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Does the proportion of immature oocytes impinge ICSI outcome?
16, 17, 18, 21, 22, X and Y) was performed by using FISH on day3 blastomeres. The donated spare embryos were reassessed by using same set of FISH probes. RESULTS: FISH results were obtained from 9, 55 and 8 embryos from group 1, 2 and 3, respectively. The rate of abnormal chromosomes was 89 %, 67% and 100% in groups 1, 2 and 3, respectively (P < 0.05). Out of 53 day3 diagnosed abnormal embryos, aneuploidy (57%) was the most common form of chromosome abnormality observed. However, out of 38 donated spare embryos, chaotic (45%) was the most common form of chromosome abnormality observed. An average of 2.3 embryos was transferred resulting in 56% of clinical pregnancy rate and 33% of implantation rate. CONCLUSION: The embryos generated from in vitro matured oocytes for 48 h (group 3) showed higher rate of chromosome abnormality compared to 24 h in vitro matured oocytes (group 2). This result suggests that when available, the first choice of embryos from in vitro matured ooyctes for transfer in an IVM cycle without aneuploidy screening should be from 24 hours matured oocytes.
P-171 Tuesday, October 26, 2010 GENE EXPRESSION PROFILE IN HUMAN CUMULUS CELLS OF IMMATURE AND MATURE OOCYTE UNDER IN VITRO MATURATION: CLINICAL APPLICATIONS. N. Frydman, L. Hesters, G. Ouandaogo, S. Assou, R. Frydman, S. Hamamah. APHP, Service d’Histologie Embryologie Cytogenetique, Service de Gynecologie obstetrique, Hoˆpital Antoine Becle`re, Clamart, France; CHU Montpellier, Institute for Research in Biotherapy, Hoˆpital Saint-Eloi, INSERM U847, Montpellier, France; CHU Montpellier, Unite Biologie Clinique d’AMP - DPI, Hoˆpital Arnaud de Villeneuve, Montpellier, France. OBJECTIVE: Oocyte competence to undergo IVF is affected by maturation conditions. Patients suffering from Polycystic Ovary Syndrome (PCOs) present hormonal dysfunctions with arrested follicle development and anovulation. For these patients we offered in vitro maturation (IVM). The aim of this study was to establish the gene expression profile of cumulus cells (CCs) derived from IVM oocytes and to identify new biomarkers for selection of competent oocytes. DESIGN: Clinical-based research using CCs (n¼23) from 13 PCOs patients (age < 35 years). Three groups of 8, 6 and 9 samples of CCs derived respectively from oocyte at germinal vesicle (CCGV), metaphase I (CCMI), metaphase II (CCMII) stages were constituted. MATERIALS AND METHODS: CCs were removed mechanically from each single oocyte after exposition to IVM media (MediCult) during 24h. After RNA extraction, samples were analyzed by whole genome U133P Affymetrix microarrays. RESULTS: A large number of genes were overexpressed in CCGV (n¼2133) and the lowest number was observed in CCMI (n¼363). Most of genes found in CCGV such as S100A6 (x3.83, p¼0.03), CAMTA2 (x2.77, p¼0.008) are involved in calcium activities. In CCMI, up-regulated genes such as RB1 (x3.09, p¼0.0002), ATP6V0E1 (x2.98, p¼0.005), ATP8A1 (x2.08, p¼0.0002) are mainly enrolled in cell cycle spindle checkpoint and protons transports. In CCMII, we observed up-regulation of anti-apoptotic [BIRC5 (x2.20, p¼0.001)], associated heat shock genes such as [HSPA4 (x2.02, p¼0.0002), HSP90A1B1 (x2.79, p¼0.0002)] and DNA repair genes [RAD17 (x1.83, p¼0.0001), RAD23A (x1.88, p¼0.02), RRAD (x2.51, p¼0.0002)]. CONCLUSION: Abnormal folliculogenesis in PCOs patients could alter the CCs expression of genes crucial for oocyte maturation, even under IVM. These data show for the first time, that anti-apoptotic gene BIRC5 encoding for survivin protein is up-regulated in human CCs from oocytes which successfully matured in vitro. Supported by: Ferring pharmaceutical company.
