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Rna high throughput sequencing of cumulus cells reveals new pathways involved in human oocyte aging
MATERIALS AND METHODS: One hundred and fifty-three oocytes (51 of GV, MI and MII, respectively) from 51 cases of normal ovulatory women and 72 oocytes (24 of GV, MI and MII, respectively) from 24 women with polycystic ovary syndrome (PCOS) were collected in the reproductive center from March to December in 2013. Nested quantitative real-time PCR was used to detect the transcript abundance of GDF9 and BMP15 in a single oocyte. RESULTS: The data were expressed as medians with 25th-75th in parentheses. The ratio of GDF9 and BMP15 mRNA expression was 4.65 (1.83 - 9.05), 8.80 (2.83 - 14.69) and 11.63 (7.69 - 15.26) in normal oocytes of GV, MI and MII, respectively. It was increased during oocyte maturation in normal oocytes (P<0.05). The results were 99.17 (19.21 - 242.82), 11.07 (1.20 - 166.01) and 2.50 (0.05 - 8.94) in PCOS oocytes of GV, MI and MII, respectively. It was reduced during oocyte maturation in PCOS oocytes (P<0.05). CONCLUSION: The ratio of GDF9 and BMP15 mRNA expression demonstrates dynamic changes in human oocytes during maturation, and the trends are different between normal and PCOS oocytes. These results suggest that the expression pattern of GDF9 and BMP15 was abnormal in PCOS oocytes during maturation. Supported by: The present study was Supported by the grants from National Natural Science Foundation of China (Grant No.81200476), Natural Science Foundation of Guangdong Province (Grant No. S2012040007770), National Doctoral Foundation of China (Grant No. 20120171120122), Medical Science and Technology Research Foundation of Guangdong Province (Grant No. B2012150). P-587 Wednesday, October 22, 2014 OOCYTES FROM WOMEN WITH DIMINISHED OVARIAN RESERVE AND OBESITY HAVE SHORTENED TELOMERES. D. M. F. Antunes,a,b K. K. Kalmbach,a F. Wang,a M. L. Seth-Smith,a Y. Kramer,a F. B. Kohlrausch,b D. L. Keefe.a aDepartment of Obstetrics and Gynecology, New York University, New York, NY; b Department of Pathology, Fluminense Federal University, Niteroi, Rio de Janeiro, Brazil. OBJECTIVE: Telomere shortening in mouse oocytes promotes genomic instability, apoptosis, spindle abnormalities and infertility. In humans, the highly correlated (R2¼98%) polar body telomere length is associated with embryo aneuploidy, fragmentation and decreased pregnancy rate. To further test the hypothesis that telomere length reflects oocyte quality we examined the relationship between oocyte telomere length and factors associated with oocyte quality, including ovarian reserve and body mass index (BMI). DESIGN: Prospective observational study. MATERIALS AND METHODS: 143 arrested oocytes (M1and M2 stages) were collected three days after retrieval from consenting women undergoing IVF at NYU Fertility Center. BMI, age, anti-M€ullerian hormone (AMH) and follicle-stimulating hormone (FSH) levels were obtained from medical records. A novel single-cell telomere length assay (SCT-pqPCR) measured telomere length relative to a reference gene (T/R) in individual oocytes (Wang et al., 2013). Mann-Whitney and Kruskal-Wallis Test were performed to compare means. RESULTS: Telomere length in oocytes from women with AMH < 0.8 ng/ml (n¼20; 1.570.47) was significantly less than that from oocytes of women with AMH > 0.8 ng/ml (n¼30; 2.840.65) p¼0.044. Oocytes from women with FSH > 10 IU (n¼21) had significantly shorter telomeres (1.270.31) than oocytes from women with FSH < 10 IU (n¼108; 2.230.27), p¼0.026. Telomeres in oocytes from older women (n¼99; 2.090.28) were shorter than those from younger women (<35 years old, n¼44) (2.730.59), but this difference did not reach significance at the sample size studied (p¼0.112). Oocytes from overweight women (n¼51; 1.340.23) had shorter telomeres than those from women with normal BMI (n¼67) (2.560.48) p¼0.029. Oocytes from underweight women (n¼11) had telomeres similar to those from women with normal BMI (2.380.62). CONCLUSION: Oocytes from women with depleted low ovarian reserve and obesity, factors known to contribute to poor oocyte quality, have shortened telomeres. Polar body telomere length, which reliably estimates oocyte telomere length, may provide a valuable assay of oocyte quality. Supported by: CAPES Brazil, Dept Ob/Gyn NYU Langone and NIH1UL1RR029893. P-588 Wednesday, October 22, 2014 RNA HIGH THROUGHPUT SEQUENCING OF CUMULUS CELLS REVEALS NEW PATHWAYS INVOLVED IN HUMAN OOCYTE AGING. E. Molinari,a P. Patrizio,b A. M. Pyle.a aDepartment of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT; b Yale Fertility Center, Yale University, New Haven, CT.
