86 BIRTH OF HEALTHY CALVES AFTER INTRAFOLLICULAR OOCYTE TRANSFER

2015 ◽  
Vol 27 (1) ◽  
pp. 136
Author(s):  
M. Hoelker ◽  
A. Kassens ◽  
E. Held ◽  
C. Wrenzycki ◽  
U. Besenfelder ◽  
...  
Keyword(s):  
Lipid Content ◽  
Ex Vivo ◽  
Survival Rates ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
In Vitro Production ◽  
Negative Effects ◽  
Uterine Flushing

The in vitro production (IVP) of bovine embryos is a well-established technique that has been available for nearly 20 years. However, there remain major differences between IVP-derived blastocysts and their in vivo-derived counterparts. Many studies have pointed out that most of these differences are due to the in vitro developmental environment. To circumvent these negative effects due to in vitro culture conditions, a new method – intrafollicular oocyte transfer (IFOT) – was established in the present study. Using modified ovum pick-up (OPU) equipment, in vitro-matured oocytes derived from slaughterhouse ovaries were injected into the dominant preovulatory follicle of synchronised heifers (follicular recipients) enabling subsequent ovulation, in vivo fertilization, and in vivo development. A total of 810 in vitro-matured oocytes were transferred into 14 heifers. Subsequently, 222 embryos (27.3%) were recovered after uterine flushing at Day 7. Based on the number of cleaved embryonic stages, 64.2% developed to the blastocyst stage, which did not differ from the IVP-derived embryos (58.2%). Interestingly, lipid content of IFOT-derived blastocysts did not differ from the fully in vivo-produced embryos, whereas IVP-derived blastocysts showed significantly higher lipid droplet accumulation compared with fully in vivo-derived and IFOT-derived blastocysts (P < 0.05). Accordingly, IFOT blastocysts showed significantly higher survival rates after cryopreservation than complete IVP-derived embryos (77% v. 10%), which might be attributed to a lower degree of lipid accumulation. In agreement, transfer of frozen-thawed IFOT blastocysts to synchronized recipients (uterine recipients) resulted in much higher pregnancy rates compared with transfer of IVP-derived blastocysts (42.1 v. 13.8%) but did not differ from frozen-thawed ex vivo blastocysts (52.4%). Of these presumed IFOT pregnancies, 7 went to term, and microsatellite analysis confirmed that 5 calves were indeed derived from IFOT, whereas 2 were caused by fertilization of the follicular recipient's own oocyte after AI. Taken together, IFOT-derived blastocysts closely resemble in vivo-derived blastocysts, confirming earlier suggestions that the ability to develop to the blastocyst stage is already determined in the matured oocyte, whereas the quality in terms of lipid content and survival rate after cryopreservation is affected by the environment thereafter. However, to the best of our knowledge, this is the first study reporting healthy calves after intrafollicular transfer of in vitro-matured oocytes.

Growth and viability of human blastocysts in vitro

2000 ◽  
Vol 8 (3) ◽  
pp. 241-287 ◽  
Author(s):  
GM Jones
Keyword(s):  
Blastocyst Stage ◽  
Culture Conditions ◽  
Cleavage Stage ◽  
Embryo Viability ◽  
Early Cleavage ◽  
Uterine Endometrium ◽  
Stage Of Development ◽  
Uterine Lavage

