- Email: [email protected]
Evaluation of human oocytes ageing by focal plane array (FPA) fourier transform infrared (FT-IR) imaging spectroscopy
Mitochondrial distribution was evaluated using MitoTracker. Oocyte ATP concentration was assessed using luciferin-luciferase biolumisescent assay kit. RESULTS: The survival rate of vw GV oocytes was 92% (270/300). GVBD in vw oocytes was delayed when compared to controls. Similarly, vw oocyte had lower maturation rates than controls (60.6% vs. 68.3%, respectively). The typical redistribution of mitochondria during maturation from the center of the oocyte into an even pattern of distribution was altered in the vw oocytes. At 24 hr, the majority (87%) of control oocytes showed even mitochondria distribution. In contrast, 37% of vw oocytes had mitochondria aggregates. Oocyte ATP concentration plummeted at 0 hr following vitrification, but gradually increased with time, and maturity reaching comparable concentration with controls at 24 hr. CONCLUSION: Despite high survival rates, vw GVoocytes showed a delay in maturation. Abnormal mitochondrial distribution and lower ATP concentration were observed following vitrification and were associated with the delayed maturation. Thus, our data indicate that vitrification-induced damage to mitochondrial bioenergetic activity correlates with impeded in vitro oocyte maturation. Supported by: Vincent Memorial Fund.
P-446 Wednesday, October 19, 2011 DEHYDROEPIANDROSTERONE SUPPLEMENTATION MAY IMPROVE OVULATION RATES BUT NOT OOCYTE QUALITY. P. T. Jimenez, A. I. Frolova, M. M. Chi, K. H. Moley. Obstetrics & Gynecology, Washington University School of Medicine, St. Louis, MO. OBJECTIVE: Multiple case series suggest that dehydroepiandrosterone (DHEA) may increase oocyte yield in women undergoing in vitro fertilization with a prior poor response to gonadotropins. ATP is a marker of oocyte metabolism and is believed to be associated with oocyte quality. The purpose of this study is to determine the effects of DHEA on oocyte yield and competence in an older-aged mouse model. DESIGN: Experimental animal study. MATERIALS AND METHODS: Nine-month-old FVB female mice were fed regular rodent chow or chow supplemented with 0.01% DHEA or 0.1% DHEA for two weeks. Mice were superovulated and serum estradiol levels were measured. Ovulation rates were determined following hCG administration. ATP levels were measured in oocytes following superovulation. RESULTS: Serum estradiol concentrations following stimulation did not differ between the control (84.2 4.4 pg/ml) and 0.01% DHEA groups (78.3 1.5 pg/ml). There was a trend for higher estradiol concentrations in the 0.1% DHEA group (108.7 10.5 pg/ml; P¼0.08). There were no statistical differences in the average ovulation rates between the three groups. However, all mice exposed to DHEA ovulated whereas the controls had higher rates of anovulation. ATP levels in single denuded oocytes were also altered. CONCLUSION: These findings suggest that DHEA supplementation does not improve estradiol concentrations in response to gonadotropins. Although DHEA may lead to slightly higher ovulation rates, oocyte metabolism is abnormal. Future studies will examine the effects of DHEA on oocyte mitochondria and apoptosis. Supported by: T32 HD040135.
sessment of DNA status (TOTO-3), the oocytes were grouped according to their maturation stage: GV, Ana/Telophase I and metaphase II. The images were captured under Confocal microscopy and processed for fluorescence density scoring. In the same conditions, other pool of oocytes were processed for western blot. ANOVA was performed for statistical analysis. RESULTS: The relative expression of pCdc2 have revealed significant differences during oocyte maturation. Thus, the expression levels were: GV (20,805 1165), telophase I (90,033 9689) and MII (1683 840) (p % 0.05). These results were validated by western blot technique. According to our results, non-activated oocytes after ICSI were considered as immature when pCdc2(Y15) had an elevated expression. Unfertilized oocytes after ICSI revealed 60.2% of activation failure with PCC (44.7% had elevated levels of pCdc2 and 15.5% had lower levels). Then, 31.6% had activation failure with sperm DNA decondensation failure (6.7% had high levels of pCdc2, and 21.9% had lower levels). Finally, three oocytes (2.9%) showed the presence of sperm in the perivitelline space, while 5.3% of cases depicted a first metaphase arrest. CONCLUSION: There is a significant increase of pCdc2 during the transition from MI to MII stages. Arrested MII oocytes had a significantly decrease of pCdc2. The oocyte activation failure with PCC is highly influenced by the oocyte cytoplasm immaturity. Supported by: CEGYR Foundation.
