- Email: [email protected]
Vitrification of human embryonic stem cells with cryovials
MATERIALS AND METHODS: The first and second meiotic divisions were studied using fluorescent and time-lapse microscopy. Controlled compression of oocytes prevented rotation of the first meiotic spindle, which remained parallel to the surface of the oolemma. This resulted in the division of the maturing oocytes into two approximately equal cells, one of which was equivalent to an enlarged first polar body. Such 2-celled oocytes were cultured with sperm after localized openings were made in their zona pellucida using laser. Fertilization and embryonic development were photographed at frequent intervals. RESULTS: Each of the two cells in the oocytes established a second meiotic spindle. Both cells were fertilizable, released a second polar body and formed a male and female pronucleus. Each cell cleaved to establish twin 2-cell embryos within the zona pellucida. After each twin embryo had cleaved to beyond the 4-cell stage, it became difficult to distinguish individual embryos due to mingling of their blastomeres. Following intimate aggregation of blastomeres a single compacted morula was established. This progressed to a blastocyst, but expansion failed to occur. In some cases, both cells were fertilized within the zona but only one produced a cleaving embryo. In such cases the developing half produced a blastocyst that contained a large neighbouring cell. CONCLUSIONS: A single oocyte may give rise to twin intra-zonal embryos that subsequently combine to form a chimera, or a mosaic embryo, containing two maternal and two paternal genetic contributions. It can be deduced that the composite embryo could contain either an XX/XX, or XX/XY, or XY/XY genotype. But the developmental potential of such chimeric entities needs to be evaluated using genetic analysis. In addition, if the original twin embryos do not combine, their separate development with fusion and sharing of trophoblasts, could give rise to monochorionic twins. Supported by: None.
Tuesday, November 11, 2008 3:30 pm O-126 MEIOTIC SPINDLE LOCALIZATION AND MICROFILAMENT DISTRIBUTION DURING OOCYTE VITRIFICATION. C.-C. Chang, Z. P. Nagy, L.-Y. Sung, H. I. Kort, X. Yang, C. X. Tian. Reproductive Biology Associates, Atlanta, GA; University of Connecticut, Storrs. OBJECTIVE: During female meiosis, most of the maternal resources are retained in the oocyte for the early developing embryo. This outcome is coordinated by asymmetrical divisions. Two significant events ensure these asymmetrical divisions occur during meiosis: first, the meiotic spindle migrates towards the cell cortex, and second, a microfilament-rich cortical domain over the spindle is formed. It is not known if cryopreservation may impact these delicate arrangements which determine these critical processes. Our objective, therefore, was to investigate the specific changes of oocyte spindle localization and microfilament distribution before, during, and after vitrification. DESIGN: Timed observations were performed before, during, and after vitrification. MATERIALS AND METHODS: Mature oocytes were collected from 8wk-old BDF1 mice with standard superovulation. Cryopreservation on all oocytes was performed by the vitrification method with 15% ethylene glycol, 15% DMSO and 0.5 M sucrose. Oocyte warming was performed after vitrification by serial dilutions in three steps using 1.0M, 0.5M, and 0M sucrose solutions. To examine the spindle, the oocytes were fixed at 5 min of equilibration solution, 1 min of vitrification solution, 0 min of warming (warming directly into fixative), and 1h after warming, and stained with propidium iodide and FITC-conjugated anti-tubulin antibody by confocal microscopy. RESULTS: A total of 75 oocytes were included in the present study. After 5 min exposure to equilibration solution, all of the meiotic spindles were positioned at the microfilament-rich domain of the oocyte’s cortex (16/16) as the control group before exposure to cryoprotectant (12/12). All oocyte spindles were still localized to microfilament-rich domain of the oocyte’s cortex in vitrification solution (15/15) with even higher concentration of cryoprotectant and the subsequent cooling process (15/15). We observed that all examined oocytes sustained consistent microtubule and microfilament structures at 1h after warming (17/17). CONCLUSIONS: This study demonstrated that the spindle localization and microfilament distribution were well conserved throughout the cooling and warming processes, even though oocytes were exposed to high concentrations of cryoprotectants and non-physiological temperatures for substantial time. This study thus indicates that oocyte cryopreservation using vitrification may be a suitable technique in regard to maintaining cytoskeletal structure. Supported by: None.