The transfer of a blastocyst established the first human clinical pregnancy following in vitro fertilization (IVF). Nine years later Cohen et al. reported pregnancies resulting from the transfer of cryopreserved human blastocysts. However, it was another six years before the first report of births resulting from the transfer of human blastocysts produced in vitro appeared in the medical literature. In the intervening period clinics have opted to transfer embryos at the early cleavage stage to the uterus, despite the fact that in vivo the embryo does not enter the uterus until two to three days later at the morula to blastocyst stage of development. The viability and potential for implantation of blastocysts is high, as indicated by the finding that more than 60% of in-vivo-derived blastocysts, recovered by uterine lavage following artificial insemination of fertile donors, implant and develop into viable fetuses when transferred to recipients. This is in stark contrast to the 10–20% of in-vitro-produced embryos transferred at the early cleavage stage of development that result in a live-birth. This reduction in viability following transfer of in-vitro-derived early cleavage stage embryos may have several possible explanations: (1) a failure of implantation due to poor synchronization between the embryo and the uterine endometrium; (2) a hostile environment in the uterus for early cleavage stage embryos; (3) sub-optimal in vitro culture conditions which result in a reduction in embryo viability; (4) the assumption that all oocytes retrieved in an IVF cycle have an equal ability to develop into viable embryos; and (5) the failure to identify the most viable embryo in a cohort. Certainly, improving culture conditions and laboratory techniques for developing high quality blastocysts routinely in vitro will not only address many of the above questions but will also improve the quality and viability of earlier stages of embryo development.

193 IMPROVED POST-THAW SURVIVAL OF BOVINE EMBRYOS PRODUCED IN SERUM-FREE IN VITRO PRODUCTION SYSTEM

2016 ◽  
Vol 28 (2) ◽  
pp. 227
Author(s):  
M. Nõmm ◽  
E. Mark ◽  
O. Sarv ◽  
S. Kõks ◽  
Ü. Jaakma
Keyword(s):  
Survival Rates ◽  
Slow Freezing ◽  
In Vitro Fertilisation ◽  
Ministry Of Education ◽  
In Vitro Production ◽  
Freezing And Thawing ◽  
Embryo Survival ◽  
Serum Free

Over a few decades the bovine in vitro embryo production (IVP) systems have been improving rapidly. Still, the goal to produce the same quality embryos in vitro as in vivo has not yet been reached. The FCS is usually added to media during IVP to provide growth factors and energy sources. Currently, serum-free culture systems are often preferred due to the lower risk of contamination and prevention of the development of large offspring syndrome. The aim of this study was to establish whether complete elimination of FCS from the bovine IVP system has an effect on blastocyst rates, embryo quality, and embryo survival rates after slow freezing. We replaced our conventional in vitro maturation (IVM) medium [tissue culture medium-199, 10% (v/v) FCS, 10 µg mL–1 epidermal growth factor (EGF), 1500 U mL–1 serum gonadotropin and chorionic gonadotropin (PG600), Na-pyruvate 0.5 mM, gentamycin sulfate 50 µg mL–1 and l-glutamine 1 mM] with SOF (SOFaaci) supplemented with 0.4% fatty acid-free BSA fraction V, 10 µg mL–1 EGF, and 1500 U mL–1 PG600. Matured cumulus-oocyte complexes (COC) from both experimental groups (total of 1145 from serum-free IVP and 687 from our conventional IVP system) were used for in vitro fertilisation and culture. Blastocyst rates were similar in the serum-free and our usual IVP protocol, 18 and 22%, respectively. Seventy-seven Grade 1 (according to IETS) Day 7 blastocysts from the serum-free IVP system and 80 Grade 1 Day 7 blastocysts from our conventional IVP system were frozen in 1.5 M ethylene glycol and 0.1 M sucrose containing cryopreservation medium. The post-thaw survival rates after 24 h of culture and evaluated as percentages of re-expanded embryos were 63.6% for the serum-free IVP and 46.3% for the conventional IVP system (P < 0.05, Z Test for 2 population proportions). These results indicate that it is possible to have a completely serum-free bovine IVP system and based on the slow freezing and thawing results the quality of serum-free IVP embryos might be better than of the embryos matured in our conventional maturation media. However, more experiments and increased sample sizes are needed to confirm the results. This study was supported by Project 3.2.0701.12–0036 of Archimedes Foundation, AP 2.4 of CCRMB, and institutional research funding (IUT 08–01) of the Estonian Ministry of Education and Research.

scholarly journals Developmental and molecular correlates of bovine preimplantation embryos

