Vitrification alters fetal development by profound differences during fetal placenta formation
ABSTRACT: Vitrification is replacing slow freezing as the most popular method for human embryo cryopreservation in clinics world-wide. Several studies demonstrated that cryopreservation alters gene expression of mammalian embryos, but none of them analysed what happen with those embryos that get implanted and follow with the gestation. The aim of this study was to evaluate the effect of vitrification technique on rabbit embryonic and fetal development by performing a transcriptomic analysis of 6 day old embryos and 14 days old fetal placentas. Effect of vitrification on late blastocyst and fetal placenta transcriptome. Four indepent replicates were performed for each condition (control and vitrified) and for both tissues (embryo and fetal placenta).
Project description:Nowadays there is a need to improve cryopreservation protocols in both human assisted reproduction and domestic animal field. We wanted to compare the transcriptomic changes induced by two commonly used cryopreservation procedures (slow freezing and vitrification) on rabbit late blastocyst before the onset of implantation. We recovered rabbit morulae at day 3 of development, freezing or vitrified and transferred them into recipient maternal tracts till day 6 of development. In this way, we wanted to analyse if different cryopreservation techniques produce different effects on gene expression that embryos are unable to get over after three days of in vivo development. After artificially insemination morulae were recovered at day 3 of development, frozen or vitrified, and then transferred to recipient does. To assess gene expression differences between frozen and vitrified the recipients were slaughtered three days after the transfer, at day 6.
Project description:Studies on embryo cryopreservation efficiency had been focused mainly on technical and embryo factors. While structural damages can be easily evaluated, physiological damages only can be estimated by analyzing their in vitro and in vivo development to later stages. In order to determine how cryopreservation process affect embryo pre-implantory development, a transcriptional microarray study has been performed comparing gene expression of 6 days old rabbit embryos, previously vitrified or frozen and transferred into recipients rabbit females, to their in vivo counterparts. For each experimental group, control, vitrified and frozen late blastocysts, total RNA was extracted from 3 pools of approximately 10 embryos and labeled with Cy3 or Cy5 dyes. Then, six competitive hybridizations were carried out including two dye-swaps to compensate dye-bias. A specifically microarray designed to study rabbit gene expression profiling, the Rabbit 44K oligonucleotide array (Agilent Technologies), was used in this study. Identification of differentially expressed transcripts from 6 day old blastocysts was achieved using the Limma algorithm, and functional annotation was performed by Blast2GO software. Compared to 6 day old in vivo derived embryos, viable vitrified embryos only present 3 differentially expressed genes, in contrast to frozen viable embryos with 24 genes upregulated and 46 genes downregulated. These results reveal that effects of cryopreservation still remain in late blastocyst pre-implantatory gene expression, and potential damage and alterations produced by vitrification and slow-freezing procedures are differentially resolved after 3 days of in vivo development. Transcriptional microarray study that compares gene expression of viable 6 day old rabbit embryos, previosly vitrified or frozen and tranferred into recipient rabbit females, to their in vivo counterparts. Experiment 1: Control embryos vs. Vitrified embryos and Experiment 2: Control embryos vs. Frozen embryos. Biological replicates used: 3 replicates for control embryos, 3 replicates for vitrified embryos and 3 replicates for frozen embryos.
Project description:High hydrostatic pressure (HHP) has been used to pre-condition embryos before essential, yet potentially detrimental procedures such as cryopreservation. However, the mechanisms for HHP are poorly understood. We treated bovine blastocysts with three different HHP (40, 60 and 80 MPa) in combination with three recovery periods (0, 1h, 2h post HHP). Re-expansion rates were significantly higher at 40 and 60 but lower at 80 MPa after vitrification-warming in the treated groups than controls. Microarray analysis revealed 399 differentially expressed transcripts, representing 254 unique genes, among different groups. Gene ontology analysis indicated that HHP at 40 and 60 MPa promoted embryo competence through down-regulation of genes in cell death and apoptosis, and up-regulation of genes in RNA processing, cellular growth and proliferation. In contrast, 80 MPa up-regulated genes in apoptosis, and down-regulated protein folding and cell cycle-related genes. Moreover, gene expression was also influenced by the length of the recovery time after HHP. The significantly over-represented categories were apoptosis and cell death in the 1h group, and protein folding, response to unfolded protein and cell cycle in the 2h group compared to 0h. Taken together, HHP promotes competence of vitrified bovine blastocysts through modest transcriptional changes. 4*3*2 design experiment. The following treatments were included: (1) Control embryos were left untreated in the incubator (one atmospheric pressure or 0.1 MPa); (2) treatment groups were assigned to 40, 60 and 80 MPa HHP for 1h at either 24°C (room temperature) or 39°C (body temperature), followed by three different recovery time periods (0, 1 and 2h) post-HHP in the holding medium. Biological replicates: 3 control replicates. Technical replicates: dye-swap.
Project description:Changes in gene expression induced by the Cryotop vitrification procedure in bovine blastocysts using Agilent EmbryoGENE microarray slides. Bovine in vitro-produced embryos at the blastocyst stage (144 to 156 hours post insemination) were vitrified using the Cryotop system and compared with non-vitrified (control) embryos. After vitrification, the embryos were warmed and cultured for an additional 4 hours. Embryos that re-expanded or developed to the expanded blastocyst stage were used for microarray analysis. Four pools of vitrified embryos were hybridized against four pools of control embryos in a dye-swap design.