S46
Abstracts
Tuesday, November 11, 2008 3:45 pm O-127 EMBRYONIC STEM CELL (ESC) PLURIPOTENCY REGULATORS HAVE NOVEL AND CRITICAL ROLES IN PRE-BLASTOCYST DEVELOPMENT. K. M. Foygel, S. H. Jun, D. E. Leong, B. Choi, W. H. Wong, M. W. M. Yao. Ob/Gyn, Stanford University School of Medicine, Stanford, CA; Applied Physics, Stanford University, Stanford, CA; Statistics, Stanford University, Stanford, CA. OBJECTIVE: Gene regulation and reprogramming during the maternalembryonic transition in the early embryo have remained elusive despite our understanding of the ESC gene network. Based on their transcript abundance, we hypothesized that the ESC pluripotency regulators, Sall4, Oct4, and Sox2, may be required for pre-blastocyst development, and their precise functions would help us to understand mechanisms controlling early embryo development. DESIGN: Gene-specific, antisense morpholino oligonucleotides (MOs) were injected into the cytoplasm at the 2PN stage to block translation and induce gene knockdown (KD) of each of Sall4Oct4, and Sox2, via steric hindrance. Maternal and embryonic transcripts are thus simultaneously targeted in a way that is not possible by conventional knockout mouse models. MATERIALS AND METHODS: We compared the developmental effect of each gene KD to its uninjected and mismatch controls inR 3 independent experiments by Student’s t-test. Protein expression was tested by immunocytochemistry. Global gene expression profiles were obtained from KD and control embryos at the 2-cell stage as per protocol (PicoPureÒ kit, Molecular Devices; Pico-ovationÒ, Nugen; GeneChipÒ Mouse Genome 430 2.0 Array, Affymetrix). RESULTS: We found that Sall4, Oct4, and Sox2 were each critical for early embryo development. Specifically, Sall4, Oct4, and Sox2 were required for development beyond the 4-cell, multicell, and morula stages, respectively. Few embryos reached the blastocyst stage, if at all. Further, successful translational block was confirmed by abrogation of nuclear signals. Finally, analyses of global expression profiles of Sall4 and Oct4 KD models suggest novel roles in cell cycle regulation, apoptosis, chromatin remodeling and epigenetic regulation, via transcriptional and posttranscriptional mechanisms. TABLE 1. Developmental Effects of Gene Knockdown Gene Knockdown Sall4 Oct4 Sox2
Stage of Arrest
% Arrest MO
% Arrest % Arrest p-value (MO Mismatch Uninjected vs mismatch)
1- to 4-cell 76.2 10.9 4.2 4.2 6.7 6.7 1- to multicell 89.6 5.8 18.9 8.1 0 multicell, morula 78.5 11.5 5.6 2.9 0
0.013 0.003 0.019
CONCLUSIONS: By combining MO-mediated gene KD and global gene expression profiling, we discovered that Sall4, Oct4, and Sox2, have novel and critical roles in normal embryo development. More broadly, we propose to apply our strategy to dissect the dynamic gene network that directs early embryo development. Supported by: NIH HD057970, HD00849, HD01249, ASRM, Organon, Women’s Health at Stanford.