P-172 Tuesday, October 26, 2010 AROMATIZATION MAINTENANCE DURING GRANULOSA-LUTEINIC CELLS CULTURE MAY PROVIDE A GOOD CO-CULTURE SYSTEM FOR IN VITRO MATURATION PROCEDURES. C. O. Campos, A. A. Vireque, J. R. Campos, M. F. S. Sa´, R. A. Ferriani, C. C. J. S. Rosa-e-Silva. Obstetrics and Gynecology, Faculty of Medicine of Ribeir~ao Preto - University of S~ao Paulo, Ribeir~ao Preto, S~ao Paulo, Brazil.
FERTILITY & STERILITYÒ
OBJECTIVE: Since granulose-luteinic cells (GC) in culture tend to luteinize on current culture systems they are representative of the corpus luteum. However, these systems may also be used in immature oocytes co-culture, so this steroidogenic pattern would not be synchronized with the oocyte stage of maturation. With the purpose of maintaining the aromatase capacity of the GC a different culture system is proposed. DESIGN: GC obtained from 11 women submitted to assisted reproduction treatment (ART) were cultured. MATERIALS AND METHODS: Human GCs were obtained during oocyte retrieval for ART. These GCs were cultured for 144 hours in alphaMEM medium with IGF-I, insulin, androstenedione and PVP-40 (Proposed medium- PM) and separately in M199 medium with FSH and fetal calf serum (Control medium- CM). The media were changed after 48, 96 and 144 h and frozen for posterior estradiol (E2) and progesterone (P4) measurement. RESULTS: GC cultured in PM secreted progressively higher concentrations of estradiol throughout the culture period, while cells cultured in CM had gradually lower estradiol production (p < 0.01). We also evaluated the estradiol/progesterone ratio to confirm the reversal of the luteinization process along the culture, this ratio was significantly higher in the PM than in the CM (48h¼0.13 0.12 vs 0.01 0.02, (p<0.01); 96h¼0.12 0.06 vs 0.01 0.01, (p<0.01); 144h¼0.90 0.87 vs 0.01 0.06, (p¼ 0.06), respectively). This decrease on E2 secretion associated with the increased on P4 secretion observed in the CM suggests luteinization and disruption of the aromatization process. Inversely, E2 concentrations increase associated with reduction in P4 synthesis with the PM suggests a maintained aromatization capacity. CONCLUSION: Aromatase activity was maintained in granulosa cells until 144 h of culture in the PM. This system presents a profile of steroid production similar to that seen during early follicular phase and might prove useful for in vitro maturation procedures. Supported by: FAPESP and FAEPA/HC-FMRP/USP, Brazil.
P-173 Tuesday, October 26, 2010 DOES THE PROPORTION OF IMMATURE OOCYTES IMPINGE ICSI OUTCOME? D. Monahan, Q. V. Neri, Z. Rosenwaks, G. D. Palermo. The Ronald O Perelman & Claudia Cohen Center for Reproductive Medicine, Weill Cornell Medical College, New York, NY. OBJECTIVE: In 9% of cycles, oocyte maturation rate is below 50% and is a reason of concern because it limits the availability of conceptuses for replacement. It may also impair the developmental competence of the mature oocyte cohort. DESIGN: We assessed the clinical outcome of cycles with varying proportions of immature oocytes. MATERIALS AND METHODS: From Sept 1993-Mar 2010, ICSI cycles (n¼7867) with R8 oocytes retrieved included women %35yo and utilizing fresh ejaculated spermatozoa with adequate semen parameters. Cycles were categorized into three groups: minimal (0-33% MII; n¼128), adequate (34-66% MII; n¼4181), and predominantly mature (67-100%; n¼4466). Moreover, only one immature stage, either GV or MI, were assessed over the mature cohort. RESULTS: The average maternal age was 31.8 3yrs. Ejaculated samples yielded a mean density of 54.2 61x106/ml, motility of 36.6 27%, and morphology of 2.8 5%. The fertilization rate was comparable between the adequate (70.0%) and predominantly mature group (74.8%), while it was impaired in the minimal cohort (62.4%, P<0.0001). The clinical pregnancy in the minimal group (31.4%) was also compromised when compared to adequate (41.9%) and predominantly mature groups (50.2%, P<0.0001) and was confirmed in the delivery rate (29.6 vs 39.5 vs 47.8%, respectively, P<0.0001). While the fertilization of the GV only group (77.5%) was higher than the MI only cohort (68.8%, P<0.001), the clinical pregnancy rate was similar. No difference in pregnancy losses was observed. CONCLUSION: It appears that a larger proportion of immature oocytes influenced developmental competence of MII siblings. The most relevant effect was exerted by the presence of MI over GV, however, only on fertilization potential. While the specific MI cohort impairment may have exerted its effect on fertilization through nuclear/cytoplasmic maturational asynchrony, an overwhelming number of immature oocytes limited the conceptuses available for transfer. Supported by: Institutional, QVN is funded by CTSC-WCMC NIH grant ULI RR024996.