Reproduction ◽  
2006 ◽  
Vol 131 (5) ◽  
pp. 895-904 ◽  
Author(s):  
Hakan Sagirkaya ◽  
Muge Misirlioglu ◽  
Abdullah Kaya ◽  
Neal L First ◽  
John J Parrish ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methyltransferase ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Preimplantation Embryos ◽  
Developmental Potential

Expression of embryonic genes is altered in different culture conditions, which influence developmental potential both during preimplantation and fetal development. The objective of this study was to define the effects of culture conditions on: bovine embryonic development to blastocyst stage, blastocyst cell number, apoptosis and expression patterns of a panel of developmentally important genes. Bovine embryos were culturedin vitroin three culture media containing amino acids, namely potassium simplex optimization medium (KSOMaa), Charles Rosenkrans 1 (CR1aa) and synthetic oviductal fluid (SOFaa). Apoptosis in blastocysts was determined by TUNEL assay and expression profiles of developmentally important genes were assayed by real-time PCR.In vivo-produced bovine blastocysts were used as controls for experiments determining gene expression patterns. While the cleavage rates did not differ, embryos cultured in SOFaa had higher rates of development to blastocyst stage (P< 0.05). Mean cell numbers and percentages of apoptotic cells per blastocyst did not differ among the groups. Expression of the heat shock protein 70 (Hsp70) gene was significantly up-regulated in both CR1aa and KSOMaa when compared with SOFaa (P< 0.001). DNA methyltransferase 3a (Dnmt3a) expression was higher in embryos cultured in CR1aa than in those cultured in SOFaa (P< 0.001). Expression of interferon tau (IF-τ) and insulin-like growth factor II receptor (Igf-2r) genes was significantly up-regulated in KSOMaa when compared with CR1aa (P< 0.001). Gene expression did not differ betweenin vivo-derived blastocysts and theirin vitro-derived counterparts. In conclusion, SOFaa supports higher development to blastocyst stage than KSOMaa and CR1aa, and the culture conditions influence gene expression.

In vitro production of horse embryos predisposes to micronucleus formation, whereas time to blastocyst formation affects likelihood of pregnancy

2019 ◽  
Vol 31 (12) ◽  
pp. 1830 ◽  
Author(s):  
Kaatje D. Ducheyne ◽  
Marilena Rizzo ◽  
Juan Cuervo-Arango ◽  
Anthony Claes ◽  
Peter F. Daels ◽  
...  
Keyword(s):  
Chromosomal Abnormalities ◽  
Morphological Characteristics ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Early Embryo ◽  
Embryo Morphology ◽  
In Vitro Production ◽  
Pregnancy Failure ◽  
Micronucleus Formation

Invitro embryo production is an increasingly popular means of breeding horses. However, success is limited by a high incidence of early embryo loss. Although there are various possible causes of pregnancy failure, chromosomal abnormalities, including aneuploidy, are important potential contributors. This study evaluated the frequency of micronucleus formation as a proxy for aneuploidy in invitro-produced (IVP) and invivo-derived horse blastocysts. Associations between IVP embryo morphology, frequency of nuclear abnormalities and the likelihood of pregnancy were investigated. IVP blastocysts exhibited a higher frequency of cells with micronuclei than invivo-derived embryos (10% vs 1% respectively; P=0.05). This indication of chromosomal instability may explain the higher incidence of pregnancy failure after transfer of IVP embryos. However, the frequency of micronuclei was not correlated with brightfield microscopic morphological characteristics. Nevertheless, IVP embryos reaching the blastocyst stage after Day 9 of invitro culture were less likely to yield a pregnancy than embryos that developed to blastocysts before Day 9 (27% vs 69%), and embryos that had expanded before transfer were more likely to undergo embryonic death than those that had not expanded (44% vs 10%). These findings indicate that current embryo culture conditions are suboptimal and that the speed of embryo development is correlated with pregnancy survival.