Tuesday, November 11, 2008 4:00 pm O-128 VITRIFICATION OF HUMAN EMBRYONIC STEM CELLS WITH CRYOVIALS. T. Li, C. Zhou, Q. Mai, Y. Shu. Reproductive Med Ctr, Department of OB&GYN, The 1st Affiliated Hospital, Sun Yat-sen University, Guangzhou, China; Reproductive Med Ctr, Department of OB&GYN, The 1st Affiliated Hospital, Sun Yat-sen University, Guangzhou, China; Reproductive Medical Ctr, Department of OB&GYN, The 1st Affiliated Hospital, Sun Yat-sen University, Guangzhou, China; Reproductive Med Ctr, Department of OB&GYN, The 1st Affiliated Hospital, Sun Yat-sen University, Guangzhou, China. OBJECTIVE: The traditionally used vitrification method is no longer suitable for the vigorously proliferating human embryonic stem cells (HES) because of mini vitrification carriers. Recently we have reported a bulk vitrification (BV) method with cell strainer, which could cryopreserve a large
Vol. 90, Suppl 1, September 2008
quantity of HES cell clumps at one time. Since the cell strainer is not specially designed for cell and tissue cryopreservation, it is still not convenient to store cell strainers. Moreover, a cell strainer is unnecessarily big for vitrification. To circumvent the limitations, a custom-made cryo-container was made to bulk vitrified HES cells and more easily handle and store the vitrified HES cells in liquid nitrogen tank. DESIGN: Academic assisted reproduction program. MATERIALS AND METHODS: A 2-ml cryovial was cut transversely into two halves. The upper half with lid was kept for later use and the bottom half was discarded. After the lid of the upper half was removed, a little piece of stainless steel mesh (70mm) was put into the cryovial to be the bottom of the new vial. HES cell clumps were harvested and transferred to homemade cryovials and cell strainers respectively; only clumps with a diameter more than 70mm were included in the study and randomly selected to be vitrified by BV method with different cryo-containers. HES cell survival, growth and pluripotency were analyzed after warming. RESULTS: Compared to cell strains, homemade cryovials are easier to be handed and stored in liquid nitrogen tank. Homemade cryovials vitrified 11721.8 cell clumps at one time (round), which was similar to those with cell strainer (13934.5). After warming, vitrified HES cells exhibited high survival rate up to 91.8%, which was comparable to bulk vitrification method with cell strainers. All survived cell clumps generated HES cell colonies. Teratomas comprising all three primordial germ layers were formed in severe combined immunodeficient mice after subcutaneous injection of postthawed, bulk-vitrified HES cell clumps, confirming the pluripotency of vitrified-warmed stem cell clump. CONCLUSIONS: This new homemade cryovial not only effectively cryopreserved a large quantity of HES cell clumps at one time, but also easily set/fixed on cane and saved space in liquid nitrogen tank, which would more satisfy routine cryopreservation of HES cell during daily culture process than vitrification with cell strainer. Supported by: National Natural Science Foundation of China (NSFC No. 30571956, 30300372) and Guangdong Natural Science Foundation (No. 063 00778).
Tuesday, November 11, 2008 4:15 pm O-129 ALTERED GENE EXPRESSION PATTERN IN MURINE BLASTOCYSTS PRODUCED BY INTRACYTOPLASMIC SPERM INJECTION (ICSI). G. Giritharan, M. W. Li, L. Delle Piane, F. Di Sebastiano, A. Donjacour, P. F. Rinaudo. Obstetrics, Gynecology & Reproductive Sciences, University of California San Francisco, San Francisco, CA; Center for Comparative Medicine, University of California Davis, Davis, CA. OBJECTIVE: The ICSI process bypasses multiple processes occurring during natural fertilization; it is thought to be more stressful to the developing embryos, with unknown long term consequences. This study was conducted to explore if preimplantation mouse embryo produced by ICSI have an altered gene expression compared to IVF generated embryos. DESIGN: Comparison of gene expression of IVF and ICSI blastocysts using microarray technology. MATERIALS AND METHODS: CF-1 female mice were superovulated and the resulting oocytes were incubated in Whitten’s Medium (WM) with sperm of B6D2F1/J mice (group 1: IVF-WM) or underwent microinjection (group 2: ICSI-WM). All embryos were cultured in WM to the blastocyst stage under 5% CO2 in humidified air at 37 C. Additionally, a cohort of ICSI embryos was cultured in KSOM medium (group 3: ICSI-KSOM). Representative blastocysts from each treatment group were stained with propidium iodide and bisbenzamide, and cell number recorded. Total RNA was extracted and 5 embryo equivalents were used for reverse transcription, linear amplification, fragmentation and biotin labeling. The samples were hybridized to Affymetrix mouse 430 2.0 GeneChip (4 replicate per group). Pair-wise comparison was conducted using GeneSpring and EASE Software. RESULTS: The unsupervised hierarchical clustering revealed that ICSI and IVF embryos clustered separately. Only 37 probe sets were different between ICSI embryos cultured in KSOM or WM, but 5876 probe sets were different among ICSI and IVF embryos cultured in WM (p<0.05). Interestingly ICSI is associated with down regulation of genes related to DNA and RNA metabolism and mitochondrial genes (oxidoreductase, transferase and electron transport activity). ICSI embryos had significantly lower number of inner cell mass cells (9.6 vs. 12.8, p<0.05) compared to IVF embryos.
