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Regional enrichment of altered sperm DNA methylation marks associated with paternal aging
swim-up(TG1/TS1), and 2 hrs post-gradient/swim-up (TG2/TS2) samples at room temperature or 37 C, later in TG2/TS2 samples after 30, 60, and 90 min of incubation in PVP at 37 C. TG and TS (RT and 37 C) aliquots after 24 hours of incubation were also assessed. RESULTS: Considerign the overall population, TUNEL levels were significantly reduced after G and S separation relative to neat values (T0¼26% vs. TG¼15% and TS¼17%). After G, in group A, TUNEL levels were significantly higher in TG2-90 hours at RT(12.8%) and TG2-30 at 37 C(12.3%). Samples did not reach abnormal levels. In group B, the levels were significantly higher in TG2+60 at RT(31.6%) and TG2-30 at 37 C(31.4%). After S, in group A, the levels were significantly higher in TS2-30 hours at RT(30.4%) and 37 C(31.5%). In group B, TUNEL levels were significantly higher in TS2 at RT(16.4%) and 37 C(17.6). After 24 hours of incubation the increase was 11.4%(RT) and 13.2%(37 C) after G. When S was carried out the increase was 15.8%(RT) and 16.3%(37 C). Patients from group B had a significant increase in age, and a lower rate of normal sperm morphology and progressive motility. CONCLUSION: Sperm DNA fragmentation significantly decreased after G and S, regardless of the initial levels of the sample. Samples with TUNELR20% were more susceptible to a significant increase in DNA fragmentation over time. Samples after G and incubated at RT were lesser susceptible to a significant increase in DNA fragmentation. These data may be relevant for sperm preparation for ICSI. Supported by: CEGYR Foundation. O-286 Tuesday, October 15, 2013 04:15 PM REGIONAL ENRICHMENT OF ALTERED SPERM DNA METHYLATION MARKS ASSOCIATED WITH PATERNAL AGING. T. G. Jenkins,a K. I. Aston,a D. Carrell.a,b aAndrology and IVF Laboratories, University of Utah, Salt Lake City, UT; bHuman Genetics, University of Utah, Salt Lake City, UT. OBJECTIVE: To identify alterations to the sperm methyome associated with age. DESIGN: 17 fertile sperm donors collected two semen samples between 9 and 21 years apart. We analyzed the methylomes of these samples to identify age associated intra-individual alterations to DNA methylation that were common among individuals. MATERIALS AND METHODS: Sperm DNA was bisulfite converted and hybridized to Illumina’s Infinium HumanMethylation 450K BeadChip microarray. A sliding window analysis was performed on the derived methylation data to identify regions of differential methylation within paired samples. We analyzed all CpGs within a 1000 bp sliding window and calculated the pseudomedian of combined values to control for outliers. Benjamini/Hochberg FDR thresholds of 13 and 40 were used to test for significance. A minimum of 2 CpG probes were required within the 1000 bp region. Wilcoxon signed rank test was used to identify significant differences in pseudomedians between young and aged samples that were consistent across individuals. RESULTS: We identified many subtle yet highly significant alterations to the sperm DNA methylome that were common among the individuals screened. With an FDR threshold of 13, there were 321 genomic regions with decreased and 54 regions of increased DNA methylation with increasing age. With a highly stringent FDR threshold of 40 there were 172 genomic regions with decreased and 12 regions with increased DNA methylation with increasing age. These altered regions were found in CpG islands and within gene bodies. CONCLUSION: Our data indicate that subtle yet significant changes to the sperm DNA methylome occur with age. Remarkably, many of the ageassociated changes to the methylome localized to the same regions of the genome across individuals. Additional studies are required to determine whether these alterations might explain some of the paternal age associated changes in fertility, pregnancy outcome and offspring health. Supported by: This work Supported by a grant from the University of Utah Center on Aging. O-287 Tuesday, October 15, 2013 04:30 PM LEVELS OF THE OOCYTE ACTIVATION FACTOR, PHOSPHOLIPASE C ZETA, IN HUMAN SPERM ARE REDUCED FOLLOWING INCUBATION WITH POLYVINYLPYRROLIDONE (PVP). S. Yelumalai,a C. Jones,a G. Mounce,a E. McVeigh,b M. Fatum,b K. Coward.a aNuffield Department of Obstetrics and Gynaecology, University of Oxford, Oxford,
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ASRM Abstracts
