- Email: [email protected]
The PDPK1 gene variants do not seem to be associated with premature ovarian failure
CONCLUSION: This study reveals that reflectance spectroscopy have a diagnostic and prognostic value to assess the torsion/detorsion injury in a rat model.
P-680 Wednesday, October 16, 2013 FATTY ACID SYNTHASE IS EXPRESSED AND ENZYMATICALLY ACTIVE IN THE MOUSE OVARY AND HUMAN GRANULOSA CELLS. J. K. Riley, J. K. Bolzenius, W. G. Lewis, E. S. Jungheim. Obstetrics and Gynecology, Washington University School of Medicine, St. Louis, MO. OBJECTIVE: Animals obtain fatty acids from their diet or via de novo lipogenesis, the enzymatic pathway in which carbohydrate is converted into fat. Fatty acid synthase (FAS) is a key enzyme in this metabolic pathway, catalyzing the synthesis of long-chain fatty acids. The goal of our study is to characterize FAS expression and enzymatic activity in mouse ovaries and human granulosa cells. DESIGN: FAS activity was assessed in gonadotropin-stimulated mouse ovarian homogenates. To identify potential sources of this enzymatic activity we determined the cell specific localization of FAS expression in the mouse ovary. As an extension of our mouse studies, FAS protein expression and enzymatic activity were characterized in human granulosa cells. MATERIALS AND METHODS: C57BL/6 mice were superovulated and euthanized 48 h post pregnant mare serum gonadotropin injection or at distinct time points post human chorionic gonadotropin injection. FAS expression was examined via immunohistochemistry and western blot. FAS activity in ovarian homogenates was measured by the rate of NADPH oxidation pre and post addition of malonyl-CoA, a FAS substrate. Finally, human granulosa cells were collected from follicular aspirates immediately following oocyte retrieval during IVF cycles. FAS activity was assessed and protein expression examined via western blot. RESULTS: FAS activity was detected in gonadotropin-stimulated mouse ovarian homogenates. Several cell types within the mouse ovary expressed FAS protein including cumulus cells, granulosa cells, theca cells and cells present within the corpus luteum. A low level of FAS protein was also detected in the oocyte. Importantly, FAS was expressed and functional in human granulosa cells. CONCLUSION: Distinct cell types within the mouse and human ovary have the ability to synthesize fatty acids through de novo lipogenesis. Future studies will elucidate the role of de novo lipogenesis in ovarian function.
P-681 Wednesday, October 16, 2013 FOLLOWING PREVIOUSLY DESCRIBED OVARIAN PHENOTYPES OF THE FMR1 GENE, METHYLATION FRACTIONS WERE SIGNIFICANTLY SKEWED BETWEEN FMR1 GENOTYPES AND SUB-GENOTYPES. D. H. Barad,a,b G. J. Latham,c S. Filipovic-Sadic,c V. A. Kushnir,a A. Shohat-Tal,a N. Gleicher.a,b aCenter for Human Reproduction, New York, NY; bFoundation for Reproductive Medicine, New York, NY; cAsuragen, Austin, TX. OBJECTIVE: Based on a normal CGG count range of n¼26-34, new FMR1 genotypes and sub-genotypes have been associated with distinct ovarian aging patterns.We attempted to assess if these genotypes and sub-genotypes vary in methylation or AGG interruption patterns. DESIGN: Prospective collaborative study between infertility center and corporation with a novel FMR1 assay (Chen et al Genet Med 2011;13:52838). MATERIALS AND METHODS: We evaluated 26 infertility patients with high performance FMR1 PCR (as reported in above reference), which assesses CGGn, intermittent AGG genotypes and methylation status of each allele. For CGGn, patients were classified as previously reported [PLoS ONE 2010;5(12): e15303] as normal (norm) if both alleles were in normal CGG range (n¼26-34), homozygous (hom) if both were outside normal range, and heterozygous (het) if one was outside normal range. Hom and het were further divided into sub-genotypes; low if the abnormal-range allele was n<26 and high if n>34. RESULTS: No significant difference in methylation percentage was observed in both FMR1 alleles for any pairwise sample combination. A statistically significant skew in methylation fraction, however, was observed between women with het-norm/low sub-genotype and women with norm
FERTILITY & STERILITYÒ
genotype (analysis of variance P¼0.0167). Specifically, methylation skew from 50% per CGG bin (suggesting perfect random X-inactivation) was 7.3% for both alleles in norm women, 19.7% in women with low and 17.3% with high sub-genotype (t test, comparisons of means: low vs. norm, P¼0.0063; high vs. norm, P¼0.0605; low vs. high, P¼0.6529). Number of AGG interruptions per CGGn sub-genotype did not differ from reference values. CONCLUSION: Observed skew in methylation fraction was the strongest with low alleles and weakest in women with norm FMR1. These findings exactly mimic a variety of previously reported different FMR1 phenotypical appearances, including IVF pregnancy chances (PLoS ONE 2010). Supported by: Foundation for Reproductive Medicine, Center for Human Reproduction, Asuragen.
