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Factors affecting the gene expression of in vitro cultured human preimplantation embryos

ABSTRACT: Study question: What is the relative effect of common environmental and biological factors on transcriptome changes during human preimplantation development? Summary answer: Developmental stage and maternal age had a larger effect on the global gene expression profile of human preimplantation embryos than the culture medium or oxygen concentration used in in vitro culture. What is known already: Studies on mouse and bovine embryos have shown that different conditions in the in vitro culture of embryos can lead to changes in transcriptome profiles. For humans, an effect of developmental stage on the transcriptome profile of embryos has been demonstrated, but studies on the effect of maternal age or culture conditions are lacking. Participants/materials, setting, methods: Embryos that developed to morula or blastocyst stage during these 2 days whose amplified mRNA passed our quality control criteria for microarray hybridization were individually examined for genome-wide gene expression (N = 37). Main results and the role of chance: Based on the number of differentially expressed genes (DEGs), developmental stage (3519 DEGs) and maternal age (1258 DEGs) had a larger effect on the global gene expression profile of human preimplantation embryos than either tested culture medium (596 DEGs) or oxygen concentration (492 DEGs) used during in vitro culture. Interactions between the factors were found, indicating that culture conditions might have a different effect depending on the developmental stage or the maternal age of the embryos. Affected pathways included metabolism, cell cycle processes and oxidative phosphorylation. Limitations, reasons for caution: Culture of embryos for only 2 days might have limited the effect on global gene expression by the investigated culture conditions. Earlier stages of development (Day 0 until Day 4) were not analyzed and these embryos might respond differently to the experimental conditions. The freezing and thawing procedures might have had an effect on gene expression. RT–PCR validation was not performed due to scarcity of the material. Wider implications of the findings: Our results show that when studying gene expression in single human preimplantation embryos under various experimental conditions, one should take into account the confounding effect of biological variables, such as developmental stage and maternal age. This makes these experiments different from gene expression experiments where these variables can be tightly controlled, for example when using cell lines.

ORGANISM(S): Homo sapiens

PROVIDER: GSE49374 | GEO | 2015/12/31

SECONDARY ACCESSION(S): PRJNA213986

REPOSITORIES: GEO

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Project description:Study question: What is the relative effect of common environmental and biological factors on transcriptome changes during human preimplantation development? Summary answer: Developmental stage and maternal age had a larger effect on the global gene expression profile of human preimplantation embryos than the culture medium or oxygen concentration used in in vitro culture. What is known already: Studies on mouse and bovine embryos have shown that different conditions in the in vitro culture of embryos can lead to changes in transcriptome profiles. For humans, an effect of developmental stage on the transcriptome profile of embryos has been demonstrated, but studies on the effect of maternal age or culture conditions are lacking. Participants/materials, setting, methods: Embryos that developed to morula or blastocyst stage during these 2 days whose amplified mRNA passed our quality control criteria for microarray hybridization were individually examined for genome-wide gene expression (N = 37). Main results and the role of chance: Based on the number of differentially expressed genes (DEGs), developmental stage (3519 DEGs) and maternal age (1258 DEGs) had a larger effect on the global gene expression profile of human preimplantation embryos than either tested culture medium (596 DEGs) or oxygen concentration (492 DEGs) used during in vitro culture. Interactions between the factors were found, indicating that culture conditions might have a different effect depending on the developmental stage or the maternal age of the embryos. Affected pathways included metabolism, cell cycle processes and oxidative phosphorylation. Limitations, reasons for caution: Culture of embryos for only 2 days might have limited the effect on global gene expression by the investigated culture conditions. Earlier stages of development (Day 0 until Day 4) were not analyzed and these embryos might respond differently to the experimental conditions. The freezing and thawing procedures might have had an effect on gene expression. RTâPCR validation was not performed due to scarcity of the material. Wider implications of the findings: Our results show that when studying gene expression in single human preimplantation embryos under various experimental conditions, one should take into account the confounding effect of biological variables, such as developmental stage and maternal age. This makes these experiments different from gene expression experiments where these variables can be tightly controlled, for example when using cell lines. Study design, size, duration: Donated, good quality, day 4 cryopreserved human preimplantation embryos (N = 89) were randomized to be cultured in one of two culture media (G5 medium or HTF medium) and one of two oxygen concentrations (5% or 20%), with stratification for maternal age. Next to these variables, developmental stage after culture was taken into account in the analysis.

Project description:Study question: Is gene expression in human preimplantation embryos affected by medium used for culturing embryos during an IVF treatment? Summary answer: Six days of in vitro culture of human preimplantation embryos resulted in a medium-dependent expression level of genes involved in apoptosis, protein degradation, metabolism and cell cycle regulation. What is known already: Several human studies have shown an effect of culture medium on embryo development, pregnancy outcome and birthweight. However, the underlying mechanisms in human embryos are still unknown. In animal blastocysts, it has been demonstrated that culture of preimplantation embryos affects gene expression. In humans, it has been found that culture medium affects gene expression of cryopreserved embryos that, after thawing, were cultured in two different media for 2 more days. Study design, size, duration: In a multicenter trial, women were randomly assigned to two culture media groups (G5 and HTF). Data on embryonic development were collected for all embryos. In one center, embryos originating from 2PN zygotes that were not selected for transfer or cryopreservation on day 2 or 3, were further cultured until day 6 and collected for this study, if couples consented. Participants/materials, setting, methods: Ten blastocysts from each study group, matched for fertilization method, maternal age and blastocyst quality, were selected and their mRNA was isolated and amplified. Embryos were individually examined for genome-wide gene expression using Agilent microarrays and PathVisio was used to identify the pathways that showed a culture medium-dependent activity. Main results and the role of chance: Expression of 951 genes differed significantly (P < 0.01) between the two study groups. Eighteen pathways, involved in apoptosis, metabolism, protein processing and cell cycle regulation, showed a significant overrepresentation of differentially expressed genes (DEGs). The DNA replication, G1 to S cell cycle control and oxidative phosphorylation pathways were upregulated in the G5 group compared with the HTF group. This is in agreement with the higher number of cells seen in G5 embryos on day 2 and 3. Limitations, reasons for caution: Despite careful matching of the embryos, it cannot be ruled out that differences observed between the study groups are affected by factors not investigated. Extrapolation of these results to embryos used for transfer demands caution as in the present study embryos that were not selected for either embryo transfer or cryopreservation have been used for the culture experiment until day 6. Wider implications of the findings: This study shows that gene expression in human preimplantation embryos is affected by the culture medium used during an IVF treatment. Furthermore, it provides insight into the biological mechanisms that are affected. The increased cell cycle regulation is reflected by a higher number of blastomeres. Whether these results are also connected to long-term effects remains unknown. However, it is not unlikely that early adaptations of the preimplantation embryo to its environment persist during fetal and post-natal development. Note: this study has been conducted using the Agilent-028004 SurePrint G3 Human GE 4x180K Microarray. This array contains the same reporters as the Agilent-028004 SurePrint G3 Human GE 8x60K Microarray. As such we have linked this entry to the A-GEOD-14550 array design, belonging to the latter.

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Factors affecting the gene expression of in vitro cultured human preimplantation embryos

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