FERTILITY & STERILITYÒ
OBJECTIVE: To investigate the impact of aging on metaphase II oocytes by means of RNA high throughput sequencing of human cumulus cells (CCs) and to provide the first comprehensive whole transcriptome signature analysis of CCs by assessing simultaneously both coding and non-coding transcripts. DESIGN: Basic research comparative study. MATERIALS AND METHODS: A total of 21 CCs were isolated from mature MII oocytes collected from patients aged <30 years (younger, n¼10) and from patients aged >40 years old (older, n¼11). Only cases of male factor infertility were considered. CCs were individually processed for RNA extraction, library preparation and sequenced on the Illumina HiSeq 2000 platform. Two different enrichment protocols were used to obtain information regarding mRNA and non-coding RNAs. The FastQC program was used for assessing overall read quality, whereas gene mapping, annotation, counting and differential expression was performed according to the programs in the Tuxedo pipeline [1]. Gene enrichment and gene ontology analysis were used to define upregulated gene networks and interactions in the two cohorts [2, 3]. Significance of differentially expressed genes was assigned when both p value and false discovery rate were <0.05. Validation was performed with qPCR. RESULTS: We found that 319 genes were differentially expressed between the younger and older cohorts. In CCs collected from the younger group, genes involved in oxidoreductase activity, extracellular matrix organization, and membrane composition were strongly upregulated (p<0.001 and FDR <0.05) compared to older women. By contrast, CCs from older patients show an overrepresentation of genes involved in the oxidative stress cascade and apoptosis (p<0.001, FDR <0.05). The whole transcriptomic analysis of CCs revealed an interesting complex of expressed transcripts, including a new set of potential long noncoding RNAs. CONCLUSION: This novel application of RNA high throughput sequencing on human cumulus cells reveals: a) The complete transcriptomic signature of CCs; b) The first RNAseq analysis to contrast younger and older patients and highlights pathways involved in diminishing female fertility. The unprecedented amount of information revealed by RNAseq will help identify markers related to oocyte aging, such as aneuploidy and reduced embryonic developmental competence. P-589 Wednesday, October 22, 2014 MITOCHONDRIAL DNA ABUNDANCE DECLINES IN HUMAN CUMULUS GRANULOSA CELLS WITH AGE. V. A. Kushnir,a Y.-G. Wu,a I.-C. Chen,a H.-J. Lee,a D. H. Barad,a,b E. Lazzaroni-Tealdi,a N. Gleicher.a,b aCenter for Human Reproduction, New York, NY; bFoundation for Reproductive Medicine, New York, NY. OBJECTIVE: Cumulus granulosa cells (cGCs) are fundamental to oocyte maturation, supporting metabolism, disposal of waste and molecular signaling. Since mitochondrial (mt) contribution to reproductive aging is poorly understood, we examined whether mtDNA abundance changes in cGCs with advancing female age. DESIGN: Prospective case control study. MATERIALS AND METHODS: We examined effects of aging on mtDNA/ nuclear DNA (nDNA) ratios using real-time PCR in human cGCs , collected at time of oocyte retrieval from 4 young oocyte donors (ages 21-30 years), 4 younger (30-37 years) and 4 older (44-46 years) infertility patients. Nuclear PCR probes targeted GAPDH, while mitochondrial PCR probes targeted ND5. RESULTS: Relative mtDNA/nDNA ratios significantly decreased with advancing female age from 9.0 in young oocyte donors to 5.2 in younger infertility patients and 2.3 in oldest infertility patients (P¼0.03). CONCLUSION: These data demonstrate evidence for significant agerelated decline in human cGCs mtDNA abundance, which can be assumed to play a role in reproductive aging. Functional studies are ongoing at our center to further elucidate mitochondrial contribution to reproductive aging. Supported by: Foundation for Reproductive Medicine, Center for Human Reproduction. P-590 Wednesday, October 22, 2014 EFFECTS OF ADVANCING FEMALE AGE ON PROLIFERATION AND GENE EXPRESSION IN CULTURED HUMAN GRANULOSA CELLS FROM POST-HCG RETRIEVED FOLLICLES. Y.-G. Wu,a H.-J. Lee,a D. H. Barad,a,b V. A. Kushnir,a E. Lazzaroni-Tealdi,a N. Gleicher.a,b aCenter for Human Reproduction, New York, NY; bFoundation for Reproductive Medicine, New York, NY. OBJECTIVE: Granulosa cells (GCs) form the follicular microenvironment, which facilitates oocyte development, supplies energy, disposes of
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FERTILITY & STERILITYÒ