P-682 Wednesday, October 16, 2013 GONADOTROPIN STIMULATION IS ASSOCIATED WITH UPREGULATION OF KISSPEPTIN RECEPTOR IN HUMAN CUMULUS GRANULOSA CELLS. R. S. Raj, P. R. Casson, Z. Merhi. Obstetrics, Gynecology and Reproductive Sciences, University of Vermont College of Medicine, Burlington, VT. OBJECTIVE: Kisspeptin (Kp) receptor (KISS-1R) has been recently detected in animal and human ovaries, where it plays a role in ovulation and oocyte maturation. Gonadotropin treatment stimulates Kp gene expression in rat ovaries, further indicating its role in ovulation. We have recently shown that Kp mRNA is not expressed by human luteinized granulosa cells (GC) and that there is an age-related upregulation in KISS-1R expression in human GC indicating a possible physiologic role for the Kp system in human follicular dynamics. The objective of this study was to test the hypothesis that gonadotropins given during controlled ovarian hyperstimulation (COH) in IVF increase the expression of KISS-1R gene in human GC, potentially impacting ovarian Kp sensitivity. DESIGN: Experimental human study. MATERIALS AND METHODS: 12 non-PCOS women (mean age¼34.6 1.5 years, mean BMI¼28.5 1.9 kg/m2) with normal ovarian reserve (defined as Day 3 FSH < 10 mIU/mL) who underwent COH followed by oocyte retrieval for IVF were enrolled. Cumulus and mural GC were collected separately. KISS-1R mRNA levels were quantified using RTPCR. Pearson correlation was used to assess the relationship between KISS-1 mRNA levels and gonadotropin dose, peak E2 level on day of hCG, # of eggs retrieved and # of days of stimulation. RESULTS: As previously shown, there was a positive correlation between age and KISS-1R mRNA levels (r ¼ 0.33, p<0.05). KISS-1R mRNA levels were positively correlated with the gonadotropin dose used in cumulus (r ¼ 0.34, p<0.05) but not in mural GC. In both cumulus and mural GC, there was no correlation between KISS-1R mRNA levels and BMI, peak E2 level, # of oocytes retrieved or # of days of stimulation. CONCLUSION: These data support the rodent studies where gonadotropin priming resulted in upregulation of KISS-1R gene expression. Alterations in Kp sensitivity in human GC might provide more insight in understanding the function of ovarian Kp signaling and its implications on ovulatory dysfunction and possibly infertility. Supported by: Internal funds.