P-448 Wednesday, October 19, 2011 EVALUATION OF HUMAN OOCYTES AGEING BY FOCAL PLANE ARRAY (FPA) FOURIER TRANSFORM INFRARED (FT-IR) IMAGING SPECTROSCOPY. G. Gioacchini, O. Carnevali, E. Giorgini, L. Vaccari, V. Bianchi, A. Borini. Tecnobios Procreazione, Centre for Reproductive Health, Bologna, Italy; Scienze del Mare, Universita Politecnica delle Marche, Ancona, Italy; Dipartimento di Idraulica, Strade, Ambiente e Chimica ISAC, Sezione Chimica, Universita Politecnica delle Marche, Ancona, Italy; SISSI Beamline, ELETTRA Synchrotron Light Laboratory, Basovizza, Trieste, Italy. OBJECTIVE: Oocytes quality may change as a consequence of normal aging and their potential be compromised in older women. The aim of this study is to investigate possible changes in macromolecules composition occurring into human oocytes at different ages. DESIGN: Supernumerary metaphaseII oocytes were analysed by the FPAFT-IR technique in patients enrolled in our in vitro fertilization program. MATERIALS AND METHODS: Oocytes were obtained after controlled ovarian stimulation from patients at different ages (32, 37 and 39 years old). Measurements were carriedout by using a Bruker VERTEX70 interferometer, generating chemical maps based on biocomponents localization. By means of multivariate analysis, it was possible to segregate the spectral data in clusters representing nucleus, cytoplasm and membrane and to analyse the relative average spectra (representative bands were reported in Table 1).
Table.1 BIOLOGICAL PROCESS
P-447 Wednesday, October 19, 2011 pCDC2 (Y15) EXPRESSION DURING OOCYTE MATURATION- IMPLICATIONS IN FERTILIZATION FAILURE AFTER ICSI. C. Alvarez Sed o, H. Uriondo, M. Lavolpe, F. Noblia, M. Baronio, F. Nodar. Centro de Estudios en Ginecologıa y Reproduccion (CEGyR), Capital Federal, Buenos Aires, Argentina. OBJECTIVE: To evaluate the expression of pCdc2 during oocyte maturation and fertilization failures after ICSI. Determine the oocyte cytoplasmic competence (pCdc2 levels) with premature sperm chromatin condensation (PCC) incidence. DESIGN: Descriptive research. MATERIALS AND METHODS: Seventy-seven supernumerary oocytes and 102 oocytes with fertilization failure after ICSI (ART treatments) were donated by informed consent for this research. Oocytes at different maturation stages and others with fertilization failure were processed for tubulin and pCdc2(Y15) immunostaining. After the as-
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Abstracts
Protein degradation Changes in protein secondary structures Carbohydrates content RNA amount Oxidative process Lipid content
BAND RATIOS (vibrational modes) 2925/2957cm-1 (CH2 / CH3) 1720-1470cm-1 (helical/bstructures) 1048/1235cm-1 (COO/ PO2) 990/970cm-1 (RNA/ DNA) 1397/1455cm-1 (COO + CH2)/ (CH2/3) 1727/1742cm-1 (cholesterol/ triglycerides and phospholipids)
32 y 37 y 39 y (a.u.) (a.u.) (a.u.) 1.00 1.00
0.97 1.42
0.77 0.69
1.00 1.00 1.00
0.45 0.48 1.08
0.42 0.31 1.19
1.00
0.42
0.26
RESULTS: Differences were found according to the patients age. In older patients was observed a degradation of the protein and changes in their secondary structure. Moreover, in aged oocytes it was evidenced a decrease in carbohydrates, RNAand lipid contents and an increase in the oxidative process.
