risk (RR) 1.53 for pregnancy, 95%CI 1.25-1.88). As for couples of non-ART treatment, PR was also higher in couples with BBT (P<0.001, 50.3% vs. 29.4%; RR 1.71, 95%CI 1.312.23). There was no significant difference in PR of ART treatment cases (P¼0.098, 57.6% vs. 44.3%; RR 1.30, 95%CI 0.94-1.79).
TABLE 1. Effect of BBT on pregnancy outcome according to treatment group Total Cases (N¼615) BBT(+) & Preg(+)/ BBT(+) & Preg(-) BBT(-) & Preg (+)/ BBT(-) & Preg(-) RR (95% CI) ARR (95% CI) NNT
Non-ART Cases (N¼460)
ART Cases (N¼155)
200/185
151/149
49/36
78/152
47/113
31/39
1.53(1.25-1.88)* 1.71(1.31-2.23)* 1.30(0.95-1.79)** -0.18(-0.26 -0.21(-0.30 -0.13(-0.29 -0.10)* -0.12)* 0.02)** 5.54 4.77 7.48
Preg: pregnancy; RR: relative risk; ARR: absolute risk reduction; NNT: number needed to treat for pregnancy; *: P<0.05; **: not significant
CONCLUSION: These data support a beneficial effect of BBT measurement in infertile couples for improving the pregnancy rate, especially in non-ART treatment cases. Recommending BBT measurement for infertility treatment is therefore justified.
P-425 Wednesday, October 24, 2012 IVF INSURANCE COVERAGE AND DONOR OOCYTE RECIPIENTS. S. B. Schon,a M. M. B. Schulte,a B. Hamilton,b R. R. Odem,a E. S. Jungheim.a aDepartment of Obstetrics and Gynecology, Washington University in St. Louis, Saint Louis, MO; bOlin Business School, Washington University in St. Louis, Saint Louis, MO. OBJECTIVE: Our clinic sits on the border between a state with a mandate for employers to provide insurance coverage for IVF using donor gametes and a state without such a mandate. Our objective was to investigate demographics and IVF outcomes in donor oocyte recipients with and without IVF insurance coverage. DESIGN: Retrospective cohort. MATERIALS AND METHODS: Medical and billing records were reviewed for women undergoing their first donor oocyte cycle in our center between 1999 and 2010. Home values were identified through an on-line real estate database used as a surrogate marker of socioeconomic status. 137 women were identified and placed into the following groups based on their IVF insurance coverage: 1) no coverage (NC) (n¼85), 2) state-mandated coverage (SMC) (n¼27), and 3) non-mandated insurance coverage (IC) (n¼25). Differences in demographics, economic factors and IVF outcomes were investigated between groups with standard bivariate statistics and Poisson regression. RESULTS: The groups did not differ in age. Home values were not different between women in the IC and SMC groups, but these women had a lower home value than women in the NC group ($168,22094804 vs. $275,188180758, P<0.01). Women in the SMC group were more likely to have previously tried autologous IVF than women in the IC and NC groups (RR¼1.98, 95% CI: 1.5-2.7). They were also more likely to have had a tubal ligation (RR 4.19, 95% CI: 1.74-10.12) and had more children than women in the other groups at the time of their donor cycle (B¼-1.9, P¼0.02). However, women with SMC were not more likely to have had a previous live birth than women in the other groups. IVF outcomes did not differ among groups. CONCLUSION: Our findings suggest state-mandated coverage for IVF using donor gametes does not influence IVF outcomes in cycles using donor oocytes, but it may increase access to and utilization of donor oocytes. Supported by: K12 NIH HD063086.
