- Email: [email protected]
Effects of androgen substrate on bovine cumulus-oocyte complexes steroidogenesis during in vitro maturation
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
MATERIALS AND METHODS: Bovine cumulus–oocyte complexes (COCs) aspirated from 2 to 8 mm follicles of slaughterhouse ovaries, were matured in (1) TCM199 in the presence of 10% FBS, 20 mg/mL FSH, 0.5 mg/mL LH (40 COCs in each treatment/replicate), or (2) as 1 but with the addition of 100 nmol/L androstenedione (A4) for 22 h at 39 degrees C in a humidified atmosphere of 5% CO2 in air. After maturation, with or without COCs medium were collected and analyzed for its content of estradol-17b and (E2) and progesterone (P4) by chemiluminescence method. RESULTS: The E2 content of culture medium increased markedly (P<0.0001) after incubation of COCs with TCM+A4 compared to TCM (17.52 1.86 ng/mL; 0.32 0.05 ng/mL, respectively). No significant difference in the P4 content (P¼0,4088) was detected into the IVM medium despite the presence of A4 (21.73 1.67 ng/mL to TCM; 21,83 1.61 ng/mL to TCM+A4). CONCLUSION: Our results suggest that decreased estradiol production by COCs in serum-supplemented IVM media is caused by deficiency of substrate rather than low cumulus cells aromatase activity. Supported by: FAPESP¼Fundac¸~ao de Amparo a Pesquisa do Estado de S~ao Paulo.
P-453 Wednesday, October 19, 2011 IDENTIFICATION OF GENE EXPRESSION OF MYH11 AND PF4V1 IN CUMULUS CELLS (CC) AFTER OVARIAN STIMULATION BY MICROARRAYS TECHNOLOGY: POSSIBLE IMPLICATIONS ON OOCYTE MATURATION. V. Garcıa-Laez, D. Beltran, J. M. De los Santos, F. J. Esteban, J. A. Martınez-Conejero, M. J. De los Santos. FIV, IVI Valencia and INCLIVA, Valencia, Spain; Universidad de Jaen, Jaen, Spain; Igenomix, Valencia, Spain. OBJECTIVE: MYH11 and PF4V1 are genes encoding for molecules related to the regulation of angiotensin II (AngII) and angiogenesis, MYH11 induces the synthesis of AngII and PF4V1 inhibits it. An endothelin-angiotensin-atrial natriuretic peptide functional system at the ovarian level is involved in oocyte maturation and ovulation and seems to be controlled by LH. Since most of the protocols used in IVF require the use of gonadotrophins is logical to think on the possible effect of ovarian stimulation on the process related to ovulation and oocyte maturation. Therefore, the objective of this study was to analyze whether ovarian stimulation may influence CC transcriptome using egg donors undergoing natural cycle (NC) compared to stimulated cycles (SC). DESIGN: Prospective experimental study. MATERIALS AND METHODS: A total of 8 egg donors were included in this study, 4 NC and 4 SC. After RNA isolation of CC with Trizol, lineal amplification was done. The cDNA were hybridized onto Microarray (Agilent). Non parametrics test were used for comparisons. Genes were considered as over or underexpressed when P<0.05 and the fold change>2. Microarrays were validated additionally in a total of 19 CC by qRT-PCR. RESULTS: When we compare SC vs NC, the transcriptome of CC showed a significant differential expression of genes involved in immune response, cell activation, cell differentiation and organ development. Among the 14 genes differentially expressed 10 of them were down regulated on SC. MHY11 and PF4V1 were shown to be associated to the process of regulation of AII. While MHY11 was down-regulated in SC, PF4V1 was up-regulated. CONCLUSION: Ovarian stimulation affects gene expression profile of CC. The differential transcription of MYH11 and PF4V1 in CC and its relationship with the regulation of AngII open the possibility of the implication of these molecules on the mechanisms related to oocyte maturation. Supported by: IMPIVA IMIDTG/2008/26 and IVI.