Vol. 96., No. 3, Supplement, September 2011
CONCLUSION: This study represents the first vibrational approach to evaluate the macromolecular changes associated with oocyte ageing. So, FPAFT-IR imaging could be considered a powerful technique to provide a biochemical fingerprint. This novel approach may represent a synergic support to evaluate oocyte quality. Supported by: University of Ancona founding 2010 to O. Carnevali and E. Giorgini.
P-449 Wednesday, October 19, 2011
TE D
IMPAIRED DNA REPAIR AS THE ROOT CAUSE OF OOCYTE AGING. K. Oktay, E. Heytens, R. Soleimani, V. Baltaci, S. Goswami. Institute for Fertility Preservation, Obstetrics & Gynecology, New York Medical College, Valhalla, NY; Cell Biology & Anatomy, New York Medical College, New York, NY; Department of Human Genetics, Istanbul Bilim University, Istanbul, TR, Turkey; Department of Biology, Yeshiva University, New York, NY.
RE
TR
AC
OBJECTIVE: The molecular mechanisms behind age-related decline in oocyte quantity and quality are unknown. Recent work indicated that women with BRCA mutations may have lower egg reserve and experience menopause earlier. Because BRCA is a key double strand DNA break (DSB) repair gene, we hypothesized that impaired DSB repair and resulting accumulation of DSBs is responsible for oocyte aging. DESIGN: Experimental. MATERIALS AND METHODS: Given the age-related decline in oocyte yield after ovarian stimulation (41 15 at 4 wks to 11 5 at 9-mo), we used ‘‘young’’ (4-wk-old) and ‘‘old’’ (9-mo-old) female FVB mice (n ¼ 24). Either primordial follicles (PDF) were assessed for DSBs by gH2AX immunostaining in ovarian sections, or GVoocytes by confocal microscopic quantification of gH2AX foci. The expression of ATM-mediated DSB repair pathway genes were analyzed by QRT-PCR in PDF captured by laser dissection (LD) as well as GVoocytes. The same was also analyzed in single human oocytes (n ¼ 20) by QRT-PCR from young (age<27) and old (age>37) subjects. RESULTS: gH2AX-positive PDF increased significantly in old mice compared to young (16 3 vs. 10 2; P¼0.002). Mean number of gH2AX foci was also significantly higher in GVoocytes of old vs. young (1,279 594 vs. 373 258; P¼0.01). By QRT-PCR from mouse GV oocytes, the expression of MRE11, a gene involved in sensing DSBs and activating repair via the ATM- pathway, was downregulated by 50-99% with age. In PDF from old mice, the expression of BRCA1 was downregulated by 77-89%. Consistent with findings in rodent oocytes, QRT-PCR of single human oocytes showed that the key genes in the DSB repair pathway was down-regulated with age. CONCLUSION: These rodent and human data support our novel hypothesis that oocyte aging is associated with accumulation of potentially lethal and mutagenic DSBs due to the impairment of DSB repair in the aging oocyte. Active and efficient DNA repair appears to be vital for oocyte health. These findings can be paradigm-shifting in understanding oocyte aging. Supported by: RO1 HD053112.