S238
ASRM Abstracts
ENVIRONMENT AND TOXICOLOGY
P-426 Wednesday, October 24, 2012 EFFECT OF FEMALE SMOKING STATUS ON ZONA PELLUCIDA THICKNESS. T. Freour, J. Lammers, C. Splingart, S. Lattes, P. Barriere. Medecine et Biologie de la Reproduction, University Hospital of Nantes, Nantes, France. OBJECTIVE: To compare zona pellucida (ZP) thickness measured with a time-lapse monitoring system (EmbryoscopeÒ) according to female smoking status. DESIGN: Retrospective study. MATERIALS AND METHODS: All ICSI cycles performed with the Embryoscope in 2011 were included. Female smoking status was recorded at interview just before IVF cycle. After injection, all oocytes were incubated in the EmbryoscopeÒ until transfer. ZP measurement was performed just after microinjection and on day 3, with a precision of 1mm. Two measures were made by 2 independant operators and the mean was considered. One or 2 embryos were selected for transfer on day 3 or 5 according to morphokinetics parameters. Pregnancy test was performed 12 days after embryo transfer, and confirmed ultrasonographically 3 weeks later. Statistical analysis was performed with Medcalc software. RESULTS: A total of 210 women were included in the study, with 1379 oocytes collected. Among them, 37 women were active smokers (226 oocytes collected) and 173 were non smokers (1153 oocytes). Mean age, ovarian stimulation parameters, number of oocytes, fertilization, conventional embryology parameters, embryo freezing and embryo transfer were not statistically different between both groups. Mean ZP thickness was not statistically different between the 2 groups, neither at the oocyte stage (16.971.97 mm in smokers and 17.472.66 mm in non smokers) nor on day 3 embryos. However, time lapse analysis showed that some early developmental events occurred significantly later in smokers than in non smokers (Polar Body extrusion, 3, 4 and 5 cells stage). Finally, ongoing pregnancy rate per cycle was significantly lower in smokers than in non smokers (22% versus 30.6%). CONCLUSION: Contrarily to what has been previously reported in the literature, we did not find a difference in ZP thickness according to female smoking status. Further studies are needed to confirm these results and decipher the mechanisms underlying tobacco induced poor embryo implantation ability in smokers patients.
OXIDATIVE STRESS
P-427 Wednesday, October 24, 2012 EXPRESSION OF LIPID OXIDATIVE STRESS-RELATED GENE ALDH3A2 (ALDEHYDE DEHYDROGENASE 3 FAMILY, MEMBER A2) IN HUMAN GRANULOSA-LUTEIN (GL) CELLS CORRELATES WITH FSH RESPONSE AND PREGNANCY. A. Palumbo,b,c R. Gonzalez-Fernandez,a O. Pe~na,a J. Hernandez,b J. Avila.a aBioquimica y Biologia Molecular, Universidad de La Laguna, La Laguna, Santa Cruz de Tenerife, Spain; bCentro de Asistencia a la Reproduccion Humana de Canarias, La Laguna, Santa Cruz de Tenerife, Spain; cObstetrics and Gynecology, New York University, New York, NY. OBJECTIVE: To study expression of ALDH3A2 as a marker of cellular oxidative stress in human GL cells and its relationship with FSH response and IVF outcome. DESIGN: Analysis of ALDH3A2 and FSH receptor (FSHR) gene expression by qRT-PCR in GL cells of pregnant versus not pregnant IVF patients. MATERIALS AND METHODS: One hundred IVF patients, including 47 pregnant (mean age 327.2) and 53 not pregnant (mean age 365.8) were studied. Ovulation induction was performed with downregulation or microflare or antagonist protocol. Gonadotrophin doses were selected based on ovarian reserve and adjusted to individual response. After ultrasound guided egg retrieval, GL cells were isolated from pooled follicular fluids from each patient using a percoll gradient and anti-CD45 immunobeads to eliminate WBCs; viability was assessed by trypan blue. ALDH3A2 and FSHR genes were measured by RT-PCR as relative expression compared to beta actin. Statistical analysis was performed with SPSS using Pearson’s Correlation coefficient, Pearson’s partial correlation and Student t-test.
Vol. 98, No. 3, Supplement, September 2012
RESULTS: Statistically significant differences in ALDH3A2 and FSHR gene expression were found between pregnant and not pregnant patients (2938309 vs 4318428, P¼0.016 and 434.5104.6 vs 177.945.6, P¼0.018 respectively). Only in not pregnant patients ALDH3A2 expression showed an age-independent positive correlation with the units of FSH administered (P<0.05), but not with FSHR expression. CONCLUSION: Higher ALDH3A2 expression in not pregnant vs pregnant patients, regardless of age and infertility origin, suggests a direct relationship between cellular oxidative stress and IVF outcome. The positive correlation between ALDH3A2 and FSH administered, independent of FSHR expression level and age, observed in not pregnant patients suggests that oxidative stress may negatively affect IVF outcome by decreasing FSHR response to FSH in GL cells. Supported by: FIS PS0900128; educational grants from Merck Serono Spain to R. G.-F. and from Canarian Agency for Research to O.P.