OBJECTIVE: To investigate the impact of aging on metaphase II oocytes by means of RNA high throughput sequencing of human cumulus cells (CCs) and to provide the first comprehensive whole transcriptome signature analysis of CCs by assessing simultaneously both coding and non-coding transcripts. DESIGN: Basic research comparative study. MATERIALS AND METHODS: A total of 21 CCs were isolated from mature MII oocytes collected from patients aged <30 years (younger, n¼10) and from patients aged >40 years old (older, n¼11). Only cases of male factor infertility were considered. CCs were individually processed for RNA extraction, library preparation and sequenced on the Illumina HiSeq 2000 platform. Two different enrichment protocols were used to obtain information regarding mRNA and non-coding RNAs. The FastQC program was used for assessing overall read quality, whereas gene mapping, annotation, counting and differential expression was performed according to the programs in the Tuxedo pipeline [1]. Gene enrichment and gene ontology analysis were used to define upregulated gene networks and interactions in the two cohorts [2, 3]. Significance of differentially expressed genes was assigned when both p value and false discovery rate were <0.05. Validation was performed with qPCR. RESULTS: We found that 319 genes were differentially expressed between the younger and older cohorts. In CCs collected from the younger group, genes involved in oxidoreductase activity, extracellular matrix organization, and membrane composition were strongly upregulated (p<0.001 and FDR <0.05) compared to older women. By contrast, CCs from older patients show an overrepresentation of genes involved in the oxidative stress cascade and apoptosis (p<0.001, FDR <0.05). The whole transcriptomic analysis of CCs revealed an interesting complex of expressed transcripts, including a new set of potential long noncoding RNAs. CONCLUSION: This novel application of RNA high throughput sequencing on human cumulus cells reveals: a) The complete transcriptomic signature of CCs; b) The first RNAseq analysis to contrast younger and older patients and highlights pathways involved in diminishing female fertility. The unprecedented amount of information revealed by RNAseq will help identify markers related to oocyte aging, such as aneuploidy and reduced embryonic developmental competence. P-589 Wednesday, October 22, 2014 MITOCHONDRIAL DNA ABUNDANCE DECLINES IN HUMAN CUMULUS GRANULOSA CELLS WITH AGE. V. A. Kushnir,a Y.-G. Wu,a I.-C. Chen,a H.-J. Lee,a D. H. Barad,a,b E. Lazzaroni-Tealdi,a N. Gleicher.a,b aCenter for Human Reproduction, New York, NY; bFoundation for Reproductive Medicine, New York, NY. OBJECTIVE: Cumulus granulosa cells (cGCs) are fundamental to oocyte maturation, supporting metabolism, disposal of waste and molecular signaling. Since mitochondrial (mt) contribution to reproductive aging is poorly understood, we examined whether mtDNA abundance changes in cGCs with advancing female age. DESIGN: Prospective case control study. MATERIALS AND METHODS: We examined effects of aging on mtDNA/ nuclear DNA (nDNA) ratios using real-time PCR in human cGCs , collected at time of oocyte retrieval from 4 young oocyte donors (ages 21-30 years), 4 younger (30-37 years) and 4 older (44-46 years) infertility patients. Nuclear PCR probes targeted GAPDH, while mitochondrial PCR probes targeted ND5. RESULTS: Relative mtDNA/nDNA ratios significantly decreased with advancing female age from 9.0 in young oocyte donors to 5.2 in younger infertility patients and 2.3 in oldest infertility patients (P¼0.03). CONCLUSION: These data demonstrate evidence for significant agerelated decline in human cGCs mtDNA abundance, which can be assumed to play a role in reproductive aging. Functional studies are ongoing at our center to further elucidate mitochondrial contribution to reproductive aging. Supported by: Foundation for Reproductive Medicine, Center for Human Reproduction. P-590 Wednesday, October 22, 2014 EFFECTS OF ADVANCING FEMALE AGE ON PROLIFERATION AND GENE EXPRESSION IN CULTURED HUMAN GRANULOSA CELLS FROM POST-HCG RETRIEVED FOLLICLES. Y.-G. Wu,a H.-J. Lee,a D. H. Barad,a,b V. A. Kushnir,a E. Lazzaroni-Tealdi,a N. Gleicher.a,b aCenter for Human Reproduction, New York, NY; bFoundation for Reproductive Medicine, New York, NY. OBJECTIVE: Granulosa cells (GCs) form the follicular microenvironment, which facilitates oocyte development, supplies energy, disposes of
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