P-450 Wednesday, October 19, 2011 ROLE OF MELATONIN IN PREVENTING HYPOCHLOROUS ACID INDUCED ALTERATIONS IN MICROTUBULE AND CHROMOSOMAL STRUCTURE IN METAPHASE-II MOUSE OOCYTES IN VITRO. J. Banerjee, D. Maitra, F. Shaeib, G. M. Saed, M. P. Diamond, H. Abu-Soud. Obstetrics and Gynecology, Division of Reproductive Endocrinology and Infertility, Wayne State University, Detroit, MI. OBJECTIVE: Recently, we have shown that melatonin, a pineal gland hormone, can prevent hypochlorous acid (HOCl) mediated protein aggregation and hemeprotein destruction. We have demonstrated that HOCl may alter metaphase-II oocyte microtubule and chromosomal alignment. The objective of the study was to test whether melatonin prevents the impairment of oocyte quality induced by HOCl in vitro. DESIGN: Dose response study. MATERIALS AND METHODS: Metaphase–II mouse oocytes, obtained commercially, were incubated in HTF for 60 minutes. Oocytes were grouped as: control, melatonin (150, 200 nmol/mL), HOCl (10, 20, 50, 100 nmol/mL) and HOCl (50 nmol/mL) pretreated with 150 and 200 nmol/mL of melatonin. Microtubule and chromosomal alignment was studied on fixed and stained oocytes and then scored by two observers based on a previously published scoring system (Fert Stert 1222,Vol 88,Suppl 2, October 2007). Pearson
FERTILITY & STERILITYÒ
Chi-square test, Fisher’s Exact test were used compare outcomes between controls and treated groups and also amongst each group. RESULTS: Poor scores for the spindle and chromosomes increased significantly at 50 nmol/mL of HOCl (P<0.001). Oocytes treated with melatonin only at 150 and 200 nmol/mL showed no changes; significant differences (P<0.001) were observed when oocytes exposed to 50 nmol/mL of HOCl were compared to oocytes pretreated with melatonin 200 nmol/mL.Fifty percent of the oocytes demonstrated good scores both in microtubule and chromosomal alterations when pretreated with melatonin at 150 nmol/mL compared to 0% in the only HOCl group. CONCLUSION: HOCl alters the metaphase-II mouse oocyte spindle and chromosomal alignment in a dose dependant manner, a potential cause of poor oocyte quality in patients with endometriosis. Melatonin prevented the HOCl-mediated spindle and chromosomal damage. Melatonin supplementation could be an attractive therapeutic option to prevent oocyte damage in endometriosis or inflammatory diseases to improve fertility and reproductive outcome. Supported by: Wayne State University.
OOCYTE MATURATION P-451 Wednesday, October 19, 2011 IVM FOR WOMEN WITH POLYCYSTIC OVARIES? A CASE-CONTROL STUDY. T. J. Child, J. Craig, K. Turner, E. McVeigh, M. Fatum, A.S. Gremeau. Oxford Fertility Unit, Oxford, Oxfordshire, United Kingdom. OBJECTIVE: The in-vitro maturation (IVM) of immature oocytes retrieved from unstimulated ovaries is a promising treatment, particularly for women with polycystic ovaries (PCO). IVM is safer, simpler and cheaper than IVF. There are no published RCTs comparing IVM and IVF and only one case-control study from 2002. The aim of this study is to compare the outcome of unstimulated IVM and routine IVF/ICSI for women with ultrasonographic PCO undergoing treatment at the Oxford Fertility Unit. DESIGN: Case-control study. MATERIALS AND METHODS: The study included 250 women with PCO undergoing their first IVM (n ¼ 125) or IVF (n ¼ 125) cycle, matched for age, infertility diagnosis and ovulatory status (ovulatory PCO or anovulatory PCOS). The primary outcome measure was live birth rate per cycle. IVM cycles were unstimulated and hCG was administered 35-40h before trans-vaginal immature oocyte retrieval. Oocytes were matured in-vitro for 24-48h before insemination by ICSI. Endometrial priming with estradiol and progesterone was commenced following oocyte retrieval and 1-2 embryos transferred on day 2-5 of embryo development. Standard long protocol IVF/ICSI was used in the control group. RESULTS: The mean age was 32.8 years in both groups. A mean (SD) of 17.4 (9.6) and 15.3 (7.4) eggs were retrieved in the IVM and IVF groups, respectively (P<0.05). 66% of IVM eggs matured in-vitro. The live birth rate per retrieval was significantly lower for IVM compared to IVF (18.4% v 42.4%; P<0.001), as were the implantation (14.2% v 37.2%; P<0.001) and clinical pregnancy rates (21.6% vs. 52.0%, P<0.001). The OHSS rate was significantly higher in the IVF group (8.8%) compared to none in the IVM group. CONCLUSION: IVM is a safer and simpler alternative to conventional IVF for women with PCO or PCOS. IVM avoids the cost of gonadotropin stimulation and the risk of OHSS, but has a significantly lower live birth rate than IVF.