132 DIFFERENCES IN RESPIRATION RATES BETWEEN IN VIVO- AND IN VITRO-PRODUCED BOVINE EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 174
Author(s):  
A. S. Lopes ◽  
S. E. Madsen ◽  
N. B. Ramsing ◽  
L. H. Larsen ◽  
T. Greve ◽  
...  
Keyword(s):  
Metabolic Stress ◽  
Average Diameter ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Outer Diameter ◽  
Bovine Embryos ◽  
Physiological Level ◽  
Respiration Rates

In vitro-produced (IVP) bovine embryos differ (e.g. morphology and physiology) from their in vivo counterparts. Oxygen consumption is an indicator of the overall metabolic activity of a single embryo. Therefore, the aim of this study was to determine and compare respiration rates of in vivo- and in vitro-produced bovine day 7 embryos. Diameters of these two embryo types were also compared. In vivo embryos (n = 28) were recovered from 8 superovulated Holstein Frisian cows on day 7 following AI, while IVP embryos (n = 160; Holm et al. 1999 Theriogenology 52, 683-700) were used on day 7 after fertilization. Embryos were measured (outer diameter) and morphologically evaluated (Quality 1 to 4, IETS Manual, 1998). Only transferable in vivo embryos were used (i.e. excluding Quality 4). Respiration rates were measured on each embryo by Nanorespirometer technology (Lopes et al. 2005 Reprod. Fertil. Develop. 17, 151). Data were analyzed using Proc Mixed, and values are presented as mean � SEM. Values with different superscripts differ significantly (P < 0.05). The average respiration rates were 0.82 � 0.06a nL/h for in vivo vs. 1.37 � 0.06b nL/h for IVP embryos. The average respiration rates for the different morphological qualities were as follows (nL/h, numbers in brackets): IVP: 2.1 � 0.08a (38), 1.37 � 0.07b (55), 1.08 � 0.07c (48) and 0.62 � 0.11d (19) for Quality 1, 2, 3, and 4, respectively. In vivo: 1.17 � 0.21b,c,e (6), 0.80 � 0.15c,d,e (12), and 0.64 � 0.16d,f (10) for Quality 1, 2, and 3, respectively. The average diameter (mm) of in vivo and IVP embryos was 0.157 � 0.002a and 0.176 � 0.002b, respectively. Respiration rates were directly related to embryo diameter; larger embryos were associated with higher respiration rates (y = 17.55 � 1.32 nL/h � mm, n = 188). Respiration rates of in vivo embryos were significantly lower than those of IVP embryos, regardless of quality. This difference could reflect an effect of the culture conditions on IVP embryos because media components affect embryo metabolism. Moreover, the different ages (day 7 for IVP vs. approximately Day 6.5 for in vivo embryos, because in vivo embryos are less than 7 days after fertilization at recovery) and stages (IVP: up to expanded blastocyst stage; in vivo: morula or early blastocyst stage) could have influenced the results and also partly explain the smaller diameter of the in vivo embryos. Finally, respiration rates decreased proportionately to the morphological quality within embryo type, indicating that morphological differences are reflected at the physiological level. In conclusion, this study further outlines metabolic differences between in vivo and IVP bovine embryos. Whether such differences are a manifestation of metabolic stress associated to the separation from the natural environment or reflect suboptimal culture conditions is yet to be determined. ASL is supported by FCT, Portugal.

211 IN VITRO CULTURE CONDITIONS AFFECT GENE EXPRESSION PATTERN OF BOVINE BLASTOCYST IN A STAGE-SPECIFIC MANNER

2013 ◽  
Vol 25 (1) ◽  
pp. 254 ◽  
Author(s):  
A. Gad ◽  
U. Besenfelder ◽  
V. Havlicek ◽  
M. Hölker ◽  
M. U. Cinar ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Death ◽  
In Vitro Culture ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Control Group ◽  
Transcriptome Profile ◽  
Specific Manner