P-683 Wednesday, October 16, 2013 THE PDPK1 GENE VARIANTS DO NOT SEEM TO BE ASSOCIATED WITH PREMATURE OVARIAN FAILURE. Q. Liu, I. Van den Veyver, W. E. Gibbons, E. Kovanci. Obstetrics and Gynecology, Baylor College of Medicine, Houston, TX. OBJECTIVE: The PDPK1 gene, located on chromosome 16p13.3, encodes 3-phosphoinositide-dependent protein kinase (PDPK1). PDPK1 signaling in oocytes preserves reproductive lifespan by maintaining the survival of ovarian primordial follicles. One animal study showed that in mice lacking the PDPK1 gene in oocytes, the majority of primordial follicles are depleted around the onset of sexual maturity, causing premature ovarian failure (POF) during early adulthood. The study further showed that PDPK1 regulate the primordial follicles through PDPK1-Akt-p70 S6 kinase 1 (S6K1)-ribosomal protein S6 (rpS6) signaling in oocytes, indicating the importance of this pathway in controlling the survival. Our goal was to
S345
investigate whether novel variants in the PDPK1 coding region were present in our study population. DESIGN: Mutation analysis. MATERIALS AND METHODS: We collected genomic DNA from predominantly North American Caucasian women with POF defined as amenorrhea prior to age 40 with elevated FSH >20 IU/L. Primer sets specific for all 14 exons and exon-intron boundaries of PDPK1 were designed for polymerase chain reaction (PCR). The PCR products were then sequenced using Sanger sequencing method on a commercial platform and systematically compared to the human genome database sequence (ENSG00000178804) using Sequencher v5.0 (Gene Codes Corporation, Ann Arbor, MI). Chromatograms were also manually examined to identify sequence variants. RESULTS: In 69 women with POF, we did not detect any mutation. Moreover, no other novel or known polymorphisms were identified in any exon of PDPK1. CONCLUSION: All 14 exons of PDPK1 are highly conserved in our study population. Therefore, PDPK1 mutations are not likely to be a common cause of sporadic POF in Caucasians. P-684 Wednesday, October 16, 2013 THE EVIDENCE OF BIOLOGICAL PROCESSES IN CUMULUS CELLS FROM PATIENTS UNDERGOING IN VITRO FERTILIZATION TREATMENT BASED ON PROTEOMICS ANALYSIS. T. C. Bonetti,a J. Tipton,b E. L. Motta,a,c M. Riboldi,c J. R. Alegretti,a I. D. Silva.a aGynecology Department, Federal University of Sao Paulo, Sao Paulo, Brazil; bProteomics and Mass Spectrometry Facility - Center of Excellence of Drug Discovery and Innovation, University of South Florida, Tampa, FL; cHuntington - Medicina Reprodutiva, Sao Paulo, Brazil. OBJECTIVE: The mechanisms which affect oocyte quality are not complete understood. Cumulus cells (CC) are highly metabolic and directly associated to oocyte development and maturation, being an import regulator of oocyte quality. The aim of this preliminary study was to evaluate the proteomic profile of CC from patients undergoing IVF treatment. DESIGN: Preliminary prospective cohort study. MATERIALS AND METHODS: This study included CC obtained from 4 patients undergoing ICSI cycles. Patients were submitted to pituitary blockage with GnRH agonist, ovarian stimulation using rFSH and final follicular maturation triggered by rhCG. Oocyte retrieval was performed 35-36 hours after trigger and CC were recovered. Standardization of samples preparation to mass spectrometry used cells disruptions, tryptic digestion and desalting processes were performed. Peptides for each sample were analyzed by nanoliquid chromatography (nLC) on-line by positive-ion micro-electrospray with a linear Ion Trap (LTQ-XL). Data was searched against human Uniprot database with MASCOT and validated with Scaffold 3.6 with a 2% false positive rate. Pathway analysis was performed by the MetaCore network building tool. RESULTS: We identified 156 proteins. Highlighted Gene Ontology processes included exocytosis, secretion by cell, coagulation and homeostasis. Moreover, IL-6 signaling, iron transport and complement and Kallikreinkinin systems were processes probably activated in CC according to proteins present. CONCLUSION: Mass spectrometry approach is high throughput methodology which allows novel functional profiles and molecular processes identification. The preliminary outcomes of this study shown some processes associated with CC metabolism, which can help understanding the molecular mechanisms of oocyte development and maturation. Further studies are being conducted to investigate the differential expression of CC proteome in different causes of infertility and also relate them with oocyte quality and IVF outcomes. Supported by: FAPESP: 2010/51877-1, FAPESP: 2010/51873-6. P-685 Wednesday, October 16, 2013