P-452 Wednesday, October 19, 2011 EFFECTS OF ANDROGEN SUBSTRATE ON BOVINE CUMULUSOOCYTE COMPLEXES STEROIDOGENESIS DURING IN VITRO MATURATION. A. C. J. S. Rosa-e-Silva, D. L. Bulgarelli, A. A. Vireque, C. P. Pitangui, M. P. Bernuci, M. F. Silva-de-Sa. Gynecology and Obstetrics, Faculty of Medicine of Ribeir~ao Preto - University of S~ao Paulo, Ribeir~ao Preto, S~ao Paulo, Brazil. OBJECTIVE: Ovarian steroids are known as important factors on the route of oocytes development. Since serum-supplemented in vitro maturation (IVM) media decreases estradiol secretion by bovine cumulus-oocyte complexes (COCs), the aim of this study was to assess whether this effect is caused by deficiency of substrate or by low cumulus cells aromatase activity. DESIGN: Prospective experimental study with bovine oocytes.
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P-446 Wednesday, October 19, 2011 DEHYDROEPIANDROSTERONE SUPPLEMENTATION MAY IMPROVE OVULATION RATES BUT NOT OOCYTE QUALITY. P. T. Jimenez, A. I. Frolova, M. M. Chi, K. H. Moley. Obstetrics & Gynecology, Washington University School of Medicine, St. Louis, MO. OBJECTIVE: Multiple case series suggest that dehydroepiandrosterone (DHEA) may increase oocyte yield in women undergoing in vitro fertilization with a prior poor response to gonadotropins. ATP is a marker of oocyte metabolism and is believed to be associated with oocyte quality. The purpose of this study is to determine the effects of DHEA on oocyte yield and competence in an older-aged mouse model. DESIGN: Experimental animal study. MATERIALS AND METHODS: Nine-month-old FVB female mice were fed regular rodent chow or chow supplemented with 0.01% DHEA or 0.1% DHEA for two weeks. Mice were superovulated and serum estradiol levels were measured. Ovulation rates were determined following hCG administration. ATP levels were measured in oocytes following superovulation. RESULTS: Serum estradiol concentrations following stimulation did not differ between the control (84.2 4.4 pg/ml) and 0.01% DHEA groups (78.3 1.5 pg/ml). There was a trend for higher estradiol concentrations in the 0.1% DHEA group (108.7 10.5 pg/ml; P¼0.08). There were no statistical differences in the average ovulation rates between the three groups. However, all mice exposed to DHEA ovulated whereas the controls had higher rates of anovulation. ATP levels in single denuded oocytes were also altered. CONCLUSION: These findings suggest that DHEA supplementation does not improve estradiol concentrations in response to gonadotropins. Although DHEA may lead to slightly higher ovulation rates, oocyte metabolism is abnormal. Future studies will examine the effects of DHEA on oocyte mitochondria and apoptosis. Supported by: T32 HD040135.
sessment of DNA status (TOTO-3), the oocytes were grouped according to their maturation stage: GV, Ana/Telophase I and metaphase II. The images were captured under Confocal microscopy and processed for fluorescence density scoring. In the same conditions, other pool of oocytes were processed for western blot. ANOVA was performed for statistical analysis. RESULTS: The relative expression of pCdc2 have revealed significant differences during oocyte maturation. Thus, the expression levels were: GV (20,805 1165), telophase I (90,033 9689) and MII (1683 840) (p % 0.05). These results were validated by western blot technique. According to our results, non-activated oocytes after ICSI were considered as immature when pCdc2(Y15) had an elevated expression. Unfertilized oocytes after ICSI revealed 60.2% of activation failure with PCC (44.7% had elevated levels of pCdc2 and 15.5% had lower levels). Then, 31.6% had activation failure with sperm DNA decondensation failure (6.7% had high levels of pCdc2, and 21.9% had lower levels). Finally, three oocytes (2.9%) showed the presence of sperm in the perivitelline space, while 5.3% of cases depicted a first metaphase arrest. CONCLUSION: There is a significant increase of pCdc2 during the transition from MI to MII stages. Arrested MII oocytes had a significantly decrease of pCdc2. The oocyte activation failure with PCC is highly influenced by the oocyte cytoplasm immaturity. Supported by: CEGYR Foundation.