ular fluids of dominants follicles which was correlated with estradiol (E2) concentration. Objective analyze the differences on gene expression between egg donors undergoing natural (NC) and ovarian stimulated cycles (OS) and their possible correlation with intrafolicular E2 and oocyte quality. DESIGN: Prospective experimental study. MATERIALS AND METHODS: A total of 8 egg donors were included in this study, 4 undergoing NC and 4 OS protocol. After RNA isolation of CC was done lineal amplification. The cDNA were hybridized onto Microarray (Agilent). Non parametrics test were used for comparisons. Genes were considered as over or under-expressed when P<0.05 and the fold change>2. Microarrays were validated in a total of 19 CC by qRT-PCR. Presence and position of meiotic spindle was evaluated by means the Oosight image software. Hormonal measurement intrafollicular E2 was made by MEIA (Microparticle enzyme immunoassay). Chi-square comparison tests were used for statistical comparisons, P<0.05 were considered statistically different. RESULTS: OS induces activation or deactivation of a certain number of genes (7 up and 10 down-regulated). Expression of ALDH1A2 in CC was over-expressed in NC (FC 4.62 and P¼0.025), showing an association with an increase on E2 intrafollicular (562.000 NC vs 241.750 pg/ml OS). However, we didn’t find any significant differences between NC and OS regarding the position and state of meiotic spindle (66.7% vs 46.7%), birefringence (1.77 vs 1.31nm) and fertilization rates (66.6% vs 59.4%), respectively. CONCLUSION: In spite of the importance on AR on cytoplasmic maturation observed in the bovine, the higher expression of ALDH1A2 found in CC from NC wasn’t correlated with parameters of oocyte quality. Supported by: IMPIVA IMIDTG/2008/26. IVI.
P-455 Wednesday, October 19, 2011 THE EFFECT OF 5 DAYS EXTENDED EXPOSURE OF IMMATURE BOVINE OOCYTES TO A COMBINATION OF MATURATION INHIBITORS ON THE IN VITRO MATURATION (IVM) OUTCOME. T. A. Farghaly, S. A. Mostafa, E. M. Khalifa, M. A. Bedaiwy, J. Goldfarb, A. Ahmady. MacDonald Fertility & IVF Center, UH Case Medical Center, Cleveland, OH; Obstetrics and Gynecology, Womens’ Health Center,Assiut Universty, Assiut, Egypt. OBJECTIVE: To test the effect of the combination of 2 of the maturation inhibitors Adenylate cyclase activator (Forskolin;A), phosphodiesterase-3 inhibitor (Cilostamide;B), and Maturation promoting factor inhibitor (Roscovitine;C) on meiotic resumption and embryonic development of bovine immature oocytes. DESIGN: Experimental Study. MATERIALS AND METHODS: The prematuration culture (PMC) consisted of M199 medium containing 10 mM of each of 2 inhibitors: (A+B), (B+C) and (A+C). The oocytes were cultured in either of these medium for 5 days and then transferred to a complete maturation media (CMM) containing 7.5 IU FSH/LH for 48 hours. Two control groups were used. In control group 1, immature oocytes were cultured in the same medium as the experimental group without any inhibitors. In the control group 2, oocytes were only cultured in the maturation medium supplemented with FSH/LH for 48 hours. Mature oocytes were then inseminated with frozen-thawed bull sperm. Fertilization, as defined by 2cell division 48h post-insemination, and blastocyst formation were monitored. Chi square test was used for statistical analysis. RESULTS: The following table showing IVM outcome of 276 oocytes after exposure to PMC:
Groups
n Meiotic arrest% Maturation% fertilization% blastulation%
DETECTION OF THE EXPRESSION OF ALDEHYDE DESHYDROGENASE 1A2 (ALDH1A2) IN CUMULUS CELLS (CC) BY DIFFERENTIAL GENE EXPRESSION ANALYSIS: IMPLICATIONS FOR MATURATION. V. Garcıa-Laez, D. Beltran, C. Albert, J. A. Horcajadas, F. J. Esteban, M. J. De los Santos. FIV, IVI Valencia and INCLIVA, Valencia, Spain; Igenomix, Paterna, Valencia, Spain; Universidad de Jaen, Jaen, Spain.
A+B B+C A+C control1 control2
54 58 51 55 55
OBJECTIVE: ALDH1A2 catalyzes the formation of retinoic acid (RA). RA is important during the formation of primordial follicles in humans and oocyte cytoplasmic maturation in the bovine. Expressions of RA receptors have been found in CC and higher RA concentration are found in follic-
Culturing oocyte in pre-maturation medium containing any combination of two of the three inhibitors had a significant higher rate of maturation in comparison to control 1 (P<0.05). The combination of B +C had higher embryonic developmental outcome in comparison to control 1(P<0.05).