Project description:We aimed to study the effects of CSF2 treatment on the methylome of the Day 15 female and male embryo. Methylomic profiling of bovine extra-embryonic membrane at Day 15 post-insemination produced by IVF. Oocytes were matured and fertilized in vitro using a single Holstein bull. After 5 days in vitro culture, the embryos were either treated with 10 ng/ml bovine recombinant CSF2 or the Control (DPBS/BSA) until Day 7. At Day 7, the embryos were transferred into recipients. At day 15 following insemination, embryos were flushed and collected for total RNA and gDNA extraction. The gender of the embryos were determined by PCR prior to microarray analysis. CSF2 males were compared to Control males while CSF2 females were compared to Control females
Project description:We aimed to study the effects of CSF2 treatment on the transcriptome of the Day 15 female and male embryo. Within the raw files, channel 1= Cy3 and channel 2= Cy5 Transcriptional profiling of bovine extra-embryonic membrane at Day 15 post-insemination produced by IVF. Oocytes were matured and fertilized in vitro using a single Holstein bull. After 5 days in vitro culture, the embryos were either treated with 10 ng/ml bovine recombinant CSF2 or the Control (DPBS/BSA) until Day 7. At Day 7, the embryos were transferred into recipients. At day 15 following insemination, embryos were flushed and collected for total RNA and gDNA extraction. The gender of the embryos were determined by PCR prior to microarray analysis. CSF2 males were compared to Control males while CSF2 females were compared to Control females. Control females were also compared to control males.
Project description:Vitrification is commonly used in the cryopreservation of mammalian blastocysts to overcome the temporal and spatial limitations of embryo transfer. Previous studies have shown that the implantation ability of vitrified blastocysts is impaired and that microRNAs (miRNAs) regulate the critical gene for embryo implantation. However, little information is available about the effect of vitrification on the miRNA transcriptome in blastocysts, which partially explains the impaired implantation ability of vitrified blastocysts. In the present study, the miRNA transcriptomes in fresh and vitrified mouse blastocysts were analyzed by microRNA Taqman assay based method, and the results were validated using quantitative real-time PCR (qRT-PCR). Then, the differentially expressed miRNAs were assessed using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Overall, 760 known mouse miRNAs were detected in the vitrified and fresh mouse blastocysts. Of these, the expression levels of five miRNAs differed significantly: in the vitrified blastocysts, four miRNAs (mmu-miR-199a-5p, mmu-miR-329-3p, mmu-miR-136-5p and mmu-miR-16-1-3p) were upregulated, and one (mmu-miR-212-3p) was downregulated. The expression levels of all miRNAs measured by the microRNA Taqman assay based method and qRT-PCR were consistent. The four upregulated miRNAs were predicted to regulate 877 candidate target genes, and the downregulated miRNA was predicted to regulate 231 genes. The biological analysis further showed that the differentially expressed miRNAs mainly regulated the implantation of embryos. In conclusion, the results of our study showed that vitrification significantly altered the miRNA transcriptome in mouse blastocysts, which may decrease the implantation potential of vitrified blastocysts. Overall design: The TaqMan® Array Rodent MicroRNA A+B Cards Set v3.0 with an Applied Biosystems 7900 HT Fast Real-time PCR system was utilized to detect the miRNA transcriptome in fresh and vitrified mouse blastocysts.6 samples were analyzed, including 3 vitrification samples and 3 fresh samples.
Project description:Some embryos display better survival potential to cryopreservation than others. The cause of such phenotype is still unclear and might be due to cell damage during cryopreservation, resulting from over-accumulation and composition of lipids. In cattle embryos, in vitro culture conditions have been shown to impact the number of lipid droplets within blastomeres. So far, the impact of breed on embryonic lipid content has not yet been studied. In this study were compared the colour, lipid droplet abundance, lipid composition, mitochondrial activity, and gene expression of in vivo collected Jersey breed embryos which are known to display poor performance post-freezing and in vivo Holstein embryos which have good cryotolerance. Holstein in vivo day 6 embryos vs Jersey in vivo day 6 embryos: 4 replicates of each breed, with dye-swap.
Project description:TWIST1, a basic helix-loop-helix transcription factor is essential for the development of cranial mesoderm and cranial neural crest-derived craniofacial structures. Our previous work showed that, in the absence of TWIST1, some cells within the cranial mesoderm adopt an abnormal epithelial configuration. Here, we show by transcriptome analysis that loss of TWIST1 in the cranial mesoderm is accompanied by a reduction in the expression of genes that are associated with cell-extracellular matrix interactions and the acquisition of mesenchymal characteristics. By comparing the transcriptional profiles of cranial mesoderm-specific Twist1 loss-of-function mutant and control mouse embryos, we identified a set of genes that are both TWIST1-dependent and predominantly expressed in the mesoderm. By ChIP-seq in a cell line model of a TWIST1-dependent mesenchymal state, we identified, among the downstream genes, three direct transcriptional targets of TWIST1: Ddr2, Pcolce and Tgfbi. Our findings show that the mesenchymal properties of the cranial mesoderm is likely to be regulated by a network of TWIST1 targets genes that influence the extracellular matrix and cell-matrix interactions, and collectively they are required for the morphogenesis of the craniofacial structures. Cranial neural crest and cranial mesoderm cells were isolated by flow sorting of GFP reporter-labelled cells collected from heads of E9.5 mouse embryos. Three replicates were independently isolated and hybridized to Illumina mouse WG v 2.0 chips
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.