P-428 Wednesday, October 24, 2012 SPHINGOSINE-1-PHOSPHATE INHIBITS HYDROGEN PEROXIDE-INDUCED GRANULOSA CELL APOPTOSIS VIA THE PI3K/ AKT SIGNALING PATHWAY. T. Nakahara, A. Iwase, M. Kondo, T. Nakamura, M. Goto, F. Kikkawa. Departments of Obstetrics and Gynecology, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan.
P-429 Wednesday, October 24, 2012 IDENTIFICATION OF SEMINAL PLASMA PROTEINS IN MEN WITH OXIDATIVE STRESS UTILIZING PROTEOMIC TOOLS. R. Sharma,a A. Agarwal,a A. J. Hamada,a S. S. Du Plessis,b S. P. Yadav,a E. Sabanegh, Jr,a a Center of Reproductive Medicine, Cleveland Clinic, Cleveland, OH; bDivision of Medical Physiology, Stellenbosch University, Tygerberg, South Africa. OBJECTIVE: Determine the relative abundance of seminal plasma proteins in ROS+ and ROS- subjects. DESIGN: 2-DGE based proteomic analysis of seminal plasma. MATERIALS AND METHODS: Semen samples collected from 20 donors and 33 infertile men according to (WHO,1999), were divided into two groups: ROS+ (>20 RLU/s/106 sperm) and ROS– (<20 RLU/s/106 sperm). Clear seminal plasma was subjected to 2-DGE for separation of proteins. Protein bands were stained and digested into peptides and identified using the LC-MS system based on Mascot scores and Sequest programs. Quantitative comparison was measured by the normalized spectral count (nSC) ratio of the ROS– negative and ROS+ groups. RESULTS: A total of 9 proteins were identified in the ROS– group. Semenogelin II precursor and semenogelin I isoform preprotein were the most abundant. A total of 11 proteins were identified in the ROS+ group and semenogelin II precursor and prolactin induced protein were the most abundant components. Four proteins were present in both groups; 4 were unique to the ROS– group and 5 were unique to the ROS+ group. The nSC ratio of the differentially expressed proteins and their function are shown in the Table.
TABLE 1. Proteomics of seminal plasma proteins in ROS+ vs. ROS– semen.
nSC Protein
ROS- group
ROS+ group
Semenogelin I isoform preprotein
0.348
0.011
31.314
Fibronectin 1 isoform 3 preprotein Prostate specific antigen isoform Prolactin-induced protein
0.091 0.057 0.003
0.000 0.007 0.002
Only in ROS+ group 8.067 2.218
Albumin preprotein
0.000
0.034
Only in ROS+ group
OBJECTIVE: Granulosa cells play an important role in follicular development. The apoptosis of granulosa cells leads to follicular atresia. Oxidative stress contributes to granulosa cell apoptosis and is one of the most important physiological inducers of cellular injury associated with aging. The age-related decline in infertility may be modulated by oxidative stress. Therefore, the salvation of granulosa cells, especially in the presence of oxidative stress, might be of the great therapeutic value in the treatment of reproductive diseases. Sphingosine 1-phosphate (S1P) is a bioactive metabolic product of sphingolipid, which regulates various molecular and cellular events, including cell growth, cell survival. The present study investigated whether S1P protects against hydrogen peroxide-induced cell death in cultured granulosa cells. DESIGN: Experimental study in an academic medical center. MATERIALS AND METHODS: Cultured luteinized granulosa cells were treated with S1P or hydrogen peroxide, PI3K inhibitor or ERK inhibitor, in various combinations. Apoptotic changes were evaluated by cell counting and immunoblotting, and compared by ANOVA. RT-PCR was performed to evaluate the expression of S1P receptor. Activated or total protein expression of AKT, ERK, caspase-3 with various treatment were assayed by immunoblotting and compared by ANOVA. RESULTS: Hydrogen peroxide induced apoptotic cell death analyzed by cell nuclear morphology and cleavage activities of caspase-3, changes that were blocked by S1P cotreatment. The RT-PCR analysis confirmed the RNA expression of S1P1, S1P2, S1P3 and S1P5 receptors. S1P induced AKT phosphorylation by 30 minutes peaked at 10 minutes. The level of phospho-ERK did not increase after S1P treatment. CONCLUSION: S1P treatment can inhibit the apoptosis of granulosa cells induced by hydrogen peroxide. The protective effect was mediated by activating the PI3K/Akt pathway. The present study suggests that S1P can be potentially useful as a therapeutic agent for protecting granulosa cells from oxidative stress.