P-454 Wednesday, October 19, 2011
S240
Abstracts
78 91 82 63 N/A
35/42(83) 47/52(90) 40/42(95) 20/35(57) 38/55(69)
15/35(43) 17/47(36) 18/40(45) 8/20(40) 8/38(24)
5/15(33) 10/17(59) 6/18(33) 1/8(13) 2/8(25)
Vol. 96., No. 3, Supplement, September 2011
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
MATERIALS AND METHODS: Bovine cumulus–oocyte complexes (COCs) aspirated from 2 to 8 mm follicles of slaughterhouse ovaries, were matured in (1) TCM199 in the presence of 10% FBS, 20 mg/mL FSH, 0.5 mg/mL LH (40 COCs in each treatment/replicate), or (2) as 1 but with the addition of 100 nmol/L androstenedione (A4) for 22 h at 39 degrees C in a humidified atmosphere of 5% CO2 in air. After maturation, with or without COCs medium were collected and analyzed for its content of estradol-17b and (E2) and progesterone (P4) by chemiluminescence method. RESULTS: The E2 content of culture medium increased markedly (P<0.0001) after incubation of COCs with TCM+A4 compared to TCM (17.52 1.86 ng/mL; 0.32 0.05 ng/mL, respectively). No significant difference in the P4 content (P¼0,4088) was detected into the IVM medium despite the presence of A4 (21.73 1.67 ng/mL to TCM; 21,83 1.61 ng/mL to TCM+A4). CONCLUSION: Our results suggest that decreased estradiol production by COCs in serum-supplemented IVM media is caused by deficiency of substrate rather than low cumulus cells aromatase activity. Supported by: FAPESP¼Fundac¸~ao de Amparo a Pesquisa do Estado de S~ao Paulo.
P-453 Wednesday, October 19, 2011 IDENTIFICATION OF GENE EXPRESSION OF MYH11 AND PF4V1 IN CUMULUS CELLS (CC) AFTER OVARIAN STIMULATION BY MICROARRAYS TECHNOLOGY: POSSIBLE IMPLICATIONS ON OOCYTE MATURATION. V. Garcıa-Laez, D. Beltran, J. M. De los Santos, F. J. Esteban, J. A. Martınez-Conejero, M. J. De los Santos. FIV, IVI Valencia and INCLIVA, Valencia, Spain; Universidad de Jaen, Jaen, Spain; Igenomix, Valencia, Spain. OBJECTIVE: MYH11 and PF4V1 are genes encoding for molecules related to the regulation of angiotensin II (AngII) and angiogenesis, MYH11 induces the synthesis of AngII and PF4V1 inhibits it. An endothelin-angiotensin-atrial natriuretic peptide functional system at the ovarian level is involved in oocyte maturation and ovulation and seems to be controlled by LH. Since most of the protocols used in IVF require the use of gonadotrophins is logical to think on the possible effect of ovarian stimulation on the process related to ovulation and oocyte maturation. Therefore, the objective of this study was to analyze whether ovarian stimulation may influence CC transcriptome using egg donors undergoing natural cycle (NC) compared to stimulated cycles (SC). DESIGN: Prospective experimental study. MATERIALS AND METHODS: A total of 8 egg donors were included in this study, 4 NC and 4 SC. After RNA isolation of CC with Trizol, lineal amplification was done. The cDNA were hybridized onto Microarray (Agilent). Non parametrics test were used for comparisons. Genes were considered as over or underexpressed when P<0.05 and the fold change>2. Microarrays were validated additionally in a total of 19 CC by qRT-PCR. RESULTS: When we compare SC vs NC, the transcriptome of CC showed a significant differential expression of genes involved in immune response, cell activation, cell differentiation and organ development. Among the 14 genes differentially expressed 10 of them were down regulated on SC. MHY11 and PF4V1 were shown to be associated to the process of regulation of AII. While MHY11 was down-regulated in SC, PF4V1 was up-regulated. CONCLUSION: Ovarian stimulation affects gene expression profile of CC. The differential transcription of MYH11 and PF4V1 in CC and its relationship with the regulation of AngII open the possibility of the implication of these molecules on the mechanisms related to oocyte maturation. Supported by: IMPIVA IMIDTG/2008/26 and IVI.