The aim of this study was to examine the effect of in vitro culture conditions at specific phases of early embryonic development on the transcriptome profile of bovine blastocysts. Simmental heifers were superovulated and artificially inseminated 2 times with the same frozen–thawed commercial bull semen. Using nonsurgical endoscopic oviductal flushing technology (Besenfelder et al. 2001 Theriogenology 55, 837–845), 6 different blastocyst groups were flushed out at different time points (2-, 4-, 8-, 16-, 32-cell and morula). After flushing, embryos cultured under in vitro conditions until the blastocyst stage. Blastocysts from each group were collected and pooled in groups of 10. Complete in vivo blastocysts were produced and used as control. A unique custom microarray (Agilent) containing 42 242 oligo probes (60-mers) was used over 6 replicates of each group v. the in vivo control group to examine the transcriptome profile of blastocysts. A clear difference in terms of the number of differentially expressed genes (DEG, fold change ≥2, false discovery rate ≤0.05) has been found between groups flushed out at 2-, 4-, and 8-cell (1714, 1918, 1292 DEG, respectively) and those flushed out at 16-, 32-cell and morula stages and cultured in vitro until blastocyst stage (311, 437, 773 DEG, respectively) compared with the complete vivo group. Ontological classification of DEG showed cell death to be the most significant function in all groups. However, the longer time embryos spent under in vitro conditions, the more the percentage of DEG involved in cell death and apoptosis processes are represented in those groups. In addition, genes related to post-translational modification and gene expression processes were significantly dysregulated in all groups. Pathway analysis revealed that protein ubiquitination pathway was the dominant pathway in the groups flushed out at 2-, 4-, and 8-cells but not in the other groups flushed at later stages compared with the in vivo control group. Moreover, retinoic acid receptor activation and apoptosis signalling pathways followed the same pattern. Embryos flushed out before the time of embryonic genome activation and subsequently cultured in vitro were highly affected by culture conditions. Overall, the results of the present study showed that despite the fact that embryos originated from the same source, in vitro culture condition affected embryo quality, measured in terms of gene expression, in a stage-specific manner.

scholarly journals Development of Preimplantation Mouse Embryos in vivo and in vitro

1982 ◽  
Vol 35 (2) ◽  
pp. 187 ◽  
Author(s):  
GM Harlow ◽  
P Quinn
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Cell Stage ◽  
Preimplantation Mouse ◽  
Development In Vitro

The culture conditions for the development in vitro of (C57BL/6 X CBA) F2 hybrid two-cell embryos to the blastocyst stage have been optimized. Commercially available pre-sterile disposable plastic culture dishes supported more reliable development than re-usable washed glass tubes. The presence of an oil layer reduced the variability in development. An average of 85 % of blastocysts developed from hybrid two-cell embryos cultured in drops of Whitten's medium under oil in plastic culture dishes in an atmosphere of 5% O2 : 5% CO2 : 90% N2 ? The time taken for the total cell number to double in embryos developing in vivo was 10 h, and in cultured embryos 17 h. Embryos cultured in vitro from the two-cell stage to blastocyst stage were retarded by 18-24 h in comparison with those remaining in vivo. Day-4 blastocysts in vivo contained 25-70 cells (mean 50) with 7-28 (mean 16) of these in the inner cell mass. Cultured blastocysts contained 19-73 cells (mean 44) with 8-34 (mean 19) of these in the inner cell mass. In the uterine environment, inner-cell-mass blastomeres divided at a faster rate than trophectoderm blastomeres and it is suggested that a long cell cycle is associated with terminal differentiation. Although cultured blastocysts and inner cell masses contained the same number of cells as blastocysts and inner cell masses in vivo, the rate of cell division in cultured inner cell masses was markedly reduced.

scholarly journals 245RELATIVE ABUNDANCE OF HSP 70 MRNA IN BOVINE EMBRYOS PRODUCED IN VITRO USING DIFFERENT EMBRYO/VOLUME RATIOS IN CULTURE

2004 ◽  
Vol 16 (2) ◽  
pp. 243
Author(s):  
A.T.D. Oliveira ◽  
C. Gebert ◽  
R.F.F. Lopes ◽  
H. Niemann ◽  
J.L. Rodrigues
Keyword(s):  
Relative Abundance ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Hatching Rate ◽  
Bovine Embryos ◽  
Blastocyst Formation ◽  
Cleavage Rate ◽  
Gene Transcripts