FERTILITY & STERILITYÒ
SC ratio
Role/function Inhibits premature capacitation and antioxidant. Unknown, oxidized form may accumulate. Low levels associated with poor motility. Aid in reducing semen hyperviscosity, immunosuppression effects. Unknown, but may be increased due to oxidation.
CONCLUSION: Protective proteins against oxidative stress are uniquely present in seminal plasma of ROS– men. ROS+ samples may exhibit seminal plasma proteins that are either under expressed or are oxidatively modified and may contribute to male infertility. Supported by: CCF. P-430 Wednesday, October 24, 2012 SPINDLE TRANSFER CAN RESTORE DEVELOPMENTAL CAPABILITY OF MAMMALIAN OOCYTES DAMAGED BY OXIDATIVE STRESS. M. Kobayashi, T. Takeuchi, A. Yoshida. Kiba Park Clinic Research Center, Koto, Tokyo, Japan. OBJECTIVE: Oxidative stress is suggested responsible for the dysfunctional mitochondria and the abnormal meiotic spindle in aged oocytes. The objective of this study was to investigate the effect of oxidative stress on cellular organelles and developmental potency of mammalian mature oocytes, and also to assess whether spindle transfer (ST) can restore the damage induced by oxidative stress. DESIGN: Changes in the meiotic spindles, mitochondrial function and developmental potency of the oxidatively damaged oocytes were studied. The ability of ST on restoring the developmental potential of the damaged oocytes was assessed. MATERIALS AND METHODS: Mouse MII oocytes were treated with 50 or 100 mM H2O2 for 15 and 30 min. Morphology of the spindle chromosome complex was studied immunohistochemically, while mitochondrial membrane potential was evaluated with JC-1 dye. The spindle of a damaged oocyte was isolated and inserted subzonally into an enucleated intact oocyte with inactivated Sendai virus. The reconstituted oocytes were ICSI fertilized and cultured for 4 days.
S239
TABLE 1. Effect of BBT on pregnancy outcome according to treatment group Total Cases (N¼615) BBT(+) & Preg(+)/ BBT(+) & Preg(-) BBT(-) & Preg (+)/ BBT(-) & Preg(-) RR (95% CI) ARR (95% CI) NNT
Non-ART Cases (N¼460)
ART Cases (N¼155)
200/185
151/149
49/36
78/152
47/113
31/39
1.53(1.25-1.88)* 1.71(1.31-2.23)* 1.30(0.95-1.79)** -0.18(-0.26 -0.21(-0.30 -0.13(-0.29 -0.10)* -0.12)* 0.02)** 5.54 4.77 7.48
Preg: pregnancy; RR: relative risk; ARR: absolute risk reduction; NNT: number needed to treat for pregnancy; *: P<0.05; **: not significant
CONCLUSION: These data support a beneficial effect of BBT measurement in infertile couples for improving the pregnancy rate, especially in non-ART treatment cases. Recommending BBT measurement for infertility treatment is therefore justified.