2003), but there have been few reports about follicle diameter at LH surge. Therefore we examined whether aging and ovarian reserve have any impact on follicle diameter at LH surge. DESIGN: Retrospective analysis of data of patients who had frozen blastocyst transfer between Jan 2007 and May 2012. MATERIALS AND METHODS: Follicle diameter was measured in 182 natural cycles of 108 patients on the day of LH surge (serum LH > 10 mIU/mL and progesterone < 1.1 ng/mL). RESULTS: Although age was significantly younger in pregnant cases than in non-pregnant cases (P¼0.0168), serum AMH or other hormone levels were not significantly different. Mean follicle diameters were not significantly different among groups stratified by age (< 30, 30-34, 35-39, > 39 yo) or AMH (< 1.0, R 1.0 and < 2.0, R 2.0 and < 4.0, R 4.0 ng/mL). However, mean follicle diameters were significantly larger in cases of LH > 20 (17.91.5 mm, meanSD) compared with those of LH % 20 (17.32.1mm) (P¼0.0254). More than 2 mm of differences were noted in follicle diameters at LH surge even in a single patient. CONCLUSION: In women with sufficient ovarian reserve which enables blastocyst cryopreservation, LH surge occurs when the follicle diameter reaches approximately 17-18 mm in many of their natural cycles. We also have to consider cycle to cycle variability of follicular size at LH surge, however, when we estimate the timing of LH surge. P-686 Wednesday, October 16, 2013 H19 EXPRESSION IS INCREASED IN CUMULUS CELLS OF PCOS PATIENTS UNDERGOING IVF. A. N. Kallen, C. Karakaya, E. Seli, Y. Huang. Department of Obstetrics, Gynecology, and Reproductive Sciences, Yale University School of Medicine, New Haven, CT. OBJECTIVE: The H19 long noncoding RNA (lncRNA) plays important roles in development and growth. We have observed that H19 knockdown in granulosa cells leads to downregulation of the key steroidogenic enzymes STAR and cyp11a1, likely in a let-7-mediated fashion. Given the established role of let-7 in glucose homeostasis, and the importance of steroid hormone production in PCOS, we sought to determine whether H19 expression was altered in women with PCOS undergoing IVF, compared to non-PCOS women. DESIGN: Experimental. MATERIALS AND METHODS: We evaluated 39 women undergoing IVF (29 non-PCOS women undergoing IVF for male factor infertility and 10 women with PCOS). At time of oocyte retrieval, cumulus cells (CCs) were collected, total RNA was isolated and cDNA was synthesized by reverse transcription. RNA levels were analyzed using quantitative real-time PCR (qRT-PCR). H19 RNA levels were normalized to those of b-tubulin and presented as relative expression levels using the comparative CT method. Statistical analysis was performed using the Student’s t-test. RESULTS: H19 expression was increased 2.8-fold in CCs of PCOS patients undergoing IVF compared to non-PCOS normal responders (p¼0.04). There were no significant differences between the two groups in terms of age (mean 29.8 in PCOS vs 33.6 in non-PCOS patients), day 3 FSH (6.38 vs 6.96), or peak estradiol level (1758.3 vs 1441.5). Notably, non-PCOS patients received higher total dose of gonadotropins (2617.5 vs 3394.6; p¼0.02). CONCLUSION: We have recently discovered that H19 regulates the activity of the let-7 family of miRNAs. In this study, we demonstrate an increase in H19 expression in CCs of PCOS patients undergoing IVF compared to controls. The possibility that ovarian H19 expression plays a role in follicular development, glucose metabolism, and/or androgen production is intriguing and may be relevant to women with PCOS, in whom impaired glucose tolerance has major implications for cardiovascular health and fertility. Supported by: Albert McKern Scholar Award. P-687 Wednesday, October 16, 2013
AVERAGE FOLLICLE SIZE AT LH SURGE IN 182 NATURAL K. Ohara,a S. Matsunaga,a M. Saito,a CYCLES. Y. Takai,a N. Hayashi,b H. Seki.a aObstetrics and Gynecology, Saitama Medical Center/Saitama Medical University, Kawagoe, Saitama, Japan; bMuse Ladies Clinic, Fujimino, Saitama, Japan.
PRESENCE OF OLFACTORY RECEPTORS IN NORMAL AND NEOPLASTIC (HEY) CELLS IN THE FEMALE REPRODUCTIVE TRACT. S. J. Mucowski,a C. Marion,b Y. Liu,b K. A. Bendikson,a F. Z. Stanczyk,a L. Dubeau.b aObstetrics and Gynecology, Keck School of Medicine of USC, Los Angeles, CA; bPathology, Keck School of Medicine of USC, Los Angeles, CA.