Oxfordshire, United Kingdom; bOxford Fertility Unit, Oxford, Oxfordshire, United Kingdom. OBJECTIVE: Intracytoplasmic sperm injection (ICSI) involves the injection of a single sperm into an oocyte in order to facilitate fertilisation. Prior to ICSI, sperm are routinely pre-treated with polyvinylpyrrolidone (PVP), a viscous polymer, to facilitate immobilization and manipulation. However, PVP can cause detrimental effects upon sperm structure and function. Here, we investigated the effect of PVP upon phospholipase C zeta (PLCz), a cytosolic sperm protein critical for oocyte activation. Abnormalities in PLCz are strongly linked to oocyte activation deficiency. However, the effect of PVP upon PLCz in human sperm remains untested. DESIGN: To investigate and compare levels of PLCz in human sperm incubated with or without PVP by quantitative immunofluorescence. MATERIALS AND METHODS: Semen samples were obtained from Oxford Fertility Unit with informed written consent. Following density gradient washing, aliquots were treated with or without 10% PVP for 30 minutes at room temperature. After fixation, samples were exposed to a PLCz antibody and positive staining visualised by fluorescent microscopy. Mean levels of PLCz were determined by image analysis and compared by One-Way Analysis of Variance (ANOVA) followed by Bonferroni PostHoc analysis. RESULTS: Levels of PLCz were significantly reduced (p < 0.05) in sperm exposed to PVP compared to controls. Furthermore, the proportion of sperm exhibiting PLCz were significantly reduced (by 20 – 100%, p < 0.05) following exposure to PVP, compared to controls. CONCLUSION: Routine exposure of human sperm to PVP may exert detrimental effect upon the levels of PLCz and may thus reduce the oocyte activation ability of such sperm. Further studies seek to investigate the mechanisms underlying these observations which may facilitate in generating refinements or alternatives to existing laboratory protocols for ICSI. Supported by: The Nuffield Department of Obstetrics and Gynaecology (University of Oxford, UK), the University of Malaya and the Ministry of Higher Education, Malaysia.
O-288 Tuesday, October 15, 2013 04:45 PM RAMAN MICROSPECTROSCOPY: A NON-DESTRUCTIVE APPROACH FOR THE ANALYSIS OF DNA IN LIVING SPERM. V. Sanchez, J. Wistuba, S. Kliesch, S. Schlatt, C. Mallidis. Centre for Reproductive Medicine and Andrology, Muenster, NRW, Germany. OBJECTIVE: To investigate whether Raman microspectroscopic assessment of nDNA has a detrimental effect on sperm vitality. DESIGN: Exposure of sperm to Raman microspectroscopic assessment. Determination of viability following measurement and evaluation of obtained spectra. MATERIALS AND METHODS: Semen was obtained from patients attending CeRA. 5uL of motile sperm recovered by ‘‘swim up’’ were mixed with 5uL SpermSlow (Origio), placed on a Suprasil slide and coverslipped. Raman spectra were obtained using a LabRAM Aramis system (Horiba Jobin Yvon) from sperm with flagellar movement. A 632.8nm He-Ne laser, confocal and hole apertures of 100uM, 600 grooves/mm diffraction grating were used for 2 acquisitions of 15 secs over a spectral range of 6001800cm-1. Similar acquisitions were obtained from a position next to the cell to measure the background and confirm that the spectral signal was from the sperm. After measurement, each cell was re-examined to determine whether there was flagellar beat. Persistence of sperm motility was considered as an indication of sustained vitality. RESULTS: Although slightly diminished, sperm motility was maintained after exposure to the laser needed for the Raman microspectroscopic measurement indicating that the cells remained alive. The obtained spectra were distinctly different from those of the surrounding medium and contained the peaks previously shown to be characteristic of sperm nDNA. CONCLUSION: Raman microspectroscopy shows great potential as a means of analysing DNA in human sperm that can then be used in assisted reproductive procedures. We have previously found that the method provides accurate and reproducible results. Here we show that it does so whilst not affecting sperm viability. Following further refinement and the determination of its consequences on sperm function and nDNA integrity, the technique may fulfill its promise as a means of assessing, selecting and making available the best possible sperm for ART. Supported by: The study was conducted using internal CeRA funds.