In spite of in vitro embryo production systems having been greatly improved over recent years, employing a variety of culture conditions (media, protein sources, gas atmosphere, etc.), we still do not know much about the real necessity of embryos to develop under the same conditions as occur in vivo. These differences between in vivo and in vitro culture at preimplantation embryonic stages can produce deviations in gene expression and in normal fetal development (large offspring syndrome). Heat shock proteins (Hsp) are engaged in cell response to regulatory signals or perturbations in the microenviroment and can be used as a sensitive indicator of stress caused by suboptimal culture conditions (Wrenzycki et al., 2001Hum. Reprod. 16, 893–901). Hsp act as chaperones in facilitating protein folding and assembly and stabilize damaged proteins to prevent aggregation of fragments, thereby allowing repair or degradation. The aim of the present study was to investigate the effects of different embryo/volume ratios on bovine embryo development and the relative abundance of Hsp 70.1 gene transcripts. In this experiment, oocytes were isolated from slaugterhouse ovaries and matured, fertilized and cultured in groups of 5, 10, 20 or 30 per each drop of 100μL. The oocytes were matured in TCM 199 supplemented with 0.4% BSA. After maturation, oocytes were fertilized in TALP medium, using frozen/thawed sperm, selected using a percoll density gradient. The zygotes were cultured to the morula or Day 7 blastocyst stage employing SOF supplemented with 0.4 % BSA. Developmental check points were cleavage rate (Day 3pi), blastocyst formation (Day 8pi) and hatching (Day 11pi). A semi-quantitative RT-PCR assay was used to determine the relative levels of gene transcripts in single embryos at morula (Day 6) and blastocyst (Day 7) stages (Wrenzycki et al., 2001 Biol. Reprod. 65, 309–317). Data of cleavage, blastocyst formation and hatching rates were analyzed using chi-square test. Relative abundance (RA) of Hsp 70.1mRNA were compared in tested groups using ANOVA followed a Tukey test. Differences at P&lt;0.05 were considered significant. Results show that no significative difference in hatching rate per blastocyst produced was detected among the four groups. Cleavage rate and blastocyst formation were significantly higher in groups with 5, 10 and 20 embryos compared with drops containing 30 embryos. Hsp transcripts were detected in morula and blastocyst stages in all groups. In morula stage, no differences were observed in the RA of Hsp 70.1mRNA among groups with 5, 10, 20 and 30 embryos cultured per drop. However, in blastocyst stage, the RA was significantly increased in the group with 20 embryos per drop as compared to the group with 5 embryos. The results show that different embryo/volume ratios in culture influence not only cleavage rate, blastocyst formation and hatching rate, but also expression of Hsp 70.1 gene. Further studies changing other culture conditions and using in vivo-derived bovine embryos will aid in elucidating which culture systems are ideal to produce bovine embryos in vitro. This research was supported by CAPES/DAAD program and CNPq.

Abstract 4: Porcine Parthenotes as a Model to Evaluate Developmental Potential of Human Inducible Pluripotent Stem Cells

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Naoko Koyano-Nakagawa ◽  
James Dutton ◽  
Mary G Garry ◽  
Daniel J Garry
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Cellular Proliferation ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Differentiation Potential ◽  
Developmental Potential ◽  
Parthenogenetic Embryos