OBJECTIVE: It was reported that women > 44 years old ovulate at a smaller follicle diameter than those < 35 years old (J Clin Endocrinol Metab,
OBJECTIVE: Olfactory receptor expression has been shown in placenta and in male reproductive organs, but little is known about its
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ASRM Abstracts
Vol. 100, No. 3, Supplement, September 2013
P-680 Wednesday, October 16, 2013 FATTY ACID SYNTHASE IS EXPRESSED AND ENZYMATICALLY ACTIVE IN THE MOUSE OVARY AND HUMAN GRANULOSA CELLS. J. K. Riley, J. K. Bolzenius, W. G. Lewis, E. S. Jungheim. Obstetrics and Gynecology, Washington University School of Medicine, St. Louis, MO. OBJECTIVE: Animals obtain fatty acids from their diet or via de novo lipogenesis, the enzymatic pathway in which carbohydrate is converted into fat. Fatty acid synthase (FAS) is a key enzyme in this metabolic pathway, catalyzing the synthesis of long-chain fatty acids. The goal of our study is to characterize FAS expression and enzymatic activity in mouse ovaries and human granulosa cells. DESIGN: FAS activity was assessed in gonadotropin-stimulated mouse ovarian homogenates. To identify potential sources of this enzymatic activity we determined the cell specific localization of FAS expression in the mouse ovary. As an extension of our mouse studies, FAS protein expression and enzymatic activity were characterized in human granulosa cells. MATERIALS AND METHODS: C57BL/6 mice were superovulated and euthanized 48 h post pregnant mare serum gonadotropin injection or at distinct time points post human chorionic gonadotropin injection. FAS expression was examined via immunohistochemistry and western blot. FAS activity in ovarian homogenates was measured by the rate of NADPH oxidation pre and post addition of malonyl-CoA, a FAS substrate. Finally, human granulosa cells were collected from follicular aspirates immediately following oocyte retrieval during IVF cycles. FAS activity was assessed and protein expression examined via western blot. RESULTS: FAS activity was detected in gonadotropin-stimulated mouse ovarian homogenates. Several cell types within the mouse ovary expressed FAS protein including cumulus cells, granulosa cells, theca cells and cells present within the corpus luteum. A low level of FAS protein was also detected in the oocyte. Importantly, FAS was expressed and functional in human granulosa cells. CONCLUSION: Distinct cell types within the mouse and human ovary have the ability to synthesize fatty acids through de novo lipogenesis. Future studies will elucidate the role of de novo lipogenesis in ovarian function.
P-681 Wednesday, October 16, 2013 FOLLOWING PREVIOUSLY DESCRIBED OVARIAN PHENOTYPES OF THE FMR1 GENE, METHYLATION FRACTIONS WERE SIGNIFICANTLY SKEWED BETWEEN FMR1 GENOTYPES AND SUB-GENOTYPES. D. H. Barad,a,b G. J. Latham,c S. Filipovic-Sadic,c V. A. Kushnir,a A. Shohat-Tal,a N. Gleicher.a,b aCenter for Human Reproduction, New York, NY; bFoundation for Reproductive Medicine, New York, NY; cAsuragen, Austin, TX. OBJECTIVE: Based on a normal CGG count range of n¼26-34, new FMR1 genotypes and sub-genotypes have been associated with distinct ovarian aging patterns.We attempted to assess if these genotypes and sub-genotypes vary in methylation or AGG interruption patterns. DESIGN: Prospective collaborative study between infertility center and corporation with a novel FMR1 assay (Chen et al Genet Med 2011;13:52838). MATERIALS AND METHODS: We evaluated 26 infertility patients with high performance FMR1 PCR (as reported in above reference), which assesses CGGn, intermittent AGG genotypes and methylation status of each allele. For CGGn, patients were classified as previously reported [PLoS ONE 2010;5(12): e15303] as normal (norm) if both alleles were in normal CGG range (n¼26-34), homozygous (hom) if both were outside normal range, and heterozygous (het) if one was outside normal range. Hom and het were further divided into sub-genotypes; low if the abnormal-range allele was n<26 and high if n>34. RESULTS: No significant difference in methylation percentage was observed in both FMR1 alleles for any pairwise sample combination. A statistically significant skew in methylation fraction, however, was observed between women with het-norm/low sub-genotype and women with norm
FERTILITY & STERILITYÒ
genotype (analysis of variance P¼0.0167). Specifically, methylation skew from 50% per CGG bin (suggesting perfect random X-inactivation) was 7.3% for both alleles in norm women, 19.7% in women with low and 17.3% with high sub-genotype (t test, comparisons of means: low vs. norm, P¼0.0063; high vs. norm, P¼0.0605; low vs. high, P¼0.6529). Number of AGG interruptions per CGGn sub-genotype did not differ from reference values. CONCLUSION: Observed skew in methylation fraction was the strongest with low alleles and weakest in women with norm FMR1. These findings exactly mimic a variety of previously reported different FMR1 phenotypical appearances, including IVF pregnancy chances (PLoS ONE 2010). Supported by: Foundation for Reproductive Medicine, Center for Human Reproduction, Asuragen.