FERTILITY & STERILITYÒ
TABLE Up-regulated Down-regulated Up-regulated Down-regulated (>2 fold) (>2 fold) (>5 fold) (>5 fold)
Comparison ICSI-WM vs. ICSI-KSOM IVF-WM vs. ICSI-WM
17 1643
20 4233
0 251
0 288
CONCLUSIONS: Blastocysts generated using ICSI have a similar gene expression pattern independently of the culture conditions used, indicating that the microinjection process has a more noticeable effect in shifting the transcriptome of the developing embryo than the culture medium used. IVF and ICSI embryos have a remarkably different gene expression patterns. Overall ICSI embryos show alteration of fundamental metabolic pathway compared to IVF generated embryos. Supported by: WRHR.
Tuesday, November 11, 2008 4:30 pm O-130 SUPPLEMENTATION OF CULTURE MEDIA WITH ESTERFIED FORMS OF PYRUVATE IMPROVES MOUSE EMBRYO DEVELOPMENT. J. E. Swain, T. B. Pool. Fertility Center of San Antonio, San Antonio, TX. OBJECTIVE: Examine embryo development following substitution of sodium pyruvate in culture media with ethyl or methyl pyruvate. DESIGN: Prospective study. MATERIALS AND METHODS: Three media were tested to determine effects of pyruvate forms on embryo development. Media 1 contained pyruvate as the sole energy source (lactate-free P1). Media 2 contained pyruvate and lactate (standard P1). Media 3 contained pyruvate, lactate, glucose and amino acids (P1-KSOM þAA). In each media, frozen/thawed 1-cell mouse embryos were randomly distributed between 1 of 4 pyruvate treatment groups: sodium pyruvate, ethyl pyruvate, methyl pyruvate, or no pyruvate. Osmolarity of all media with equalized if necessary by adjusting NaCl levels. Embryos were cultured for 96h in 5% CO2 in air and development graded every 24h. Developmental data were arc sin transformed, analyzed using GLM ANOVA, and statistical differences determined using Bonferroni multiple comparisons test. RESULTS: In lactate-free P1, both ethyl and methyl pyruvate resulted in advanced embryo development at 48, 72 and 96h compared to sodium pyruvate. No differences in blastocyst cell number were observed. In standard P1, no developmental differences were observed between pyruvate forms over 96h. Similarly, no differences were observed in development in P1-KSOM þAA. TABLE 1. Lactate-Free P1 Embryo Development 24h 48h R 72h R 96h Total 96h Hatching Total Cleavage 8-Cell Morula Blastocyst Blastocyst Blastocyst Cell # Sodium Pyruvate n¼49 Ethyl Pyruvate n¼49 Methyl Pyruvate n¼49 No Pyruvate n¼49
100a 100a 100a 11b
22a 50b 49b 0c
59a 89b 81b 0c
19a 60b 54b 0a
0a 7a 8a 0a
31.5a 37.4a 36.5a –
Different superscripts within a column represent statistical differences, P<0.05.
