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FMR1 gene CGG repeat number variation within the normal range is not predictive of embryonic aneuploidy rate (AR) in IVF
OBJECTIVE: The aim of this study was to identify lipids biomarkers in follicular fluid (FF) samples from women with poor ovarian response. DESIGN: Observational Case-Control Study. MATERIALS AND METHODS: An observational case–control study was carried out including women submitted to controlled ovarian stimulation for in vitro fertilization with a poor ovarian response (PR group) and controls with normal ovarian response to controlled ovarian stimulation (NR group), both with a maximum age of 35 years. All women were submitted to a same controlled ovarian stimulation protocol for in vitro fertilization and FF was collected after ultrasound-guided ovarian aspiration. We report direct analysis by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of FF of poor responders and controls. An approach that combines MALDI–MS, principal component analysis (PCA) and Volcano analysis is described with the aim of generating follicular fluid classification models based on lipid profile patterns. The over expressed lipids in each studied group obtained by MALDI–MS were identified with the web-based resources. RESULTS: A total of 10 lipids were identified as differentially expressed between the PR and NR groups. Of these ions, 3 belong to phosphatidylcholines subclass (PC) and they were more present in the NR group. The other lipids for poor responders were presented in four main lipid subclasses: Phosphatidylethanolamines (PE), Phosphatidylglycerols (PG), Phosphatidylinositols (PI) and Diacylglycerols (DAG). CONCLUSION: The distinctive lipids might be involved in hormonal response, oocyte production and quality. They may additionally be useful in functioning as biomarkers for diagnostics, as well as for therapeutic intervention in poor responder women. Changes in lipid profiles may be closely related to the action of specific lipids in altering ovarian response, leading to increase of inflammatory response, oxidative stress and apoptosis. Supported by: Coordenac¸~ao de Aperfeic¸oamento de Pessoal de Nıvel Superior.
P-320 Wednesday, October 24, 2012 RADIXIN REGULATES GAMMA-AMINOBUTYRIC ACID-A RECEPTORS IN HUMAN GRANULOSA CELLS. L. Hou T. M. Yalcinkaya. Dept. of Obstetrics and Gynecology, Wake Forest School of Medicine, Winston-Salem, NC. OBJECTIVE: Gamma-aminobutyric acid (GABA) acts as an inhibitory transmitter in mature central nervous system. GABA/GABA-A receptors are also present in non-neural tissue, including ovary. We have shown a receptor specific effect of GABA-A on aromatase (CYP19) induction in cultured human granulosa cells (GC), which requires the presence of insulin. The 81-kDa actin-binding protein radixin (RDX) from ezrin/radixin/moesin family has been shown to directly link the alpha-5 subunit of GABA-A receptor to the cytoskeleton acts as membrane organizer and linker. In this study, we investigate the role of RDX in GC regarding its response to GABA-A agonists. DESIGN: In vitro experiment. MATERIALS AND METHODS: GC from oocyte retrievals were transfected after 72 h culture with small interfering RNA (siRNA) for either RDX, GAPDH or with negative control siRNAs. After 16 h, medium was replaced, then GC were treated with muscimol or carrier in serum-free medium for 24 h. mRNA levels were then determined by qRT-PCR. Expression of RDX in GC was detected by Western. E2 secretion was evaluated by RIA. Silencer Select Negative Control siRNA No.1 was used as negative control. RESULTS: RDX siRNAs downregulated the mRNA and protein (>85%), inhibited E2 secretion that muscimol induced in GC. GABA-A a5 expression is suppressed and GABA-A b3 expression was increased with RDX siRNA. Knockdown of RDX in GC resulted in decreasing E2 secretion in muscimol treated granulosa cells. RDX siRNA treatment decreased expression of CYP19, and muscimol treatment in RDX siRNA transfected GC did not increase the production of E2 or CYP19 mRNA. Similarly, expression of GABA-A receptor a5 (43 + 12%), r3 (62 + 22%) and glutamate decarboxylase-1 (GAD1) (35 + 15%) mRNA were decreased by RDX siRNA, while expression of GABA b3 (172 + 16%) were increased. CONCLUSION: RDC is expressed by GC. Using siRNA–mediated knockdown, we identified RDX as an essential component of GABA-A receptor assembly which results in induction of CYP19 expression, among other effects. Supported by: Greiss Fund.
