- Email: [email protected]
Effects of seminal glutathione peroxidase and its related non-enzymatic antioxidants on sperm DNA integrety and semen quality
Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY. OBJECTIVE: To test the ability of a biomarker-based assay to diagnose sperm ability to undergo capacitation and fertilize. We tested GM1 localization, a ganglioside important for sperm maturation, as a biomarker to quantify the sub-population that could respond to stimuli and become fertilization competent. DESIGN: Sperm GM1 localization was assessed in basal and capacitating media, from semen samples of consenting men. Localization patterns were compared between men with normal semen parameters to those with recurrent IUI failure. MATERIALS AND METHODS: Following sperm selection, samples from 35 men were processed for GM1 localization via fluorescence microscopy. Patterns were scored in at least 200 sperm incubated under both standard and capacitating conditions. Semen parameters were evaluated using WHO 2010 criteria. Based on preliminary data and that human sperm capacitation occurs within 4 hrs, we used timepoints of 1-3 hrs. RESULTS: Semen assay values were 53.916million/ml with a motility of 48.46% for control men (n¼20) and 48.918million/ml with motility 47.67% for those that failed IUI (n¼15). Baseline GM1 pattern scores for the control were 13.5%, 18.2%, and 25.0% at 1, 2 and 3hrs. In capacitating medium, pattern scores were 21.7%, 26.9%, and 34.5%. Sperm from men with recurrent IUI failure had baseline pattern scores of 9.1%, 13.4%, and 19.2%, and only 14.2%, 19.5%, and 23.9% in response to capacitating conditions. CONCLUSION: The dynamic profiling of sperm membrane changes during capacitation in standard incubation conditions versus in the presence of stimuli for capacitation predicts gamete competence. In men with IUI failure, in spite of the apparently normal semen parameters, a lower score for standard and capacitating profile was observed. This assay providing indication for sperm function, may aid clinicians towards the appropriate ART method. Supported by: BioAccelerate NYC Prize.
P-269 Tuesday, October 15, 2013 DOES SPERM DNA INTEGRITY PREDICT IVF OUTCOMES? D. E. Marchesi, Y. Pan, W. Di, E. Hawkins, A. Hershlag. Center for Human Reproduction, North Shore-LIJ Health System, Manhasset, NY. OBJECTIVE: To determine whether a correlation exists between the incidence of sperm DNA damage in semen, as measured by the Toluidine Blue Assay (TBA), and IVF/ICSI treatment outcomes. DESIGN: Semen samples from 420 patients undergoing IVF/ICSI treatment were assessed for DNA integrity, pre- and post-processing, using TBA which identifies poor quality sperm DNA as darkly stained. The proportion of high quality embryos transferred, implantation (fetal heart per embryo transferred) and live birth rates were available and assessed for 195 of these cases. MATERIALS AND METHODS: Semen was smeared on microscope slides and air dried in the raw form and after preparation by 3-layer density gradient centrifugation. Slides were fixed and stained with 0.05% Toluidine blue. At least two-hundred sperm heads per slide were examined under oil immersion light microscopy at 1000x by 2 technologists. Data were analyzed using multivariate logistic regression (Spearman Correlation, Wicoxon TwoSample, and Kruskal-Wallis testing). RESULTS: Preliminary results are summarized in Table 1. The incidence of DNA damage in processed sperm (1.5% 3.5%) was significantly lower than that in pre-processed specimens (5.7% 5.6%; p<0.05). There was no statistically significant positive or negative correlation between DNA integrity and the proportion of high quality embryos, embryo implantation or live birth rate.