P-682 Wednesday, October 16, 2013 GONADOTROPIN STIMULATION IS ASSOCIATED WITH UPREGULATION OF KISSPEPTIN RECEPTOR IN HUMAN CUMULUS GRANULOSA CELLS. R. S. Raj, P. R. Casson, Z. Merhi. Obstetrics, Gynecology and Reproductive Sciences, University of Vermont College of Medicine, Burlington, VT. OBJECTIVE: Kisspeptin (Kp) receptor (KISS-1R) has been recently detected in animal and human ovaries, where it plays a role in ovulation and oocyte maturation. Gonadotropin treatment stimulates Kp gene expression in rat ovaries, further indicating its role in ovulation. We have recently shown that Kp mRNA is not expressed by human luteinized granulosa cells (GC) and that there is an age-related upregulation in KISS-1R expression in human GC indicating a possible physiologic role for the Kp system in human follicular dynamics. The objective of this study was to test the hypothesis that gonadotropins given during controlled ovarian hyperstimulation (COH) in IVF increase the expression of KISS-1R gene in human GC, potentially impacting ovarian Kp sensitivity. DESIGN: Experimental human study. MATERIALS AND METHODS: 12 non-PCOS women (mean age¼34.6 1.5 years, mean BMI¼28.5 1.9 kg/m2) with normal ovarian reserve (defined as Day 3 FSH < 10 mIU/mL) who underwent COH followed by oocyte retrieval for IVF were enrolled. Cumulus and mural GC were collected separately. KISS-1R mRNA levels were quantified using RTPCR. Pearson correlation was used to assess the relationship between KISS-1 mRNA levels and gonadotropin dose, peak E2 level on day of hCG, # of eggs retrieved and # of days of stimulation. RESULTS: As previously shown, there was a positive correlation between age and KISS-1R mRNA levels (r ¼ 0.33, p<0.05). KISS-1R mRNA levels were positively correlated with the gonadotropin dose used in cumulus (r ¼ 0.34, p<0.05) but not in mural GC. In both cumulus and mural GC, there was no correlation between KISS-1R mRNA levels and BMI, peak E2 level, # of oocytes retrieved or # of days of stimulation. CONCLUSION: These data support the rodent studies where gonadotropin priming resulted in upregulation of KISS-1R gene expression. Alterations in Kp sensitivity in human GC might provide more insight in understanding the function of ovarian Kp signaling and its implications on ovulatory dysfunction and possibly infertility. Supported by: Internal funds.