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P-171 Tuesday, October 26, 2010 GENE EXPRESSION PROFILE IN HUMAN CUMULUS CELLS OF IMMATURE AND MATURE OOCYTE UNDER IN VITRO MATURATION: CLINICAL APPLICATIONS. N. Frydman, L. Hesters, G. Ouandaogo, S. Assou, R. Frydman, S. Hamamah. APHP, Service d’Histologie Embryologie Cytogenetique, Service de Gynecologie obstetrique, Hoˆpital Antoine Becle`re, Clamart, France; CHU Montpellier, Institute for Research in Biotherapy, Hoˆpital Saint-Eloi, INSERM U847, Montpellier, France; CHU Montpellier, Unite Biologie Clinique d’AMP - DPI, Hoˆpital Arnaud de Villeneuve, Montpellier, France. OBJECTIVE: Oocyte competence to undergo IVF is affected by maturation conditions. Patients suffering from Polycystic Ovary Syndrome (PCOs) present hormonal dysfunctions with arrested follicle development and anovulation. For these patients we offered in vitro maturation (IVM). The aim of this study was to establish the gene expression profile of cumulus cells (CCs) derived from IVM oocytes and to identify new biomarkers for selection of competent oocytes. DESIGN: Clinical-based research using CCs (n¼23) from 13 PCOs patients (age < 35 years). Three groups of 8, 6 and 9 samples of CCs derived respectively from oocyte at germinal vesicle (CCGV), metaphase I (CCMI), metaphase II (CCMII) stages were constituted. MATERIALS AND METHODS: CCs were removed mechanically from each single oocyte after exposition to IVM media (MediCult) during 24h. After RNA extraction, samples were analyzed by whole genome U133P Affymetrix microarrays. RESULTS: A large number of genes were overexpressed in CCGV (n¼2133) and the lowest number was observed in CCMI (n¼363). Most of genes found in CCGV such as S100A6 (x3.83, p¼0.03), CAMTA2 (x2.77, p¼0.008) are involved in calcium activities. In CCMI, up-regulated genes such as RB1 (x3.09, p¼0.0002), ATP6V0E1 (x2.98, p¼0.005), ATP8A1 (x2.08, p¼0.0002) are mainly enrolled in cell cycle spindle checkpoint and protons transports. In CCMII, we observed up-regulation of anti-apoptotic [BIRC5 (x2.20, p¼0.001)], associated heat shock genes such as [HSPA4 (x2.02, p¼0.0002), HSP90A1B1 (x2.79, p¼0.0002)] and DNA repair genes [RAD17 (x1.83, p¼0.0001), RAD23A (x1.88, p¼0.02), RRAD (x2.51, p¼0.0002)]. CONCLUSION: Abnormal folliculogenesis in PCOs patients could alter the CCs expression of genes crucial for oocyte maturation, even under IVM. These data show for the first time, that anti-apoptotic gene BIRC5 encoding for survivin protein is up-regulated in human CCs from oocytes which successfully matured in vitro. Supported by: Ferring pharmaceutical company.
P-172 Tuesday, October 26, 2010 AROMATIZATION MAINTENANCE DURING GRANULOSA-LUTEINIC CELLS CULTURE MAY PROVIDE A GOOD CO-CULTURE SYSTEM FOR IN VITRO MATURATION PROCEDURES. C. O. Campos, A. A. Vireque, J. R. Campos, M. F. S. Sa´, R. A. Ferriani, C. C. J. S. Rosa-e-Silva. Obstetrics and Gynecology, Faculty of Medicine of Ribeir~ao Preto - University of S~ao Paulo, Ribeir~ao Preto, S~ao Paulo, Brazil.