CONCLUSION: The mean frequency of DNA damage in sperm in this unselected patient population is well below the reported threshold of 30%. Sperm processing by gradient centrifugation enriches sperm with good DNA integrity. No correlation was found between the incidence of DNA damage and IVF/ICSI outcomes; this finding argues against indiscreminant application of TBA or other sperm DNA integrity tests. P-270 Tuesday, October 15, 2013 CORRELATION BETWEEN SPERM HYALURONAN BINDING ASSAY (HBA), SPERM DNA FRAGMENTATION AND SPERM HIGH MAGNIFICATION. K. Lattes, R. Lafuente, G. Lopez, M. Brassesco. Andrology Lab, CIRH. Clınica Corachan. ANACER, Barcelona, Spain. OBJECTIVE: The aim of this study is to assess the correlation between HBA and two other male diagnostic tests. DESIGN: Observational prospective study. MATERIALS AND METHODS: The study includes 41 samples analysed at CIRH, Barcelona (Spain) following the protocol suggested by the manufacturer. Mean age of male patients was 35 years old. Sperm DNA fragmentation was assessed using the Halosperm kit (Halotech DNA SL, Madrid, Spain). To assess sperm morphology by high magnification we considered spermatozoa from groups III and IV, those spermatozoa with high vacuolization measured by inverted microscopy (Leica AM6000) at more than 6000x magnification. RESULTS: HBA test presents a higher negative correlation with high magnification (r¼0.16) being statistically significant (p¼0.012). On the contrary, the results of the HBA test are correlated positively with DNA fragmentation (r¼0.05) although not statistically significant (p>0.05). CONCLUSION: The obtained results show that samples enriched with spermatozoa of bad morphology present low percentage of positive HBA. Furthermore, the integrity of the DNA does not seem to be related directly to the degree of sperm maturity. The lack of correlation could be due to the sample size or, maybe because the outsourcing of the hyaluronate receptor is made regardless of the degree of DNA integrity. It would be desirable to increase the number of samples to verify this hypothesis. It is necessary to deepen the relationship between the three sperm parameters to assess their predictive power. P-271 Tuesday, October 15, 2013 EFFECTS OF SEMINAL GLUTATHIONE PEROXIDASE AND ITS RELATED NON-ENZYMATIC ANTIOXIDANTS ON SPERM DNA INTEGRETY AND SEMEN QUALITY. M. Ajina, F. Atig, I. J. Zidi, W. Denguezli, H. Khairi, A. Saad. University Hospitol Farhat Hached, Unity of Reproductive Medicine, Sousse, Tunisia. OBJECTIVE: The purpose of this study was to evaluate the levels of seminal oxidative biomarkers and sperm DNA fragmentation in Tunisian fertile and infertile men in order to assess their impact on semen quality. DESIGN: Prospective study of about 115 patients. MATERIALS AND METHODS: Semen samples from 115 infertile patients and 50 fertile men (normozoospermics) were analyzed for DNA fragmentation by TUNEL assay. Glutathione peroxidase activity, glutathione amounts and malondialdehyde concentrations were measured spectrophotometrically; however selenium levels were detected by furnace atomic absorption spectrophotometry. RESULTS: The most relevant findings of this work were: (1) a reduced antioxidant status was observed in seminal plasma of infertile men collected for this investigation. Indeed, seminal Glutathione peroxidase activity showed a significant elevation in controls when compared to the Oligozoospermics (P¼0.001). Selenium concentrations in the three abnormal groups
TABLE 1.
Pre-processing
High Quality Embryos (%) Implantation (%) Live Birth Rate (%)
FERTILITY & STERILITYÒ
Post-processing
>5.7% dark
<5.7% dark
p-value
>1.5% dark
<1.5% dark
p-value
36.6 21.9 33.3
45.4 15.3 23.8
0.85 0.09 0.09
42.9 24.7 35.1
42.9 14.9 24.1
0.73 0.42 0.4
S225
were significantly decreased when compared with fertile men (P<0.001). Significant increase of total glutathione mean were found in normozoospermics (55.3721.70 mmol/l) compared to oligozoospermics (41.777.02 mmol/l; P¼0.002) and teratozoospermics (44.8517.44 mmol/l; P¼0.01). (2) Fertile group established highly significant decline of seminal lipid peroxidation and sperm DNA fragmentation levels compared to the abnormal groups. The mean of sperm DNA fragmentation was 12.234.73% in the fertile group and the difference was highly significant (P<0.001) when compared to the all pathologic groups. (3) Positive correlations were found between enhanced seminal antioxidant profile and sperm DNA integrity. Nevertheless, seminal Malondialdehyde concentrations and sperm DNA fragmentation were correlated to poor semen quality. CONCLUSION: These data suggested that routine evaluation of seminal oxidative biomarkers and sperm DNA fragmentation in infertile men becomes recommended as a reliable prognostic tool for male infertility evaluation.
were transferred to the maturation medium to acquiring the nuclear competence to reach the second metaphase (MII) (N¼4 for each FSH group). Data were analyzed using Student’s t-tests. RESULTS: Follicles grown in the presence of low-FSH produced a greater percentage of viable retrieved oocytes than high-FSH (41 and 19%, respectively, P < 0.001). The diameters of oocytes retrieved were higher in the low than the high-FSH group (90.86 +/- 4.35, and 70.77 +/- 8.01 um, respectively, P < 0.05). A follicle grown in the presence of low-FSH produced an MII oocyte. CONCLUSION: The preliminary data present herein suggest that low FSH dose provide an adequate hormonal milieu for the in vitro development of bovine preantral follicles. Supported by: This project received financial support from two governamental brazilian agencies FAPESP (Fundac¸~ao de Amparo a Pesquisa do Estado de S~ao Paulo) and CNPq (Conselho Nacional de Desenvolvimento Cientıfico e Tecnologico).