ular fluids of dominants follicles which was correlated with estradiol (E2) concentration. Objective analyze the differences on gene expression between egg donors undergoing natural (NC) and ovarian stimulated cycles (OS) and their possible correlation with intrafolicular E2 and oocyte quality. DESIGN: Prospective experimental study. MATERIALS AND METHODS: A total of 8 egg donors were included in this study, 4 undergoing NC and 4 OS protocol. After RNA isolation of CC was done lineal amplification. The cDNA were hybridized onto Microarray (Agilent). Non parametrics test were used for comparisons. Genes were considered as over or under-expressed when P<0.05 and the fold change>2. Microarrays were validated in a total of 19 CC by qRT-PCR. Presence and position of meiotic spindle was evaluated by means the Oosight image software. Hormonal measurement intrafollicular E2 was made by MEIA (Microparticle enzyme immunoassay). Chi-square comparison tests were used for statistical comparisons, P<0.05 were considered statistically different. RESULTS: OS induces activation or deactivation of a certain number of genes (7 up and 10 down-regulated). Expression of ALDH1A2 in CC was over-expressed in NC (FC 4.62 and P¼0.025), showing an association with an increase on E2 intrafollicular (562.000 NC vs 241.750 pg/ml OS). However, we didn’t find any significant differences between NC and OS regarding the position and state of meiotic spindle (66.7% vs 46.7%), birefringence (1.77 vs 1.31nm) and fertilization rates (66.6% vs 59.4%), respectively. CONCLUSION: In spite of the importance on AR on cytoplasmic maturation observed in the bovine, the higher expression of ALDH1A2 found in CC from NC wasn’t correlated with parameters of oocyte quality. Supported by: IMPIVA IMIDTG/2008/26. IVI.
P-455 Wednesday, October 19, 2011 THE EFFECT OF 5 DAYS EXTENDED EXPOSURE OF IMMATURE BOVINE OOCYTES TO A COMBINATION OF MATURATION INHIBITORS ON THE IN VITRO MATURATION (IVM) OUTCOME. T. A. Farghaly, S. A. Mostafa, E. M. Khalifa, M. A. Bedaiwy, J. Goldfarb, A. Ahmady. MacDonald Fertility & IVF Center, UH Case Medical Center, Cleveland, OH; Obstetrics and Gynecology, Womens’ Health Center,Assiut Universty, Assiut, Egypt. OBJECTIVE: To test the effect of the combination of 2 of the maturation inhibitors Adenylate cyclase activator (Forskolin;A), phosphodiesterase-3 inhibitor (Cilostamide;B), and Maturation promoting factor inhibitor (Roscovitine;C) on meiotic resumption and embryonic development of bovine immature oocytes. DESIGN: Experimental Study. MATERIALS AND METHODS: The prematuration culture (PMC) consisted of M199 medium containing 10 mM of each of 2 inhibitors: (A+B), (B+C) and (A+C). The oocytes were cultured in either of these medium for 5 days and then transferred to a complete maturation media (CMM) containing 7.5 IU FSH/LH for 48 hours. Two control groups were used. In control group 1, immature oocytes were cultured in the same medium as the experimental group without any inhibitors. In the control group 2, oocytes were only cultured in the maturation medium supplemented with FSH/LH for 48 hours. Mature oocytes were then inseminated with frozen-thawed bull sperm. Fertilization, as defined by 2cell division 48h post-insemination, and blastocyst formation were monitored. Chi square test was used for statistical analysis. RESULTS: The following table showing IVM outcome of 276 oocytes after exposure to PMC:
Groups
n Meiotic arrest% Maturation% fertilization% blastulation%
DETECTION OF THE EXPRESSION OF ALDEHYDE DESHYDROGENASE 1A2 (ALDH1A2) IN CUMULUS CELLS (CC) BY DIFFERENTIAL GENE EXPRESSION ANALYSIS: IMPLICATIONS FOR MATURATION. V. Garcıa-Laez, D. Beltran, C. Albert, J. A. Horcajadas, F. J. Esteban, M. J. De los Santos. FIV, IVI Valencia and INCLIVA, Valencia, Spain; Igenomix, Paterna, Valencia, Spain; Universidad de Jaen, Jaen, Spain.
A+B B+C A+C control1 control2
54 58 51 55 55
OBJECTIVE: ALDH1A2 catalyzes the formation of retinoic acid (RA). RA is important during the formation of primordial follicles in humans and oocyte cytoplasmic maturation in the bovine. Expressions of RA receptors have been found in CC and higher RA concentration are found in follic-
Culturing oocyte in pre-maturation medium containing any combination of two of the three inhibitors had a significant higher rate of maturation in comparison to control 1 (P<0.05). The combination of B +C had higher embryonic developmental outcome in comparison to control 1(P<0.05).
P-454 Wednesday, October 19, 2011
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Abstracts
78 91 82 63 N/A
35/42(83) 47/52(90) 40/42(95) 20/35(57) 38/55(69)
15/35(43) 17/47(36) 18/40(45) 8/20(40) 8/38(24)
5/15(33) 10/17(59) 6/18(33) 1/8(13) 2/8(25)
Vol. 96., No. 3, Supplement, September 2011