P-683 Wednesday, October 16, 2013 THE PDPK1 GENE VARIANTS DO NOT SEEM TO BE ASSOCIATED WITH PREMATURE OVARIAN FAILURE. Q. Liu, I. Van den Veyver, W. E. Gibbons, E. Kovanci. Obstetrics and Gynecology, Baylor College of Medicine, Houston, TX. OBJECTIVE: The PDPK1 gene, located on chromosome 16p13.3, encodes 3-phosphoinositide-dependent protein kinase (PDPK1). PDPK1 signaling in oocytes preserves reproductive lifespan by maintaining the survival of ovarian primordial follicles. One animal study showed that in mice lacking the PDPK1 gene in oocytes, the majority of primordial follicles are depleted around the onset of sexual maturity, causing premature ovarian failure (POF) during early adulthood. The study further showed that PDPK1 regulate the primordial follicles through PDPK1-Akt-p70 S6 kinase 1 (S6K1)-ribosomal protein S6 (rpS6) signaling in oocytes, indicating the importance of this pathway in controlling the survival. Our goal was to
S345
investigate whether novel variants in the PDPK1 coding region were present in our study population. DESIGN: Mutation analysis. MATERIALS AND METHODS: We collected genomic DNA from predominantly North American Caucasian women with POF defined as amenorrhea prior to age 40 with elevated FSH >20 IU/L. Primer sets specific for all 14 exons and exon-intron boundaries of PDPK1 were designed for polymerase chain reaction (PCR). The PCR products were then sequenced using Sanger sequencing method on a commercial platform and systematically compared to the human genome database sequence (ENSG00000178804) using Sequencher v5.0 (Gene Codes Corporation, Ann Arbor, MI). Chromatograms were also manually examined to identify sequence variants. RESULTS: In 69 women with POF, we did not detect any mutation. Moreover, no other novel or known polymorphisms were identified in any exon of PDPK1. CONCLUSION: All 14 exons of PDPK1 are highly conserved in our study population. Therefore, PDPK1 mutations are not likely to be a common cause of sporadic POF in Caucasians. P-684 Wednesday, October 16, 2013 THE EVIDENCE OF BIOLOGICAL PROCESSES IN CUMULUS CELLS FROM PATIENTS UNDERGOING IN VITRO FERTILIZATION TREATMENT BASED ON PROTEOMICS ANALYSIS. T. C. Bonetti,a J. Tipton,b E. L. Motta,a,c M. Riboldi,c J. R. Alegretti,a I. D. Silva.a aGynecology Department, Federal University of Sao Paulo, Sao Paulo, Brazil; bProteomics and Mass Spectrometry Facility - Center of Excellence of Drug Discovery and Innovation, University of South Florida, Tampa, FL; cHuntington - Medicina Reprodutiva, Sao Paulo, Brazil. OBJECTIVE: The mechanisms which affect oocyte quality are not complete understood. Cumulus cells (CC) are highly metabolic and directly associated to oocyte development and maturation, being an import regulator of oocyte quality. The aim of this preliminary study was to evaluate the proteomic profile of CC from patients undergoing IVF treatment. DESIGN: Preliminary prospective cohort study. MATERIALS AND METHODS: This study included CC obtained from 4 patients undergoing ICSI cycles. Patients were submitted to pituitary blockage with GnRH agonist, ovarian stimulation using rFSH and final follicular maturation triggered by rhCG. Oocyte retrieval was performed 35-36 hours after trigger and CC were recovered. Standardization of samples preparation to mass spectrometry used cells disruptions, tryptic digestion and desalting processes were performed. Peptides for each sample were analyzed by nanoliquid chromatography (nLC) on-line by positive-ion micro-electrospray with a linear Ion Trap (LTQ-XL). Data was searched against human Uniprot database with MASCOT and validated with Scaffold 3.6 with a 2% false positive rate. Pathway analysis was performed by the MetaCore network building tool. RESULTS: We identified 156 proteins. Highlighted Gene Ontology processes included exocytosis, secretion by cell, coagulation and homeostasis. Moreover, IL-6 signaling, iron transport and complement and Kallikreinkinin systems were processes probably activated in CC according to proteins present. CONCLUSION: Mass spectrometry approach is high throughput methodology which allows novel functional profiles and molecular processes identification. The preliminary outcomes of this study shown some processes associated with CC metabolism, which can help understanding the molecular mechanisms of oocyte development and maturation. Further studies are being conducted to investigate the differential expression of CC proteome in different causes of infertility and also relate them with oocyte quality and IVF outcomes. Supported by: FAPESP: 2010/51877-1, FAPESP: 2010/51873-6. P-685 Wednesday, October 16, 2013