P-425 Wednesday, October 24, 2012 IVF INSURANCE COVERAGE AND DONOR OOCYTE RECIPIENTS. S. B. Schon,a M. M. B. Schulte,a B. Hamilton,b R. R. Odem,a E. S. Jungheim.a aDepartment of Obstetrics and Gynecology, Washington University in St. Louis, Saint Louis, MO; bOlin Business School, Washington University in St. Louis, Saint Louis, MO. OBJECTIVE: Our clinic sits on the border between a state with a mandate for employers to provide insurance coverage for IVF using donor gametes and a state without such a mandate. Our objective was to investigate demographics and IVF outcomes in donor oocyte recipients with and without IVF insurance coverage. DESIGN: Retrospective cohort. MATERIALS AND METHODS: Medical and billing records were reviewed for women undergoing their first donor oocyte cycle in our center between 1999 and 2010. Home values were identified through an on-line real estate database used as a surrogate marker of socioeconomic status. 137 women were identified and placed into the following groups based on their IVF insurance coverage: 1) no coverage (NC) (n¼85), 2) state-mandated coverage (SMC) (n¼27), and 3) non-mandated insurance coverage (IC) (n¼25). Differences in demographics, economic factors and IVF outcomes were investigated between groups with standard bivariate statistics and Poisson regression. RESULTS: The groups did not differ in age. Home values were not different between women in the IC and SMC groups, but these women had a lower home value than women in the NC group ($168,22094804 vs. $275,188180758, P<0.01). Women in the SMC group were more likely to have previously tried autologous IVF than women in the IC and NC groups (RR¼1.98, 95% CI: 1.5-2.7). They were also more likely to have had a tubal ligation (RR 4.19, 95% CI: 1.74-10.12) and had more children than women in the other groups at the time of their donor cycle (B¼-1.9, P¼0.02). However, women with SMC were not more likely to have had a previous live birth than women in the other groups. IVF outcomes did not differ among groups. CONCLUSION: Our findings suggest state-mandated coverage for IVF using donor gametes does not influence IVF outcomes in cycles using donor oocytes, but it may increase access to and utilization of donor oocytes. Supported by: K12 NIH HD063086.
S238
ASRM Abstracts
ENVIRONMENT AND TOXICOLOGY
P-426 Wednesday, October 24, 2012 EFFECT OF FEMALE SMOKING STATUS ON ZONA PELLUCIDA THICKNESS. T. Freour, J. Lammers, C. Splingart, S. Lattes, P. Barriere. Medecine et Biologie de la Reproduction, University Hospital of Nantes, Nantes, France. OBJECTIVE: To compare zona pellucida (ZP) thickness measured with a time-lapse monitoring system (EmbryoscopeÒ) according to female smoking status. DESIGN: Retrospective study. MATERIALS AND METHODS: All ICSI cycles performed with the Embryoscope in 2011 were included. Female smoking status was recorded at interview just before IVF cycle. After injection, all oocytes were incubated in the EmbryoscopeÒ until transfer. ZP measurement was performed just after microinjection and on day 3, with a precision of 1mm. Two measures were made by 2 independant operators and the mean was considered. One or 2 embryos were selected for transfer on day 3 or 5 according to morphokinetics parameters. Pregnancy test was performed 12 days after embryo transfer, and confirmed ultrasonographically 3 weeks later. Statistical analysis was performed with Medcalc software. RESULTS: A total of 210 women were included in the study, with 1379 oocytes collected. Among them, 37 women were active smokers (226 oocytes collected) and 173 were non smokers (1153 oocytes). Mean age, ovarian stimulation parameters, number of oocytes, fertilization, conventional embryology parameters, embryo freezing and embryo transfer were not statistically different between both groups. Mean ZP thickness was not statistically different between the 2 groups, neither at the oocyte stage (16.971.97 mm in smokers and 17.472.66 mm in non smokers) nor on day 3 embryos. However, time lapse analysis showed that some early developmental events occurred significantly later in smokers than in non smokers (Polar Body extrusion, 3, 4 and 5 cells stage). Finally, ongoing pregnancy rate per cycle was significantly lower in smokers than in non smokers (22% versus 30.6%). CONCLUSION: Contrarily to what has been previously reported in the literature, we did not find a difference in ZP thickness according to female smoking status. Further studies are needed to confirm these results and decipher the mechanisms underlying tobacco induced poor embryo implantation ability in smokers patients.