FERTILITY & STERILITYÒ
P-321 Wednesday, October 24, 2012 FMR1 GENE CGG REPEAT NUMBER VARIATION WITHIN THE NORMAL RANGE IS NOT PREDICTIVE OF EMBRYONIC ANEUPLOIDY RATE (AR) IN IVF. K. H. Hong,a,b B. Devkota,a E. Forman,a O. Bendarsky,a N. Treff,a R. Scott.a aReproductive Endocrinology, Reproductive Medicine Associates of New Jersey, Morristown, NJ; bObstetrics, Gynecology & Reproductive Sciences, UMDNJ-Robert Wood Johnson Medical School, New Brunswick, NJ. OBJECTIVE: FMR1 gene CGG repeat number in the Fragile X premutation range is known to be associated with primary ovarian insufficiency. It has been suggested that more repeats within the range of normal (<45) can be correlated with other markers of ovarian aging. This study seeks to determine if CGG repeat number is correlated with embryonic aneuploidy rate. DESIGN: Retrospective. MATERIALS AND METHODS: Patients who had embryonic aneuploidy screening and also donated their DNA for research were studied. FMR1 repeats for each patient were determined by a reference lab (Labcorp). Initially, the number of CGG repeats in the low copy, high copy, or combined values were compared to aneuploidy rate. Next, the CGG repeats for each copy were designated as low, normal, or high using published thresholds and patients grouped into the resulting pair (low-low, low-normal, etc) and compared for aneuploidy rate. Finally, patients were grouped by aneuploidy rate and repeat levels compared. Comparisons done via ANOVA. RESULTS: 689 patients underwent analysis. There was no relationship between the low repeat copy and aneuploidy rate (P¼0.47). Similarly, high copy and the sum were unrelated to aneuploidy rate (P¼0.15).When grouped by repeat level as published, there were no differences in aneuploidy rates. Finally, CGG repeat numbers for either copy were equivalent in patients with a high, moderate, and low prevalences of aneuploidy (P¼0.31). FMR1 CGG repeat number combinations and Aneuploidy Rate
AR (%) high-high high-normal high-low normal-normal normal-low low-low
49.6 47.8 35.3 42.8 47.1 52.5
normal: 26-34 CGG repeats, low: <26 repeats, high: >34 repeats.
CONCLUSION: Variation of CGG repeat levels below 45 copies does not prognosticate changes in oocyte quality associated with embryonic aneuploidy. Like most biologic parameters, variation within the normal range appears clinically insignificant.
P-322 Wednesday, October 24, 2012 THE PRESENCE OF MATURE DENDRITIC CELLS WITHIN HUMAN OVARIAN FOLLICULAR FLUID CORRELATES WITH INCREASED BLOOD FLOW TO THE FOLLICLE. O. Fainaru, M. Geva, S. Hantisteanu, M. Michaeli, M. Hallak, A. Ellenbogen. IVF Unit and Laboratory for Reproductive Immunology, Obstetrics and Gynecology, Hillel Yaffe Medical Center, Hadera, Israel. OBJECTIVE: Dendritic cells (DCs) are bone marrow derived immune cells that have been shown to promote angiogenesis. We sought to determine whether the presence of DCs in ovarian follicular fluid (FF) and/or their maturation state is correlated with follicular blood flow. DESIGN: Observational human cohort study. MATERIALS AND METHODS: We studied 21 patients undergoing IVF in our institution. The first aspirated dominant follicle in each ovary was measured and blood flow parameters were analyzed using pulsed doppler ultrasound. FF samples (n¼36) were then collected, immunostained and analyzed by flow cytometry. DCs were identified as CD45+CD11c+HLADR+
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P-320 Wednesday, October 24, 2012 RADIXIN REGULATES GAMMA-AMINOBUTYRIC ACID-A RECEPTORS IN HUMAN GRANULOSA CELLS. L. Hou T. M. Yalcinkaya. Dept. of Obstetrics and Gynecology, Wake Forest School of Medicine, Winston-Salem, NC. OBJECTIVE: Gamma-aminobutyric acid (GABA) acts as an inhibitory transmitter in mature central nervous system. GABA/GABA-A receptors are also present in non-neural tissue, including ovary. We have shown a receptor specific effect of GABA-A on aromatase (CYP19) induction in cultured human granulosa cells (GC), which requires the presence of insulin. The 81-kDa actin-binding protein radixin (RDX) from ezrin/radixin/moesin family has been shown to directly link the alpha-5 subunit of GABA-A receptor to the cytoskeleton acts as membrane organizer and linker. In this study, we investigate the role of RDX in GC regarding its response to GABA-A agonists. DESIGN: In vitro experiment. MATERIALS AND METHODS: GC from oocyte retrievals were transfected after 72 h culture with small interfering RNA (siRNA) for either RDX, GAPDH or with negative control siRNAs. After 16 h, medium was replaced, then GC were treated with muscimol or carrier in serum-free medium for 24 h. mRNA levels were then determined by qRT-PCR. Expression of RDX in GC was detected by Western. E2 secretion was evaluated by RIA. Silencer Select Negative Control siRNA No.1 was used as negative control. RESULTS: RDX siRNAs downregulated the mRNA and protein (>85%), inhibited E2 secretion that muscimol induced in GC. GABA-A a5 expression is suppressed and GABA-A b3 expression was increased with RDX siRNA. Knockdown of RDX in GC resulted in decreasing E2 secretion in muscimol treated granulosa cells. RDX siRNA treatment decreased expression of CYP19, and muscimol treatment in RDX siRNA transfected GC did not increase the production of E2 or CYP19 mRNA. Similarly, expression of GABA-A receptor a5 (43 + 12%), r3 (62 + 22%) and glutamate decarboxylase-1 (GAD1) (35 + 15%) mRNA were decreased by RDX siRNA, while expression of GABA b3 (172 + 16%) were increased. CONCLUSION: RDC is expressed by GC. Using siRNA–mediated knockdown, we identified RDX as an essential component of GABA-A receptor assembly which results in induction of CYP19 expression, among other effects. Supported by: Greiss Fund.