P-448 Wednesday, October 19, 2011 EVALUATION OF HUMAN OOCYTES AGEING BY FOCAL PLANE ARRAY (FPA) FOURIER TRANSFORM INFRARED (FT-IR) IMAGING SPECTROSCOPY. G. Gioacchini, O. Carnevali, E. Giorgini, L. Vaccari, V. Bianchi, A. Borini. Tecnobios Procreazione, Centre for Reproductive Health, Bologna, Italy; Scienze del Mare, Universita Politecnica delle Marche, Ancona, Italy; Dipartimento di Idraulica, Strade, Ambiente e Chimica ISAC, Sezione Chimica, Universita Politecnica delle Marche, Ancona, Italy; SISSI Beamline, ELETTRA Synchrotron Light Laboratory, Basovizza, Trieste, Italy. OBJECTIVE: Oocytes quality may change as a consequence of normal aging and their potential be compromised in older women. The aim of this study is to investigate possible changes in macromolecules composition occurring into human oocytes at different ages. DESIGN: Supernumerary metaphaseII oocytes were analysed by the FPAFT-IR technique in patients enrolled in our in vitro fertilization program. MATERIALS AND METHODS: Oocytes were obtained after controlled ovarian stimulation from patients at different ages (32, 37 and 39 years old). Measurements were carriedout by using a Bruker VERTEX70 interferometer, generating chemical maps based on biocomponents localization. By means of multivariate analysis, it was possible to segregate the spectral data in clusters representing nucleus, cytoplasm and membrane and to analyse the relative average spectra (representative bands were reported in Table 1).
Table.1 BIOLOGICAL PROCESS
P-447 Wednesday, October 19, 2011 pCDC2 (Y15) EXPRESSION DURING OOCYTE MATURATION- IMPLICATIONS IN FERTILIZATION FAILURE AFTER ICSI. C. Alvarez Sed o, H. Uriondo, M. Lavolpe, F. Noblia, M. Baronio, F. Nodar. Centro de Estudios en Ginecologıa y Reproduccion (CEGyR), Capital Federal, Buenos Aires, Argentina. OBJECTIVE: To evaluate the expression of pCdc2 during oocyte maturation and fertilization failures after ICSI. Determine the oocyte cytoplasmic competence (pCdc2 levels) with premature sperm chromatin condensation (PCC) incidence. DESIGN: Descriptive research. MATERIALS AND METHODS: Seventy-seven supernumerary oocytes and 102 oocytes with fertilization failure after ICSI (ART treatments) were donated by informed consent for this research. Oocytes at different maturation stages and others with fertilization failure were processed for tubulin and pCdc2(Y15) immunostaining. After the as-
S238
Abstracts
Protein degradation Changes in protein secondary structures Carbohydrates content RNA amount Oxidative process Lipid content
BAND RATIOS (vibrational modes) 2925/2957cm-1 (CH2 / CH3) 1720-1470cm-1 (helical/bstructures) 1048/1235cm-1 (COO/ PO2) 990/970cm-1 (RNA/ DNA) 1397/1455cm-1 (COO + CH2)/ (CH2/3) 1727/1742cm-1 (cholesterol/ triglycerides and phospholipids)
32 y 37 y 39 y (a.u.) (a.u.) (a.u.) 1.00 1.00
0.97 1.42
0.77 0.69
1.00 1.00 1.00
0.45 0.48 1.08
0.42 0.31 1.19
1.00
0.42
0.26
RESULTS: Differences were found according to the patients age. In older patients was observed a degradation of the protein and changes in their secondary structure. Moreover, in aged oocytes it was evidenced a decrease in carbohydrates, RNAand lipid contents and an increase in the oxidative process.
Vol. 96., No. 3, Supplement, September 2011
CONCLUSION: This study represents the first vibrational approach to evaluate the macromolecular changes associated with oocyte ageing. So, FPAFT-IR imaging could be considered a powerful technique to provide a biochemical fingerprint. This novel approach may represent a synergic support to evaluate oocyte quality. Supported by: University of Ancona founding 2010 to O. Carnevali and E. Giorgini.
P-449 Wednesday, October 19, 2011
TE D
IMPAIRED DNA REPAIR AS THE ROOT CAUSE OF OOCYTE AGING. K. Oktay, E. Heytens, R. Soleimani, V. Baltaci, S. Goswami. Institute for Fertility Preservation, Obstetrics & Gynecology, New York Medical College, Valhalla, NY; Cell Biology & Anatomy, New York Medical College, New York, NY; Department of Human Genetics, Istanbul Bilim University, Istanbul, TR, Turkey; Department of Biology, Yeshiva University, New York, NY.