CONCLUSIONS: These data indicate esterfied forms of pyruvate may benefit mouse embryo development. Current culture media formulations include sodium pyruvate, however, aqueous solutions of pyruvate can be unstable and result in formation of byproducts that interfere with key steps of the TCA cycle, as well as limit ability to scavenge ROS. Ethyl and methyl pyruvate appear to be more stable and have proven superior to sodium pyruvate in protecting somatic cell systems from varying paradigms of redox stress and damage. Furthermore, these esterfied forms of pryuvate are more membrane permeable than sodium pyruvate, and are thus thought to more readily enter the mitochondria and stimulate NADH/NADPH production. Future studies will examine effects of esterfied pyruvate on embryo metabolism. Supported by: None.
S47
Tuesday, November 11, 2008 3:30 pm O-126 MEIOTIC SPINDLE LOCALIZATION AND MICROFILAMENT DISTRIBUTION DURING OOCYTE VITRIFICATION. C.-C. Chang, Z. P. Nagy, L.-Y. Sung, H. I. Kort, X. Yang, C. X. Tian. Reproductive Biology Associates, Atlanta, GA; University of Connecticut, Storrs. OBJECTIVE: During female meiosis, most of the maternal resources are retained in the oocyte for the early developing embryo. This outcome is coordinated by asymmetrical divisions. Two significant events ensure these asymmetrical divisions occur during meiosis: first, the meiotic spindle migrates towards the cell cortex, and second, a microfilament-rich cortical domain over the spindle is formed. It is not known if cryopreservation may impact these delicate arrangements which determine these critical processes. Our objective, therefore, was to investigate the specific changes of oocyte spindle localization and microfilament distribution before, during, and after vitrification. DESIGN: Timed observations were performed before, during, and after vitrification. MATERIALS AND METHODS: Mature oocytes were collected from 8wk-old BDF1 mice with standard superovulation. Cryopreservation on all oocytes was performed by the vitrification method with 15% ethylene glycol, 15% DMSO and 0.5 M sucrose. Oocyte warming was performed after vitrification by serial dilutions in three steps using 1.0M, 0.5M, and 0M sucrose solutions. To examine the spindle, the oocytes were fixed at 5 min of equilibration solution, 1 min of vitrification solution, 0 min of warming (warming directly into fixative), and 1h after warming, and stained with propidium iodide and FITC-conjugated anti-tubulin antibody by confocal microscopy. RESULTS: A total of 75 oocytes were included in the present study. After 5 min exposure to equilibration solution, all of the meiotic spindles were positioned at the microfilament-rich domain of the oocyte’s cortex (16/16) as the control group before exposure to cryoprotectant (12/12). All oocyte spindles were still localized to microfilament-rich domain of the oocyte’s cortex in vitrification solution (15/15) with even higher concentration of cryoprotectant and the subsequent cooling process (15/15). We observed that all examined oocytes sustained consistent microtubule and microfilament structures at 1h after warming (17/17). CONCLUSIONS: This study demonstrated that the spindle localization and microfilament distribution were well conserved throughout the cooling and warming processes, even though oocytes were exposed to high concentrations of cryoprotectants and non-physiological temperatures for substantial time. This study thus indicates that oocyte cryopreservation using vitrification may be a suitable technique in regard to maintaining cytoskeletal structure. Supported by: None.