FERTILITY & STERILITYÒ
P-321 Wednesday, October 24, 2012 FMR1 GENE CGG REPEAT NUMBER VARIATION WITHIN THE NORMAL RANGE IS NOT PREDICTIVE OF EMBRYONIC ANEUPLOIDY RATE (AR) IN IVF. K. H. Hong,a,b B. Devkota,a E. Forman,a O. Bendarsky,a N. Treff,a R. Scott.a aReproductive Endocrinology, Reproductive Medicine Associates of New Jersey, Morristown, NJ; bObstetrics, Gynecology & Reproductive Sciences, UMDNJ-Robert Wood Johnson Medical School, New Brunswick, NJ. OBJECTIVE: FMR1 gene CGG repeat number in the Fragile X premutation range is known to be associated with primary ovarian insufficiency. It has been suggested that more repeats within the range of normal (<45) can be correlated with other markers of ovarian aging. This study seeks to determine if CGG repeat number is correlated with embryonic aneuploidy rate. DESIGN: Retrospective. MATERIALS AND METHODS: Patients who had embryonic aneuploidy screening and also donated their DNA for research were studied. FMR1 repeats for each patient were determined by a reference lab (Labcorp). Initially, the number of CGG repeats in the low copy, high copy, or combined values were compared to aneuploidy rate. Next, the CGG repeats for each copy were designated as low, normal, or high using published thresholds and patients grouped into the resulting pair (low-low, low-normal, etc) and compared for aneuploidy rate. Finally, patients were grouped by aneuploidy rate and repeat levels compared. Comparisons done via ANOVA. RESULTS: 689 patients underwent analysis. There was no relationship between the low repeat copy and aneuploidy rate (P¼0.47). Similarly, high copy and the sum were unrelated to aneuploidy rate (P¼0.15).When grouped by repeat level as published, there were no differences in aneuploidy rates. Finally, CGG repeat numbers for either copy were equivalent in patients with a high, moderate, and low prevalences of aneuploidy (P¼0.31). FMR1 CGG repeat number combinations and Aneuploidy Rate
AR (%) high-high high-normal high-low normal-normal normal-low low-low
49.6 47.8 35.3 42.8 47.1 52.5
normal: 26-34 CGG repeats, low: <26 repeats, high: >34 repeats.
CONCLUSION: Variation of CGG repeat levels below 45 copies does not prognosticate changes in oocyte quality associated with embryonic aneuploidy. Like most biologic parameters, variation within the normal range appears clinically insignificant.
P-322 Wednesday, October 24, 2012 THE PRESENCE OF MATURE DENDRITIC CELLS WITHIN HUMAN OVARIAN FOLLICULAR FLUID CORRELATES WITH INCREASED BLOOD FLOW TO THE FOLLICLE. O. Fainaru, M. Geva, S. Hantisteanu, M. Michaeli, M. Hallak, A. Ellenbogen. IVF Unit and Laboratory for Reproductive Immunology, Obstetrics and Gynecology, Hillel Yaffe Medical Center, Hadera, Israel. OBJECTIVE: Dendritic cells (DCs) are bone marrow derived immune cells that have been shown to promote angiogenesis. We sought to determine whether the presence of DCs in ovarian follicular fluid (FF) and/or their maturation state is correlated with follicular blood flow. DESIGN: Observational human cohort study. MATERIALS AND METHODS: We studied 21 patients undergoing IVF in our institution. The first aspirated dominant follicle in each ovary was measured and blood flow parameters were analyzed using pulsed doppler ultrasound. FF samples (n¼36) were then collected, immunostained and analyzed by flow cytometry. DCs were identified as CD45+CD11c+HLADR+
S207