Vol. 100, No. 3, Supplement, September 2013
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ASRM Abstracts
Oxfordshire, United Kingdom; bOxford Fertility Unit, Oxford, Oxfordshire, United Kingdom. OBJECTIVE: Intracytoplasmic sperm injection (ICSI) involves the injection of a single sperm into an oocyte in order to facilitate fertilisation. Prior to ICSI, sperm are routinely pre-treated with polyvinylpyrrolidone (PVP), a viscous polymer, to facilitate immobilization and manipulation. However, PVP can cause detrimental effects upon sperm structure and function. Here, we investigated the effect of PVP upon phospholipase C zeta (PLCz), a cytosolic sperm protein critical for oocyte activation. Abnormalities in PLCz are strongly linked to oocyte activation deficiency. However, the effect of PVP upon PLCz in human sperm remains untested. DESIGN: To investigate and compare levels of PLCz in human sperm incubated with or without PVP by quantitative immunofluorescence. MATERIALS AND METHODS: Semen samples were obtained from Oxford Fertility Unit with informed written consent. Following density gradient washing, aliquots were treated with or without 10% PVP for 30 minutes at room temperature. After fixation, samples were exposed to a PLCz antibody and positive staining visualised by fluorescent microscopy. Mean levels of PLCz were determined by image analysis and compared by One-Way Analysis of Variance (ANOVA) followed by Bonferroni PostHoc analysis. RESULTS: Levels of PLCz were significantly reduced (p < 0.05) in sperm exposed to PVP compared to controls. Furthermore, the proportion of sperm exhibiting PLCz were significantly reduced (by 20 – 100%, p < 0.05) following exposure to PVP, compared to controls. CONCLUSION: Routine exposure of human sperm to PVP may exert detrimental effect upon the levels of PLCz and may thus reduce the oocyte activation ability of such sperm. Further studies seek to investigate the mechanisms underlying these observations which may facilitate in generating refinements or alternatives to existing laboratory protocols for ICSI. Supported by: The Nuffield Department of Obstetrics and Gynaecology (University of Oxford, UK), the University of Malaya and the Ministry of Higher Education, Malaysia.
O-288 Tuesday, October 15, 2013 04:45 PM RAMAN MICROSPECTROSCOPY: A NON-DESTRUCTIVE APPROACH FOR THE ANALYSIS OF DNA IN LIVING SPERM. V. Sanchez, J. Wistuba, S. Kliesch, S. Schlatt, C. Mallidis. Centre for Reproductive Medicine and Andrology, Muenster, NRW, Germany. OBJECTIVE: To investigate whether Raman microspectroscopic assessment of nDNA has a detrimental effect on sperm vitality. DESIGN: Exposure of sperm to Raman microspectroscopic assessment. Determination of viability following measurement and evaluation of obtained spectra. MATERIALS AND METHODS: Semen was obtained from patients attending CeRA. 5uL of motile sperm recovered by ‘‘swim up’’ were mixed with 5uL SpermSlow (Origio), placed on a Suprasil slide and coverslipped. Raman spectra were obtained using a LabRAM Aramis system (Horiba Jobin Yvon) from sperm with flagellar movement. A 632.8nm He-Ne laser, confocal and hole apertures of 100uM, 600 grooves/mm diffraction grating were used for 2 acquisitions of 15 secs over a spectral range of 6001800cm-1. Similar acquisitions were obtained from a position next to the cell to measure the background and confirm that the spectral signal was from the sperm. After measurement, each cell was re-examined to determine whether there was flagellar beat. Persistence of sperm motility was considered as an indication of sustained vitality. RESULTS: Although slightly diminished, sperm motility was maintained after exposure to the laser needed for the Raman microspectroscopic measurement indicating that the cells remained alive. The obtained spectra were distinctly different from those of the surrounding medium and contained the peaks previously shown to be characteristic of sperm nDNA. CONCLUSION: Raman microspectroscopy shows great potential as a means of analysing DNA in human sperm that can then be used in assisted reproductive procedures. We have previously found that the method provides accurate and reproducible results. Here we show that it does so whilst not affecting sperm viability. Following further refinement and the determination of its consequences on sperm function and nDNA integrity, the technique may fulfill its promise as a means of assessing, selecting and making available the best possible sperm for ART. Supported by: The study was conducted using internal CeRA funds.
Vol. 100, No. 3, Supplement, September 2013