RE
TR
AC
OBJECTIVE: The molecular mechanisms behind age-related decline in oocyte quantity and quality are unknown. Recent work indicated that women with BRCA mutations may have lower egg reserve and experience menopause earlier. Because BRCA is a key double strand DNA break (DSB) repair gene, we hypothesized that impaired DSB repair and resulting accumulation of DSBs is responsible for oocyte aging. DESIGN: Experimental. MATERIALS AND METHODS: Given the age-related decline in oocyte yield after ovarian stimulation (41 15 at 4 wks to 11 5 at 9-mo), we used ‘‘young’’ (4-wk-old) and ‘‘old’’ (9-mo-old) female FVB mice (n ¼ 24). Either primordial follicles (PDF) were assessed for DSBs by gH2AX immunostaining in ovarian sections, or GVoocytes by confocal microscopic quantification of gH2AX foci. The expression of ATM-mediated DSB repair pathway genes were analyzed by QRT-PCR in PDF captured by laser dissection (LD) as well as GVoocytes. The same was also analyzed in single human oocytes (n ¼ 20) by QRT-PCR from young (age<27) and old (age>37) subjects. RESULTS: gH2AX-positive PDF increased significantly in old mice compared to young (16 3 vs. 10 2; P¼0.002). Mean number of gH2AX foci was also significantly higher in GVoocytes of old vs. young (1,279 594 vs. 373 258; P¼0.01). By QRT-PCR from mouse GV oocytes, the expression of MRE11, a gene involved in sensing DSBs and activating repair via the ATM- pathway, was downregulated by 50-99% with age. In PDF from old mice, the expression of BRCA1 was downregulated by 77-89%. Consistent with findings in rodent oocytes, QRT-PCR of single human oocytes showed that the key genes in the DSB repair pathway was down-regulated with age. CONCLUSION: These rodent and human data support our novel hypothesis that oocyte aging is associated with accumulation of potentially lethal and mutagenic DSBs due to the impairment of DSB repair in the aging oocyte. Active and efficient DNA repair appears to be vital for oocyte health. These findings can be paradigm-shifting in understanding oocyte aging. Supported by: RO1 HD053112.
P-450 Wednesday, October 19, 2011 ROLE OF MELATONIN IN PREVENTING HYPOCHLOROUS ACID INDUCED ALTERATIONS IN MICROTUBULE AND CHROMOSOMAL STRUCTURE IN METAPHASE-II MOUSE OOCYTES IN VITRO. J. Banerjee, D. Maitra, F. Shaeib, G. M. Saed, M. P. Diamond, H. Abu-Soud. Obstetrics and Gynecology, Division of Reproductive Endocrinology and Infertility, Wayne State University, Detroit, MI. OBJECTIVE: Recently, we have shown that melatonin, a pineal gland hormone, can prevent hypochlorous acid (HOCl) mediated protein aggregation and hemeprotein destruction. We have demonstrated that HOCl may alter metaphase-II oocyte microtubule and chromosomal alignment. The objective of the study was to test whether melatonin prevents the impairment of oocyte quality induced by HOCl in vitro. DESIGN: Dose response study. MATERIALS AND METHODS: Metaphase–II mouse oocytes, obtained commercially, were incubated in HTF for 60 minutes. Oocytes were grouped as: control, melatonin (150, 200 nmol/mL), HOCl (10, 20, 50, 100 nmol/mL) and HOCl (50 nmol/mL) pretreated with 150 and 200 nmol/mL of melatonin. Microtubule and chromosomal alignment was studied on fixed and stained oocytes and then scored by two observers based on a previously published scoring system (Fert Stert 1222,Vol 88,Suppl 2, October 2007). Pearson
FERTILITY & STERILITYÒ
Chi-square test, Fisher’s Exact test were used compare outcomes between controls and treated groups and also amongst each group. RESULTS: Poor scores for the spindle and chromosomes increased significantly at 50 nmol/mL of HOCl (P<0.001). Oocytes treated with melatonin only at 150 and 200 nmol/mL showed no changes; significant differences (P<0.001) were observed when oocytes exposed to 50 nmol/mL of HOCl were compared to oocytes pretreated with melatonin 200 nmol/mL.Fifty percent of the oocytes demonstrated good scores both in microtubule and chromosomal alterations when pretreated with melatonin at 150 nmol/mL compared to 0% in the only HOCl group. CONCLUSION: HOCl alters the metaphase-II mouse oocyte spindle and chromosomal alignment in a dose dependant manner, a potential cause of poor oocyte quality in patients with endometriosis. Melatonin prevented the HOCl-mediated spindle and chromosomal damage. Melatonin supplementation could be an attractive therapeutic option to prevent oocyte damage in endometriosis or inflammatory diseases to improve fertility and reproductive outcome. Supported by: Wayne State University.