The use of human induced pluripotent stem cells (hiPSCs) has tremendous potential for regenerative medicine by providing an unlimited source of personalized cells. A number of protocols have been established for efficient differentiation of hiPSCs to the desired lineage in vitro, such as cardiomyocytes and blood. However, the field lacks an in vivo system to evaluate the differentiation potential and quality of hiPSCs. Developmental potential of stem cells derived from experimental animals can be readily assessed by generating blastocyst chimeras and examination of the contribution to the embryos, or by the potential of teratoma formation. However, this is not possible in the case of humans. As a potential solution for this issue, we examined whether porcine parthenotes could be used as an experimental model to test the developmental potential of the hiPSCs. Parthenotes are generated by electrical activation of the oocytes collected at the abattoir and will develop up to gestational day 53 if transferred to a pseudo-pregnant sow. The embryonic culture conditions have also been established and the zygotes can develop normally to the expanded blastocyst stage (day 7 post fertilization/activation), in vitro. We took advantage of this in vitro system and examined the ability of hiPSCs to proliferate and integrate into the parthenogenetic embryos. Parthenogenetic embryos were injected with ten undifferentiated hiPSCs at day 4 (8 cell ~ morula stage) and cultured up to 72 hours. During this period, parthenotes underwent blastocoel cavity formation and hatching. Cell tracing experiments demonstrated that hiPSCs proliferated and integrated into the parthenotes. They retained pluripotency marker expression during this period. hiPSCs and their derivatives were found both in trophoectoderm and embryo proper. We further observed that the hiPSCs underwent cellular proliferation and promoted developmental progression of the parthenote in vitro. In summary, the porcine parthenote model system is an efficient high throughput system to examine the developmental capacity of human stem cell populations.

PS02.174: THE ACTION OF A NOVEL RADIOSENSITISER WITHIN THE OESOPHAGEAL ADENOCARCINOMA TUMOUR MICROENVIROMENT

2018 ◽  
Vol 31 (Supplement_1) ◽  
pp. 171-171
Author(s):  
Amy Buckley ◽  
Niamh Lynam-Lennon ◽  
Susan Kennedy ◽  
Aoife Cannon ◽  
Dermott O’Toole ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Survival Rates ◽  
Oesophageal Adenocarcinoma ◽  
Naive Patient ◽  
Human Patient ◽  
Hypoxic Conditions ◽  
Treatment Naïve

Abstract Background Oesophageal Cancer (OAC) is an aggressive disease with survival rates of ∼15-20%. Current therapeutic regimes focus on neo-adjuvant therapy (chemo-radiation) prior to surgery. Unfortunately, only 20–30% of patients show a beneficial response, with 70–80% of patients as non-responders. This major clinical challenge of treatment resistance reinforces the need for the identification of novel treatments which can act as radio-sensitisers in the neo-adjuvant setting. Methods Through a drug screening approach in-vivo, we have identified a novel anti-angiogenic and anti-metabolic compound, 11B_CC8 with radiosensitising activity. The ability of 11B_CC8 to act as an anti-metabolic agent under hypoxia was evaluated using the XFe24 Seahorse analyser and the Don Whitley i2 workstation. The ability of 11B_CC8 to radiosensitise our isogenic OAC cells under hypoxic conditions was assessed by clonogenic assay. The effect of 11B_CC8 on inflammatory, metabolic and angiogenic protein secretions from OAC treatment naïve tumour conditioned media (TCM) was evaluated by multiplex ELISA. Fresh treatment naïve patient biopsies were screened for their metabolic activity using the XFe24 seahorse analyser at baseline and post- treatment with 11B_CC8. The elucidation of the possible mechanism of action of 11B_CC8 was evaluated by Mass-Spectrometry. Results Our novel anti-angiogenic and anti-metabolic agent can enhance radiosensitivity in our isogenic model of OAC under both normoxic and hypoxic (0.5% O 2) conditions. 11B_CC8 significantly reduces oxygen consumption rate (OCR) under normoxic but not hypoxic conditions. Ex-vivo, 11B_CC8 significantly reduced the secretion of IL1β (P = 0.0117). Real-time ex-vivo metabolic rate analysis of our treatment naïve OAC biopsies showed significantly elevated OCR, when compared to Extracellular Acidification rate, a measure of glycolysis (P = 0.0059). Treatment with 11B_CC8 produced a reduction in OCR (P = 0.0039). Conclusion Our novel anti-angiogenic and anti-metabolic agent can enhance radiosensitivity in-vitro under both normoxic and hypoxic conditions. Ex-vivo, treatment naïve OAC human patient samples, 11B_CC8 can significantly reduce the secretion of IL1β and altered metabolic programming, specifically oxidative phosphorylation in human explants. Disclosure All authors have declared no conflicts of interest.

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