P-272 Tuesday, October 15, 2013
P-274 Tuesday, October 15, 2013
AN INVESTIGATION OF DNA FRAGMENTATION AND MORPHOLOGICAL CHANGES CAUSED BY BACTERIA AND FUNGI IN HUMAN SPERMATOZOA. S. Sathiyanarayanan, D. Dwaraknath. Fertility, Nova IVI Fertility Centre, Chennai, India.
EFFECT OF VITRIFICATION ON MITOCHONDRIAL AND MICROVILLUS STRUCTURE OF HUMAN MATURE OOCYTES. S. B. Han, J. X. Liu, G. N. Huang. Chongqing Maternity and Children Health Care Hospital, Chongqing, China.
OBJECTIVE: The main aim of this study verifies the prevalence of semen Bacterial contamination and whether the contamination could cause the DNA fragmentation towards decreased sperm quality. MATERIALS AND METHODS: Requirements -Glass wares, Toludine blue staining, Papanicolaus staining. Culture media, Semen. RESULTS: 30 semen specimens to assess the sperm DNA fragmentation. Out of 30 samples, 20 samples were found to have defective DNA. Out of 30 semen samples, 17 samples were found to have defective sperm morphology (Head, mid piece, cytoplasmic droplet and tail) .All the 30 semen specimens were plated on culture media like blood agar. Nutrient agar, macconkey agar and sabourauds dextrose agar. It showed bacterial growth on 20 out of 30 semen samples.An in-vitro study of isolated Candida albicans from the semen, the resultsshowed the attachment of Candida albicans to spermatozoa through head. CONCLUSION: In conclusion, Candida albicans did not affect sperm parameters, but increased sperm chromatin packaging damage and apoptosis that might have caused fertilization failure after assisted reproduction treatment in this coulples. The DNA fragmentation and morphological changes of the spermatozoa might adversely affect the reproductive outcomes. Burrello,N., A.E.Calogero, A.Perdichizzi, M.D.Salmeri, R.Agata and Vicari,E. 2004. Inhibitation of Oocyte fertilization by assisted reproductive techniques and increased sperm DNA fragmentation in the presence of Candida albicans, a case report, Reprod. Biomed. online.8(5):569-73. Supported by: Ahmadi,A., 1999. Fertilizing ability of DNA-damaged spermatozoa. J.Exp. zool.284(6):696-704.
OBJECTIVE: To explore the effect of vitrification on mitochondrial and microvillus structure of human mature oocyte. DESIGN: Experimental study. MATERIALS AND METHODS: MII oocytes were obtained from clinical IVF cycles when the oocytes were failed to fertilization within 24 h after insemination in Chongqing Reproduction and Genetics Institute. All oocytes were randomly divided into 4 groups: non-frozen (fresh group), cultured for 0 h (0 h group), 2 h (2 h group) and 4 h (4 h group) after vitrification/thawing. Then oocytes were fixed for TEM analysis, oocyte fixation was performed in 1.5% glutaraldehyde in PBS solution. Post-fixed with 1% osmium tetroxide in PBS. Oocytes were then embedded in small blocks of 2% agar of about 5 51 mm in size, dehydrated in ascending series of ethanol, immersed in propylene oxide for solvent substitution, embedded in Epon 812 and Ultrathin, then cut with diamond knife. Examined and photographed by Transmission Electron Microscopes operating at 80 KV. RESULTS: The ultrastructure of human oocytes could be detected by TEM. Compared with fresh group, the structure of mitochondrial changed obviously in vitrification/thawing groups, in 0 h and 2 h group most of mitochondrial have tiny gaps at the ridge of mitochondrial and some of them also have vacuoles. In 4 h groups the tiny gaps at the ridge of mitochondrial disappeared, but vacuoles persistent existence. The shape of microvillus also changed obviously in vitrification oocytes compared with fresh oocytes. In 0 h and 2 h group, lots of microvillus appeared edema and the morphology of rest microvillus brush border became passivity. In 4 h groups the microvillus edema disappeared, and the microvillus passivity persistent existence. CONCLUSION: Our results suggest that vitrification injured mitochondrial and microvillus structures of human mature oocyte, which may be lead to the decrease of oocytes development potential, and affect the outcome of human assisted reproduction technology.