S46
Abstracts
Tuesday, November 11, 2008 3:45 pm O-127 EMBRYONIC STEM CELL (ESC) PLURIPOTENCY REGULATORS HAVE NOVEL AND CRITICAL ROLES IN PRE-BLASTOCYST DEVELOPMENT. K. M. Foygel, S. H. Jun, D. E. Leong, B. Choi, W. H. Wong, M. W. M. Yao. Ob/Gyn, Stanford University School of Medicine, Stanford, CA; Applied Physics, Stanford University, Stanford, CA; Statistics, Stanford University, Stanford, CA. OBJECTIVE: Gene regulation and reprogramming during the maternalembryonic transition in the early embryo have remained elusive despite our understanding of the ESC gene network. Based on their transcript abundance, we hypothesized that the ESC pluripotency regulators, Sall4, Oct4, and Sox2, may be required for pre-blastocyst development, and their precise functions would help us to understand mechanisms controlling early embryo development. DESIGN: Gene-specific, antisense morpholino oligonucleotides (MOs) were injected into the cytoplasm at the 2PN stage to block translation and induce gene knockdown (KD) of each of Sall4Oct4, and Sox2, via steric hindrance. Maternal and embryonic transcripts are thus simultaneously targeted in a way that is not possible by conventional knockout mouse models. MATERIALS AND METHODS: We compared the developmental effect of each gene KD to its uninjected and mismatch controls inR 3 independent experiments by Student’s t-test. Protein expression was tested by immunocytochemistry. Global gene expression profiles were obtained from KD and control embryos at the 2-cell stage as per protocol (PicoPureÒ kit, Molecular Devices; Pico-ovationÒ, Nugen; GeneChipÒ Mouse Genome 430 2.0 Array, Affymetrix). RESULTS: We found that Sall4, Oct4, and Sox2 were each critical for early embryo development. Specifically, Sall4, Oct4, and Sox2 were required for development beyond the 4-cell, multicell, and morula stages, respectively. Few embryos reached the blastocyst stage, if at all. Further, successful translational block was confirmed by abrogation of nuclear signals. Finally, analyses of global expression profiles of Sall4 and Oct4 KD models suggest novel roles in cell cycle regulation, apoptosis, chromatin remodeling and epigenetic regulation, via transcriptional and posttranscriptional mechanisms. TABLE 1. Developmental Effects of Gene Knockdown Gene Knockdown Sall4 Oct4 Sox2
Stage of Arrest
% Arrest MO
% Arrest % Arrest p-value (MO Mismatch Uninjected vs mismatch)
1- to 4-cell 76.2 10.9 4.2 4.2 6.7 6.7 1- to multicell 89.6 5.8 18.9 8.1 0 multicell, morula 78.5 11.5 5.6 2.9 0
0.013 0.003 0.019
CONCLUSIONS: By combining MO-mediated gene KD and global gene expression profiling, we discovered that Sall4, Oct4, and Sox2, have novel and critical roles in normal embryo development. More broadly, we propose to apply our strategy to dissect the dynamic gene network that directs early embryo development. Supported by: NIH HD057970, HD00849, HD01249, ASRM, Organon, Women’s Health at Stanford.
Tuesday, November 11, 2008 4:00 pm O-128 VITRIFICATION OF HUMAN EMBRYONIC STEM CELLS WITH CRYOVIALS. T. Li, C. Zhou, Q. Mai, Y. Shu. Reproductive Med Ctr, Department of OB&GYN, The 1st Affiliated Hospital, Sun Yat-sen University, Guangzhou, China; Reproductive Med Ctr, Department of OB&GYN, The 1st Affiliated Hospital, Sun Yat-sen University, Guangzhou, China; Reproductive Medical Ctr, Department of OB&GYN, The 1st Affiliated Hospital, Sun Yat-sen University, Guangzhou, China; Reproductive Med Ctr, Department of OB&GYN, The 1st Affiliated Hospital, Sun Yat-sen University, Guangzhou, China. OBJECTIVE: The traditionally used vitrification method is no longer suitable for the vigorously proliferating human embryonic stem cells (HES) because of mini vitrification carriers. Recently we have reported a bulk vitrification (BV) method with cell strainer, which could cryopreserve a large
Vol. 90, Suppl 1, September 2008