OXIDATIVE STRESS
P-427 Wednesday, October 24, 2012 EXPRESSION OF LIPID OXIDATIVE STRESS-RELATED GENE ALDH3A2 (ALDEHYDE DEHYDROGENASE 3 FAMILY, MEMBER A2) IN HUMAN GRANULOSA-LUTEIN (GL) CELLS CORRELATES WITH FSH RESPONSE AND PREGNANCY. A. Palumbo,b,c R. Gonzalez-Fernandez,a O. Pe~na,a J. Hernandez,b J. Avila.a aBioquimica y Biologia Molecular, Universidad de La Laguna, La Laguna, Santa Cruz de Tenerife, Spain; bCentro de Asistencia a la Reproduccion Humana de Canarias, La Laguna, Santa Cruz de Tenerife, Spain; cObstetrics and Gynecology, New York University, New York, NY. OBJECTIVE: To study expression of ALDH3A2 as a marker of cellular oxidative stress in human GL cells and its relationship with FSH response and IVF outcome. DESIGN: Analysis of ALDH3A2 and FSH receptor (FSHR) gene expression by qRT-PCR in GL cells of pregnant versus not pregnant IVF patients. MATERIALS AND METHODS: One hundred IVF patients, including 47 pregnant (mean age 327.2) and 53 not pregnant (mean age 365.8) were studied. Ovulation induction was performed with downregulation or microflare or antagonist protocol. Gonadotrophin doses were selected based on ovarian reserve and adjusted to individual response. After ultrasound guided egg retrieval, GL cells were isolated from pooled follicular fluids from each patient using a percoll gradient and anti-CD45 immunobeads to eliminate WBCs; viability was assessed by trypan blue. ALDH3A2 and FSHR genes were measured by RT-PCR as relative expression compared to beta actin. Statistical analysis was performed with SPSS using Pearson’s Correlation coefficient, Pearson’s partial correlation and Student t-test.
Vol. 98, No. 3, Supplement, September 2012
RESULTS: Statistically significant differences in ALDH3A2 and FSHR gene expression were found between pregnant and not pregnant patients (2938309 vs 4318428, P¼0.016 and 434.5104.6 vs 177.945.6, P¼0.018 respectively). Only in not pregnant patients ALDH3A2 expression showed an age-independent positive correlation with the units of FSH administered (P<0.05), but not with FSHR expression. CONCLUSION: Higher ALDH3A2 expression in not pregnant vs pregnant patients, regardless of age and infertility origin, suggests a direct relationship between cellular oxidative stress and IVF outcome. The positive correlation between ALDH3A2 and FSH administered, independent of FSHR expression level and age, observed in not pregnant patients suggests that oxidative stress may negatively affect IVF outcome by decreasing FSHR response to FSH in GL cells. Supported by: FIS PS0900128; educational grants from Merck Serono Spain to R. G.-F. and from Canarian Agency for Research to O.P.
P-428 Wednesday, October 24, 2012 SPHINGOSINE-1-PHOSPHATE INHIBITS HYDROGEN PEROXIDE-INDUCED GRANULOSA CELL APOPTOSIS VIA THE PI3K/ AKT SIGNALING PATHWAY. T. Nakahara, A. Iwase, M. Kondo, T. Nakamura, M. Goto, F. Kikkawa. Departments of Obstetrics and Gynecology, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan.
P-429 Wednesday, October 24, 2012 IDENTIFICATION OF SEMINAL PLASMA PROTEINS IN MEN WITH OXIDATIVE STRESS UTILIZING PROTEOMIC TOOLS. R. Sharma,a A. Agarwal,a A. J. Hamada,a S. S. Du Plessis,b S. P. Yadav,a E. Sabanegh, Jr,a a Center of Reproductive Medicine, Cleveland Clinic, Cleveland, OH; bDivision of Medical Physiology, Stellenbosch University, Tygerberg, South Africa. OBJECTIVE: Determine the relative abundance of seminal plasma proteins in ROS+ and ROS- subjects. DESIGN: 2-DGE based proteomic analysis of seminal plasma. MATERIALS AND METHODS: Semen samples collected from 20 donors and 33 infertile men according to (WHO,1999), were divided into two groups: ROS+ (>20 RLU/s/106 sperm) and ROS– (<20 RLU/s/106 sperm). Clear seminal plasma was subjected to 2-DGE for separation of proteins. Protein bands were stained and digested into peptides and identified using the LC-MS system based on Mascot scores and Sequest programs. Quantitative comparison was measured by the normalized spectral count (nSC) ratio of the ROS– negative and ROS+ groups. RESULTS: A total of 9 proteins were identified in the ROS– group. Semenogelin II precursor and semenogelin I isoform preprotein were the most abundant. A total of 11 proteins were identified in the ROS+ group and semenogelin II precursor and prolactin induced protein were the most abundant components. Four proteins were present in both groups; 4 were unique to the ROS– group and 5 were unique to the ROS+ group. The nSC ratio of the differentially expressed proteins and their function are shown in the Table.