OOCYTE BIOLOGY P-273 Tuesday, October 15, 2013
P-275 Tuesday, October 15, 2013
EFFECT OF FOLLICLE-STIMULATING HORMONE (FSH) DOSE ON THE MATURATION OF BOVINE OOCYTES FROM PREANTRAL FOLLICLES GROWN IN ALGINATE-EXTRACELLULAR MATRIX GELS: A PILOT STUDY. A. C. J. S. Rosa-e-Silva, L. A. Batista, C. G. Gervasio, J. R. Campos, M. F. Silva-de-Sa, M. P. Bernuci. Gynecology and Obstetrics, Faculty of Medicine of Ribeir~ao Preto- University of S~ao Paulo, Ribeir~ao Preto, S~ao Paulo, Brazil.
A COMPARISON OF CLINICAL OUTCOMES AS A RESULT OF TRANSFER OF EMBRYOS DERIVED FROM OOCYTES WITH DIFFERENT STAGES OF MATURITY AT RETRIEVAL, IN hCGPRIMED IVM CYCLES. W.-Y. Son, J. Chung, D. Ezgi, S. Reinblatt, M. Dahan, H. Holzer. Dept. OB/GYN, MUHC Reproductive Center, Montreal, QC, Canada.
OBJECTIVE: In the present study we aimed to examine the effect of two different doses of FSH on the in vitro developmental competence of bovine preantral follicles. DESIGN: Prospective experimental study using the bovine as a model. MATERIALS AND METHODS: Mechanically isolated preantral follicles (150 to 250 mm in diameter) obtained from fresh ovarian tissue slices were encapsulated in 0.25% (w/v) calcium alginate-beads and cultured for 21 days in the presence of low (3 ng/mL; n ¼ 34) or high (100 ng/mL; n ¼ 37) doses of FSH. Follicle developmental competence was evaluated on the basis of the number and diameter of viable oocyte retrieved. Oocyte retrieval of the grown follicles was performed and oocytes with cumulus cells
S226
ASRM Abstracts
OBJECTIVE: It is known that in-vivo matured oocytes can be retrieved along with immature oocytes following hCG-primed IVM cycles, in humans. Since embryos derived from oocytes matured in vivo and in vitro are transferred simultaneously, there is limited information on the reproductive potential of these different oocytes. Therefore, the purpose of this study is to examine success with transferred embryos derived as a function of the oocyte maturity at the time of collection in hCG-primed IVM cycles. DESIGN: Retrospective cohort study. MATERIALS AND METHODS: Research ethics approval was obtained. 242 hCG-primed IVM cycles that underwent transfer (ET) of embryos produced from oocytes matured either in-vivo or in-vitro from 2006-2012 at
Vol. 100, No. 3, Supplement, September 2013
P-269 Tuesday, October 15, 2013 DOES SPERM DNA INTEGRITY PREDICT IVF OUTCOMES? D. E. Marchesi, Y. Pan, W. Di, E. Hawkins, A. Hershlag. Center for Human Reproduction, North Shore-LIJ Health System, Manhasset, NY. OBJECTIVE: To determine whether a correlation exists between the incidence of sperm DNA damage in semen, as measured by the Toluidine Blue Assay (TBA), and IVF/ICSI treatment outcomes. DESIGN: Semen samples from 420 patients undergoing IVF/ICSI treatment were assessed for DNA integrity, pre- and post-processing, using TBA which identifies poor quality sperm DNA as darkly stained. The proportion of high quality embryos transferred, implantation (fetal heart per embryo transferred) and live birth rates were available and assessed for 195 of these cases. MATERIALS AND METHODS: Semen was smeared on microscope slides and air dried in the raw form and after preparation by 3-layer density gradient centrifugation. Slides were fixed and stained with 0.05% Toluidine blue. At least two-hundred sperm heads per slide were examined under oil immersion light microscopy at 1000x by 2 technologists. Data were analyzed using multivariate logistic regression (Spearman Correlation, Wicoxon TwoSample, and Kruskal-Wallis testing). RESULTS: Preliminary results are summarized in Table 1. The incidence of DNA damage in processed sperm (1.5% 3.5%) was significantly lower than that in pre-processed specimens (5.7% 5.6%; p<0.05). There was no statistically significant positive or negative correlation between DNA integrity and the proportion of high quality embryos, embryo implantation or live birth rate.