OOCYTE MATURATION P-451 Wednesday, October 19, 2011 IVM FOR WOMEN WITH POLYCYSTIC OVARIES? A CASE-CONTROL STUDY. T. J. Child, J. Craig, K. Turner, E. McVeigh, M. Fatum, A.S. Gremeau. Oxford Fertility Unit, Oxford, Oxfordshire, United Kingdom. OBJECTIVE: The in-vitro maturation (IVM) of immature oocytes retrieved from unstimulated ovaries is a promising treatment, particularly for women with polycystic ovaries (PCO). IVM is safer, simpler and cheaper than IVF. There are no published RCTs comparing IVM and IVF and only one case-control study from 2002. The aim of this study is to compare the outcome of unstimulated IVM and routine IVF/ICSI for women with ultrasonographic PCO undergoing treatment at the Oxford Fertility Unit. DESIGN: Case-control study. MATERIALS AND METHODS: The study included 250 women with PCO undergoing their first IVM (n ¼ 125) or IVF (n ¼ 125) cycle, matched for age, infertility diagnosis and ovulatory status (ovulatory PCO or anovulatory PCOS). The primary outcome measure was live birth rate per cycle. IVM cycles were unstimulated and hCG was administered 35-40h before trans-vaginal immature oocyte retrieval. Oocytes were matured in-vitro for 24-48h before insemination by ICSI. Endometrial priming with estradiol and progesterone was commenced following oocyte retrieval and 1-2 embryos transferred on day 2-5 of embryo development. Standard long protocol IVF/ICSI was used in the control group. RESULTS: The mean age was 32.8 years in both groups. A mean (SD) of 17.4 (9.6) and 15.3 (7.4) eggs were retrieved in the IVM and IVF groups, respectively (P<0.05). 66% of IVM eggs matured in-vitro. The live birth rate per retrieval was significantly lower for IVM compared to IVF (18.4% v 42.4%; P<0.001), as were the implantation (14.2% v 37.2%; P<0.001) and clinical pregnancy rates (21.6% vs. 52.0%, P<0.001). The OHSS rate was significantly higher in the IVF group (8.8%) compared to none in the IVM group. CONCLUSION: IVM is a safer and simpler alternative to conventional IVF for women with PCO or PCOS. IVM avoids the cost of gonadotropin stimulation and the risk of OHSS, but has a significantly lower live birth rate than IVF.
P-452 Wednesday, October 19, 2011 EFFECTS OF ANDROGEN SUBSTRATE ON BOVINE CUMULUSOOCYTE COMPLEXES STEROIDOGENESIS DURING IN VITRO MATURATION. A. C. J. S. Rosa-e-Silva, D. L. Bulgarelli, A. A. Vireque, C. P. Pitangui, M. P. Bernuci, M. F. Silva-de-Sa. Gynecology and Obstetrics, Faculty of Medicine of Ribeir~ao Preto - University of S~ao Paulo, Ribeir~ao Preto, S~ao Paulo, Brazil. OBJECTIVE: Ovarian steroids are known as important factors on the route of oocytes development. Since serum-supplemented in vitro maturation (IVM) media decreases estradiol secretion by bovine cumulus-oocyte complexes (COCs), the aim of this study was to assess whether this effect is caused by deficiency of substrate or by low cumulus cells aromatase activity. DESIGN: Prospective experimental study with bovine oocytes.
S239