quantity of HES cell clumps at one time. Since the cell strainer is not specially designed for cell and tissue cryopreservation, it is still not convenient to store cell strainers. Moreover, a cell strainer is unnecessarily big for vitrification. To circumvent the limitations, a custom-made cryo-container was made to bulk vitrified HES cells and more easily handle and store the vitrified HES cells in liquid nitrogen tank. DESIGN: Academic assisted reproduction program. MATERIALS AND METHODS: A 2-ml cryovial was cut transversely into two halves. The upper half with lid was kept for later use and the bottom half was discarded. After the lid of the upper half was removed, a little piece of stainless steel mesh (70mm) was put into the cryovial to be the bottom of the new vial. HES cell clumps were harvested and transferred to homemade cryovials and cell strainers respectively; only clumps with a diameter more than 70mm were included in the study and randomly selected to be vitrified by BV method with different cryo-containers. HES cell survival, growth and pluripotency were analyzed after warming. RESULTS: Compared to cell strains, homemade cryovials are easier to be handed and stored in liquid nitrogen tank. Homemade cryovials vitrified 11721.8 cell clumps at one time (round), which was similar to those with cell strainer (13934.5). After warming, vitrified HES cells exhibited high survival rate up to 91.8%, which was comparable to bulk vitrification method with cell strainers. All survived cell clumps generated HES cell colonies. Teratomas comprising all three primordial germ layers were formed in severe combined immunodeficient mice after subcutaneous injection of postthawed, bulk-vitrified HES cell clumps, confirming the pluripotency of vitrified-warmed stem cell clump. CONCLUSIONS: This new homemade cryovial not only effectively cryopreserved a large quantity of HES cell clumps at one time, but also easily set/fixed on cane and saved space in liquid nitrogen tank, which would more satisfy routine cryopreservation of HES cell during daily culture process than vitrification with cell strainer. Supported by: National Natural Science Foundation of China (NSFC No. 30571956, 30300372) and Guangdong Natural Science Foundation (No. 063 00778).
Tuesday, November 11, 2008 4:15 pm O-129 ALTERED GENE EXPRESSION PATTERN IN MURINE BLASTOCYSTS PRODUCED BY INTRACYTOPLASMIC SPERM INJECTION (ICSI). G. Giritharan, M. W. Li, L. Delle Piane, F. Di Sebastiano, A. Donjacour, P. F. Rinaudo. Obstetrics, Gynecology & Reproductive Sciences, University of California San Francisco, San Francisco, CA; Center for Comparative Medicine, University of California Davis, Davis, CA. OBJECTIVE: The ICSI process bypasses multiple processes occurring during natural fertilization; it is thought to be more stressful to the developing embryos, with unknown long term consequences. This study was conducted to explore if preimplantation mouse embryo produced by ICSI have an altered gene expression compared to IVF generated embryos. DESIGN: Comparison of gene expression of IVF and ICSI blastocysts using microarray technology. MATERIALS AND METHODS: CF-1 female mice were superovulated and the resulting oocytes were incubated in Whitten’s Medium (WM) with sperm of B6D2F1/J mice (group 1: IVF-WM) or underwent microinjection (group 2: ICSI-WM). All embryos were cultured in WM to the blastocyst stage under 5% CO2 in humidified air at 37 C. Additionally, a cohort of ICSI embryos was cultured in KSOM medium (group 3: ICSI-KSOM). Representative blastocysts from each treatment group were stained with propidium iodide and bisbenzamide, and cell number recorded. Total RNA was extracted and 5 embryo equivalents were used for reverse transcription, linear amplification, fragmentation and biotin labeling. The samples were hybridized to Affymetrix mouse 430 2.0 GeneChip (4 replicate per group). Pair-wise comparison was conducted using GeneSpring and EASE Software. RESULTS: The unsupervised hierarchical clustering revealed that ICSI and IVF embryos clustered separately. Only 37 probe sets were different between ICSI embryos cultured in KSOM or WM, but 5876 probe sets were different among ICSI and IVF embryos cultured in WM (p<0.05). Interestingly ICSI is associated with down regulation of genes related to DNA and RNA metabolism and mitochondrial genes (oxidoreductase, transferase and electron transport activity). ICSI embryos had significantly lower number of inner cell mass cells (9.6 vs. 12.8, p<0.05) compared to IVF embryos.