CONCLUSION: The mean frequency of DNA damage in sperm in this unselected patient population is well below the reported threshold of 30%. Sperm processing by gradient centrifugation enriches sperm with good DNA integrity. No correlation was found between the incidence of DNA damage and IVF/ICSI outcomes; this finding argues against indiscreminant application of TBA or other sperm DNA integrity tests. P-270 Tuesday, October 15, 2013 CORRELATION BETWEEN SPERM HYALURONAN BINDING ASSAY (HBA), SPERM DNA FRAGMENTATION AND SPERM HIGH MAGNIFICATION. K. Lattes, R. Lafuente, G. Lopez, M. Brassesco. Andrology Lab, CIRH. Clınica Corachan. ANACER, Barcelona, Spain. OBJECTIVE: The aim of this study is to assess the correlation between HBA and two other male diagnostic tests. DESIGN: Observational prospective study. MATERIALS AND METHODS: The study includes 41 samples analysed at CIRH, Barcelona (Spain) following the protocol suggested by the manufacturer. Mean age of male patients was 35 years old. Sperm DNA fragmentation was assessed using the Halosperm kit (Halotech DNA SL, Madrid, Spain). To assess sperm morphology by high magnification we considered spermatozoa from groups III and IV, those spermatozoa with high vacuolization measured by inverted microscopy (Leica AM6000) at more than 6000x magnification. RESULTS: HBA test presents a higher negative correlation with high magnification (r¼0.16) being statistically significant (p¼0.012). On the contrary, the results of the HBA test are correlated positively with DNA fragmentation (r¼0.05) although not statistically significant (p>0.05). CONCLUSION: The obtained results show that samples enriched with spermatozoa of bad morphology present low percentage of positive HBA. Furthermore, the integrity of the DNA does not seem to be related directly to the degree of sperm maturity. The lack of correlation could be due to the sample size or, maybe because the outsourcing of the hyaluronate receptor is made regardless of the degree of DNA integrity. It would be desirable to increase the number of samples to verify this hypothesis. It is necessary to deepen the relationship between the three sperm parameters to assess their predictive power. P-271 Tuesday, October 15, 2013 EFFECTS OF SEMINAL GLUTATHIONE PEROXIDASE AND ITS RELATED NON-ENZYMATIC ANTIOXIDANTS ON SPERM DNA INTEGRETY AND SEMEN QUALITY. M. Ajina, F. Atig, I. J. Zidi, W. Denguezli, H. Khairi, A. Saad. University Hospitol Farhat Hached, Unity of Reproductive Medicine, Sousse, Tunisia. OBJECTIVE: The purpose of this study was to evaluate the levels of seminal oxidative biomarkers and sperm DNA fragmentation in Tunisian fertile and infertile men in order to assess their impact on semen quality. DESIGN: Prospective study of about 115 patients. MATERIALS AND METHODS: Semen samples from 115 infertile patients and 50 fertile men (normozoospermics) were analyzed for DNA fragmentation by TUNEL assay. Glutathione peroxidase activity, glutathione amounts and malondialdehyde concentrations were measured spectrophotometrically; however selenium levels were detected by furnace atomic absorption spectrophotometry. RESULTS: The most relevant findings of this work were: (1) a reduced antioxidant status was observed in seminal plasma of infertile men collected for this investigation. Indeed, seminal Glutathione peroxidase activity showed a significant elevation in controls when compared to the Oligozoospermics (P¼0.001). Selenium concentrations in the three abnormal groups
TABLE 1.