2003), but there have been few reports about follicle diameter at LH surge. Therefore we examined whether aging and ovarian reserve have any impact on follicle diameter at LH surge. DESIGN: Retrospective analysis of data of patients who had frozen blastocyst transfer between Jan 2007 and May 2012. MATERIALS AND METHODS: Follicle diameter was measured in 182 natural cycles of 108 patients on the day of LH surge (serum LH > 10 mIU/mL and progesterone < 1.1 ng/mL). RESULTS: Although age was significantly younger in pregnant cases than in non-pregnant cases (P¼0.0168), serum AMH or other hormone levels were not significantly different. Mean follicle diameters were not significantly different among groups stratified by age (< 30, 30-34, 35-39, > 39 yo) or AMH (< 1.0, R 1.0 and < 2.0, R 2.0 and < 4.0, R 4.0 ng/mL). However, mean follicle diameters were significantly larger in cases of LH > 20 (17.91.5 mm, meanSD) compared with those of LH % 20 (17.32.1mm) (P¼0.0254). More than 2 mm of differences were noted in follicle diameters at LH surge even in a single patient. CONCLUSION: In women with sufficient ovarian reserve which enables blastocyst cryopreservation, LH surge occurs when the follicle diameter reaches approximately 17-18 mm in many of their natural cycles. We also have to consider cycle to cycle variability of follicular size at LH surge, however, when we estimate the timing of LH surge. P-686 Wednesday, October 16, 2013 H19 EXPRESSION IS INCREASED IN CUMULUS CELLS OF PCOS PATIENTS UNDERGOING IVF. A. N. Kallen, C. Karakaya, E. Seli, Y. Huang. Department of Obstetrics, Gynecology, and Reproductive Sciences, Yale University School of Medicine, New Haven, CT. OBJECTIVE: The H19 long noncoding RNA (lncRNA) plays important roles in development and growth. We have observed that H19 knockdown in granulosa cells leads to downregulation of the key steroidogenic enzymes STAR and cyp11a1, likely in a let-7-mediated fashion. Given the established role of let-7 in glucose homeostasis, and the importance of steroid hormone production in PCOS, we sought to determine whether H19 expression was altered in women with PCOS undergoing IVF, compared to non-PCOS women. DESIGN: Experimental. MATERIALS AND METHODS: We evaluated 39 women undergoing IVF (29 non-PCOS women undergoing IVF for male factor infertility and 10 women with PCOS). At time of oocyte retrieval, cumulus cells (CCs) were collected, total RNA was isolated and cDNA was synthesized by reverse transcription. RNA levels were analyzed using quantitative real-time PCR (qRT-PCR). H19 RNA levels were normalized to those of b-tubulin and presented as relative expression levels using the comparative CT method. Statistical analysis was performed using the Student’s t-test. RESULTS: H19 expression was increased 2.8-fold in CCs of PCOS patients undergoing IVF compared to non-PCOS normal responders (p¼0.04). There were no significant differences between the two groups in terms of age (mean 29.8 in PCOS vs 33.6 in non-PCOS patients), day 3 FSH (6.38 vs 6.96), or peak estradiol level (1758.3 vs 1441.5). Notably, non-PCOS patients received higher total dose of gonadotropins (2617.5 vs 3394.6; p¼0.02). CONCLUSION: We have recently discovered that H19 regulates the activity of the let-7 family of miRNAs. In this study, we demonstrate an increase in H19 expression in CCs of PCOS patients undergoing IVF compared to controls. The possibility that ovarian H19 expression plays a role in follicular development, glucose metabolism, and/or androgen production is intriguing and may be relevant to women with PCOS, in whom impaired glucose tolerance has major implications for cardiovascular health and fertility. Supported by: Albert McKern Scholar Award. P-687 Wednesday, October 16, 2013
AVERAGE FOLLICLE SIZE AT LH SURGE IN 182 NATURAL K. Ohara,a S. Matsunaga,a M. Saito,a CYCLES. Y. Takai,a N. Hayashi,b H. Seki.a aObstetrics and Gynecology, Saitama Medical Center/Saitama Medical University, Kawagoe, Saitama, Japan; bMuse Ladies Clinic, Fujimino, Saitama, Japan.
PRESENCE OF OLFACTORY RECEPTORS IN NORMAL AND NEOPLASTIC (HEY) CELLS IN THE FEMALE REPRODUCTIVE TRACT. S. J. Mucowski,a C. Marion,b Y. Liu,b K. A. Bendikson,a F. Z. Stanczyk,a L. Dubeau.b aObstetrics and Gynecology, Keck School of Medicine of USC, Los Angeles, CA; bPathology, Keck School of Medicine of USC, Los Angeles, CA.
OBJECTIVE: It was reported that women > 44 years old ovulate at a smaller follicle diameter than those < 35 years old (J Clin Endocrinol Metab,
OBJECTIVE: Olfactory receptor expression has been shown in placenta and in male reproductive organs, but little is known about its
S346
ASRM Abstracts
Vol. 100, No. 3, Supplement, September 2013