FERTILITY & STERILITYÒ
TABLE Up-regulated Down-regulated Up-regulated Down-regulated (>2 fold) (>2 fold) (>5 fold) (>5 fold)
Comparison ICSI-WM vs. ICSI-KSOM IVF-WM vs. ICSI-WM
17 1643
20 4233
0 251
0 288
CONCLUSIONS: Blastocysts generated using ICSI have a similar gene expression pattern independently of the culture conditions used, indicating that the microinjection process has a more noticeable effect in shifting the transcriptome of the developing embryo than the culture medium used. IVF and ICSI embryos have a remarkably different gene expression patterns. Overall ICSI embryos show alteration of fundamental metabolic pathway compared to IVF generated embryos. Supported by: WRHR.
Tuesday, November 11, 2008 4:30 pm O-130 SUPPLEMENTATION OF CULTURE MEDIA WITH ESTERFIED FORMS OF PYRUVATE IMPROVES MOUSE EMBRYO DEVELOPMENT. J. E. Swain, T. B. Pool. Fertility Center of San Antonio, San Antonio, TX. OBJECTIVE: Examine embryo development following substitution of sodium pyruvate in culture media with ethyl or methyl pyruvate. DESIGN: Prospective study. MATERIALS AND METHODS: Three media were tested to determine effects of pyruvate forms on embryo development. Media 1 contained pyruvate as the sole energy source (lactate-free P1). Media 2 contained pyruvate and lactate (standard P1). Media 3 contained pyruvate, lactate, glucose and amino acids (P1-KSOM þAA). In each media, frozen/thawed 1-cell mouse embryos were randomly distributed between 1 of 4 pyruvate treatment groups: sodium pyruvate, ethyl pyruvate, methyl pyruvate, or no pyruvate. Osmolarity of all media with equalized if necessary by adjusting NaCl levels. Embryos were cultured for 96h in 5% CO2 in air and development graded every 24h. Developmental data were arc sin transformed, analyzed using GLM ANOVA, and statistical differences determined using Bonferroni multiple comparisons test. RESULTS: In lactate-free P1, both ethyl and methyl pyruvate resulted in advanced embryo development at 48, 72 and 96h compared to sodium pyruvate. No differences in blastocyst cell number were observed. In standard P1, no developmental differences were observed between pyruvate forms over 96h. Similarly, no differences were observed in development in P1-KSOM þAA. TABLE 1. Lactate-Free P1 Embryo Development 24h 48h R 72h R 96h Total 96h Hatching Total Cleavage 8-Cell Morula Blastocyst Blastocyst Blastocyst Cell # Sodium Pyruvate n¼49 Ethyl Pyruvate n¼49 Methyl Pyruvate n¼49 No Pyruvate n¼49
100a 100a 100a 11b
22a 50b 49b 0c
59a 89b 81b 0c
19a 60b 54b 0a
0a 7a 8a 0a
31.5a 37.4a 36.5a –
Different superscripts within a column represent statistical differences, P<0.05.
CONCLUSIONS: These data indicate esterfied forms of pyruvate may benefit mouse embryo development. Current culture media formulations include sodium pyruvate, however, aqueous solutions of pyruvate can be unstable and result in formation of byproducts that interfere with key steps of the TCA cycle, as well as limit ability to scavenge ROS. Ethyl and methyl pyruvate appear to be more stable and have proven superior to sodium pyruvate in protecting somatic cell systems from varying paradigms of redox stress and damage. Furthermore, these esterfied forms of pryuvate are more membrane permeable than sodium pyruvate, and are thus thought to more readily enter the mitochondria and stimulate NADH/NADPH production. Future studies will examine effects of esterfied pyruvate on embryo metabolism. Supported by: None.
S47