FERTILITY & STERILITYÒ
OBJECTIVE: Since granulose-luteinic cells (GC) in culture tend to luteinize on current culture systems they are representative of the corpus luteum. However, these systems may also be used in immature oocytes co-culture, so this steroidogenic pattern would not be synchronized with the oocyte stage of maturation. With the purpose of maintaining the aromatase capacity of the GC a different culture system is proposed. DESIGN: GC obtained from 11 women submitted to assisted reproduction treatment (ART) were cultured. MATERIALS AND METHODS: Human GCs were obtained during oocyte retrieval for ART. These GCs were cultured for 144 hours in alphaMEM medium with IGF-I, insulin, androstenedione and PVP-40 (Proposed medium- PM) and separately in M199 medium with FSH and fetal calf serum (Control medium- CM). The media were changed after 48, 96 and 144 h and frozen for posterior estradiol (E2) and progesterone (P4) measurement. RESULTS: GC cultured in PM secreted progressively higher concentrations of estradiol throughout the culture period, while cells cultured in CM had gradually lower estradiol production (p < 0.01). We also evaluated the estradiol/progesterone ratio to confirm the reversal of the luteinization process along the culture, this ratio was significantly higher in the PM than in the CM (48h¼0.13 0.12 vs 0.01 0.02, (p<0.01); 96h¼0.12 0.06 vs 0.01 0.01, (p<0.01); 144h¼0.90 0.87 vs 0.01 0.06, (p¼ 0.06), respectively). This decrease on E2 secretion associated with the increased on P4 secretion observed in the CM suggests luteinization and disruption of the aromatization process. Inversely, E2 concentrations increase associated with reduction in P4 synthesis with the PM suggests a maintained aromatization capacity. CONCLUSION: Aromatase activity was maintained in granulosa cells until 144 h of culture in the PM. This system presents a profile of steroid production similar to that seen during early follicular phase and might prove useful for in vitro maturation procedures. Supported by: FAPESP and FAEPA/HC-FMRP/USP, Brazil.
P-173 Tuesday, October 26, 2010 DOES THE PROPORTION OF IMMATURE OOCYTES IMPINGE ICSI OUTCOME? D. Monahan, Q. V. Neri, Z. Rosenwaks, G. D. Palermo. The Ronald O Perelman & Claudia Cohen Center for Reproductive Medicine, Weill Cornell Medical College, New York, NY. OBJECTIVE: In 9% of cycles, oocyte maturation rate is below 50% and is a reason of concern because it limits the availability of conceptuses for replacement. It may also impair the developmental competence of the mature oocyte cohort. DESIGN: We assessed the clinical outcome of cycles with varying proportions of immature oocytes. MATERIALS AND METHODS: From Sept 1993-Mar 2010, ICSI cycles (n¼7867) with R8 oocytes retrieved included women %35yo and utilizing fresh ejaculated spermatozoa with adequate semen parameters. Cycles were categorized into three groups: minimal (0-33% MII; n¼128), adequate (34-66% MII; n¼4181), and predominantly mature (67-100%; n¼4466). Moreover, only one immature stage, either GV or MI, were assessed over the mature cohort. RESULTS: The average maternal age was 31.8 3yrs. Ejaculated samples yielded a mean density of 54.2 61x106/ml, motility of 36.6 27%, and morphology of 2.8 5%. The fertilization rate was comparable between the adequate (70.0%) and predominantly mature group (74.8%), while it was impaired in the minimal cohort (62.4%, P<0.0001). The clinical pregnancy in the minimal group (31.4%) was also compromised when compared to adequate (41.9%) and predominantly mature groups (50.2%, P<0.0001) and was confirmed in the delivery rate (29.6 vs 39.5 vs 47.8%, respectively, P<0.0001). While the fertilization of the GV only group (77.5%) was higher than the MI only cohort (68.8%, P<0.001), the clinical pregnancy rate was similar. No difference in pregnancy losses was observed. CONCLUSION: It appears that a larger proportion of immature oocytes influenced developmental competence of MII siblings. The most relevant effect was exerted by the presence of MI over GV, however, only on fertilization potential. While the specific MI cohort impairment may have exerted its effect on fertilization through nuclear/cytoplasmic maturational asynchrony, an overwhelming number of immature oocytes limited the conceptuses available for transfer. Supported by: Institutional, QVN is funded by CTSC-WCMC NIH grant ULI RR024996.
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