Pre-processing
High Quality Embryos (%) Implantation (%) Live Birth Rate (%)
FERTILITY & STERILITYÒ
Post-processing
>5.7% dark
<5.7% dark
p-value
>1.5% dark
<1.5% dark
p-value
36.6 21.9 33.3
45.4 15.3 23.8
0.85 0.09 0.09
42.9 24.7 35.1
42.9 14.9 24.1
0.73 0.42 0.4
S225
were significantly decreased when compared with fertile men (P<0.001). Significant increase of total glutathione mean were found in normozoospermics (55.3721.70 mmol/l) compared to oligozoospermics (41.777.02 mmol/l; P¼0.002) and teratozoospermics (44.8517.44 mmol/l; P¼0.01). (2) Fertile group established highly significant decline of seminal lipid peroxidation and sperm DNA fragmentation levels compared to the abnormal groups. The mean of sperm DNA fragmentation was 12.234.73% in the fertile group and the difference was highly significant (P<0.001) when compared to the all pathologic groups. (3) Positive correlations were found between enhanced seminal antioxidant profile and sperm DNA integrity. Nevertheless, seminal Malondialdehyde concentrations and sperm DNA fragmentation were correlated to poor semen quality. CONCLUSION: These data suggested that routine evaluation of seminal oxidative biomarkers and sperm DNA fragmentation in infertile men becomes recommended as a reliable prognostic tool for male infertility evaluation.
were transferred to the maturation medium to acquiring the nuclear competence to reach the second metaphase (MII) (N¼4 for each FSH group). Data were analyzed using Student’s t-tests. RESULTS: Follicles grown in the presence of low-FSH produced a greater percentage of viable retrieved oocytes than high-FSH (41 and 19%, respectively, P < 0.001). The diameters of oocytes retrieved were higher in the low than the high-FSH group (90.86 +/- 4.35, and 70.77 +/- 8.01 um, respectively, P < 0.05). A follicle grown in the presence of low-FSH produced an MII oocyte. CONCLUSION: The preliminary data present herein suggest that low FSH dose provide an adequate hormonal milieu for the in vitro development of bovine preantral follicles. Supported by: This project received financial support from two governamental brazilian agencies FAPESP (Fundac¸~ao de Amparo a Pesquisa do Estado de S~ao Paulo) and CNPq (Conselho Nacional de Desenvolvimento Cientıfico e Tecnologico).
P-272 Tuesday, October 15, 2013
P-274 Tuesday, October 15, 2013
AN INVESTIGATION OF DNA FRAGMENTATION AND MORPHOLOGICAL CHANGES CAUSED BY BACTERIA AND FUNGI IN HUMAN SPERMATOZOA. S. Sathiyanarayanan, D. Dwaraknath. Fertility, Nova IVI Fertility Centre, Chennai, India.
EFFECT OF VITRIFICATION ON MITOCHONDRIAL AND MICROVILLUS STRUCTURE OF HUMAN MATURE OOCYTES. S. B. Han, J. X. Liu, G. N. Huang. Chongqing Maternity and Children Health Care Hospital, Chongqing, China.
OBJECTIVE: The main aim of this study verifies the prevalence of semen Bacterial contamination and whether the contamination could cause the DNA fragmentation towards decreased sperm quality. MATERIALS AND METHODS: Requirements -Glass wares, Toludine blue staining, Papanicolaus staining. Culture media, Semen. RESULTS: 30 semen specimens to assess the sperm DNA fragmentation. Out of 30 samples, 20 samples were found to have defective DNA. Out of 30 semen samples, 17 samples were found to have defective sperm morphology (Head, mid piece, cytoplasmic droplet and tail) .All the 30 semen specimens were plated on culture media like blood agar. Nutrient agar, macconkey agar and sabourauds dextrose agar. It showed bacterial growth on 20 out of 30 semen samples.An in-vitro study of isolated Candida albicans from the semen, the resultsshowed the attachment of Candida albicans to spermatozoa through head. CONCLUSION: In conclusion, Candida albicans did not affect sperm parameters, but increased sperm chromatin packaging damage and apoptosis that might have caused fertilization failure after assisted reproduction treatment in this coulples. The DNA fragmentation and morphological changes of the spermatozoa might adversely affect the reproductive outcomes. Burrello,N., A.E.Calogero, A.Perdichizzi, M.D.Salmeri, R.Agata and Vicari,E. 2004. Inhibitation of Oocyte fertilization by assisted reproductive techniques and increased sperm DNA fragmentation in the presence of Candida albicans, a case report, Reprod. Biomed. online.8(5):569-73. Supported by: Ahmadi,A., 1999. Fertilizing ability of DNA-damaged spermatozoa. J.Exp. zool.284(6):696-704.