TABLE 1. Proteomics of seminal plasma proteins in ROS+ vs. ROS– semen.
nSC Protein
ROS- group
ROS+ group
Semenogelin I isoform preprotein
0.348
0.011
31.314
Fibronectin 1 isoform 3 preprotein Prostate specific antigen isoform Prolactin-induced protein
0.091 0.057 0.003
0.000 0.007 0.002
Only in ROS+ group 8.067 2.218
Albumin preprotein
0.000
0.034
Only in ROS+ group
OBJECTIVE: Granulosa cells play an important role in follicular development. The apoptosis of granulosa cells leads to follicular atresia. Oxidative stress contributes to granulosa cell apoptosis and is one of the most important physiological inducers of cellular injury associated with aging. The age-related decline in infertility may be modulated by oxidative stress. Therefore, the salvation of granulosa cells, especially in the presence of oxidative stress, might be of the great therapeutic value in the treatment of reproductive diseases. Sphingosine 1-phosphate (S1P) is a bioactive metabolic product of sphingolipid, which regulates various molecular and cellular events, including cell growth, cell survival. The present study investigated whether S1P protects against hydrogen peroxide-induced cell death in cultured granulosa cells. DESIGN: Experimental study in an academic medical center. MATERIALS AND METHODS: Cultured luteinized granulosa cells were treated with S1P or hydrogen peroxide, PI3K inhibitor or ERK inhibitor, in various combinations. Apoptotic changes were evaluated by cell counting and immunoblotting, and compared by ANOVA. RT-PCR was performed to evaluate the expression of S1P receptor. Activated or total protein expression of AKT, ERK, caspase-3 with various treatment were assayed by immunoblotting and compared by ANOVA. RESULTS: Hydrogen peroxide induced apoptotic cell death analyzed by cell nuclear morphology and cleavage activities of caspase-3, changes that were blocked by S1P cotreatment. The RT-PCR analysis confirmed the RNA expression of S1P1, S1P2, S1P3 and S1P5 receptors. S1P induced AKT phosphorylation by 30 minutes peaked at 10 minutes. The level of phospho-ERK did not increase after S1P treatment. CONCLUSION: S1P treatment can inhibit the apoptosis of granulosa cells induced by hydrogen peroxide. The protective effect was mediated by activating the PI3K/Akt pathway. The present study suggests that S1P can be potentially useful as a therapeutic agent for protecting granulosa cells from oxidative stress.
FERTILITY & STERILITYÒ
SC ratio
Role/function Inhibits premature capacitation and antioxidant. Unknown, oxidized form may accumulate. Low levels associated with poor motility. Aid in reducing semen hyperviscosity, immunosuppression effects. Unknown, but may be increased due to oxidation.
CONCLUSION: Protective proteins against oxidative stress are uniquely present in seminal plasma of ROS– men. ROS+ samples may exhibit seminal plasma proteins that are either under expressed or are oxidatively modified and may contribute to male infertility. Supported by: CCF. P-430 Wednesday, October 24, 2012 SPINDLE TRANSFER CAN RESTORE DEVELOPMENTAL CAPABILITY OF MAMMALIAN OOCYTES DAMAGED BY OXIDATIVE STRESS. M. Kobayashi, T. Takeuchi, A. Yoshida. Kiba Park Clinic Research Center, Koto, Tokyo, Japan. OBJECTIVE: Oxidative stress is suggested responsible for the dysfunctional mitochondria and the abnormal meiotic spindle in aged oocytes. The objective of this study was to investigate the effect of oxidative stress on cellular organelles and developmental potency of mammalian mature oocytes, and also to assess whether spindle transfer (ST) can restore the damage induced by oxidative stress. DESIGN: Changes in the meiotic spindles, mitochondrial function and developmental potency of the oxidatively damaged oocytes were studied. The ability of ST on restoring the developmental potential of the damaged oocytes was assessed. MATERIALS AND METHODS: Mouse MII oocytes were treated with 50 or 100 mM H2O2 for 15 and 30 min. Morphology of the spindle chromosome complex was studied immunohistochemically, while mitochondrial membrane potential was evaluated with JC-1 dye. The spindle of a damaged oocyte was isolated and inserted subzonally into an enucleated intact oocyte with inactivated Sendai virus. The reconstituted oocytes were ICSI fertilized and cultured for 4 days.
S239