OBJECTIVE: To explore the effect of vitrification on mitochondrial and microvillus structure of human mature oocyte. DESIGN: Experimental study. MATERIALS AND METHODS: MII oocytes were obtained from clinical IVF cycles when the oocytes were failed to fertilization within 24 h after insemination in Chongqing Reproduction and Genetics Institute. All oocytes were randomly divided into 4 groups: non-frozen (fresh group), cultured for 0 h (0 h group), 2 h (2 h group) and 4 h (4 h group) after vitrification/thawing. Then oocytes were fixed for TEM analysis, oocyte fixation was performed in 1.5% glutaraldehyde in PBS solution. Post-fixed with 1% osmium tetroxide in PBS. Oocytes were then embedded in small blocks of 2% agar of about 5 51 mm in size, dehydrated in ascending series of ethanol, immersed in propylene oxide for solvent substitution, embedded in Epon 812 and Ultrathin, then cut with diamond knife. Examined and photographed by Transmission Electron Microscopes operating at 80 KV. RESULTS: The ultrastructure of human oocytes could be detected by TEM. Compared with fresh group, the structure of mitochondrial changed obviously in vitrification/thawing groups, in 0 h and 2 h group most of mitochondrial have tiny gaps at the ridge of mitochondrial and some of them also have vacuoles. In 4 h groups the tiny gaps at the ridge of mitochondrial disappeared, but vacuoles persistent existence. The shape of microvillus also changed obviously in vitrification oocytes compared with fresh oocytes. In 0 h and 2 h group, lots of microvillus appeared edema and the morphology of rest microvillus brush border became passivity. In 4 h groups the microvillus edema disappeared, and the microvillus passivity persistent existence. CONCLUSION: Our results suggest that vitrification injured mitochondrial and microvillus structures of human mature oocyte, which may be lead to the decrease of oocytes development potential, and affect the outcome of human assisted reproduction technology.
OOCYTE BIOLOGY P-273 Tuesday, October 15, 2013
P-275 Tuesday, October 15, 2013
EFFECT OF FOLLICLE-STIMULATING HORMONE (FSH) DOSE ON THE MATURATION OF BOVINE OOCYTES FROM PREANTRAL FOLLICLES GROWN IN ALGINATE-EXTRACELLULAR MATRIX GELS: A PILOT STUDY. A. C. J. S. Rosa-e-Silva, L. A. Batista, C. G. Gervasio, J. R. Campos, M. F. Silva-de-Sa, M. P. Bernuci. Gynecology and Obstetrics, Faculty of Medicine of Ribeir~ao Preto- University of S~ao Paulo, Ribeir~ao Preto, S~ao Paulo, Brazil.
A COMPARISON OF CLINICAL OUTCOMES AS A RESULT OF TRANSFER OF EMBRYOS DERIVED FROM OOCYTES WITH DIFFERENT STAGES OF MATURITY AT RETRIEVAL, IN hCGPRIMED IVM CYCLES. W.-Y. Son, J. Chung, D. Ezgi, S. Reinblatt, M. Dahan, H. Holzer. Dept. OB/GYN, MUHC Reproductive Center, Montreal, QC, Canada.
OBJECTIVE: In the present study we aimed to examine the effect of two different doses of FSH on the in vitro developmental competence of bovine preantral follicles. DESIGN: Prospective experimental study using the bovine as a model. MATERIALS AND METHODS: Mechanically isolated preantral follicles (150 to 250 mm in diameter) obtained from fresh ovarian tissue slices were encapsulated in 0.25% (w/v) calcium alginate-beads and cultured for 21 days in the presence of low (3 ng/mL; n ¼ 34) or high (100 ng/mL; n ¼ 37) doses of FSH. Follicle developmental competence was evaluated on the basis of the number and diameter of viable oocyte retrieved. Oocyte retrieval of the grown follicles was performed and oocytes with cumulus cells
S226
ASRM Abstracts
OBJECTIVE: It is known that in-vivo matured oocytes can be retrieved along with immature oocytes following hCG-primed IVM cycles, in humans. Since embryos derived from oocytes matured in vivo and in vitro are transferred simultaneously, there is limited information on the reproductive potential of these different oocytes. Therefore, the purpose of this study is to examine success with transferred embryos derived as a function of the oocyte maturity at the time of collection in hCG-primed IVM cycles. DESIGN: Retrospective cohort study. MATERIALS AND METHODS: Research ethics approval was obtained. 242 hCG-primed IVM cycles that underwent transfer (ET) of embryos produced from oocytes matured either in-vivo or in-vitro from 2006-2012 at
Vol. 100, No. 3, Supplement, September 2013