The effect of embryo biopsy on global patterns of gene expression in the mouse blastocyst
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE11206 | GEO | 2008/04/22
SECONDARY ACCESSION(S): PRJNA106809
REPOSITORIES: GEO
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Project description:Preimplantation Genetic Testing (PGT), which encompasses both Preimplantation Genetic Diagnosis (PGD) and Preimplantation Genetic Screening (PGS), is a form of prenatal screening done on embryos conceived through assisted reproduction techniques (ART) prior to the initiation of pregnancy to ensure that only select embryos are used for transfer. PGT is typically performed on 8-cell embryos derived from either in vitro fertilization or intracytoplasmic sperm injection (ICSI) followed by extended culture. PGT requires a highly invasive embryo biopsy procedure that involves 1) incubating embryos in divalent-cation-deficient medium to disrupt cell adhesion, 2) breaching the protective zona pellucida with acid Tyrodeâs, laser drilling, or mechanical force and 3) aspirating one or two blastomeres. In this study we developed a mouse model of the embryo biopsy procedure inherent to PGT to determine the effect of various aspects of the procedure (incubation in Ca2+/Mg2+-free medium (CMF), acid Tyrodeâs treatment, blastomere aspiration), performed individually or in combination, on global patterns of gene expression in the resulting blastocysts. Experiment Overall Design: The comprehensive embryo biopsy procedure was done as outlined below and was modified according to the specific experimental treatment being performed. Experiment Overall Design: Eight-cell embryos with rounded blastomeres and no fragmentation were initially incubated in CMF-Chatot-Ziomek-Brinster medium for 5 min at 37oC in a humidified atmosphere of 5% CO2 in air to loosen cell-cell contacts and then transferred to CMF-Dulbeccoâs Phosphate-Buffered Saline. The biopsies were done on the stage of a Nikon Eclipse TE2000 microscope equipped with Hoffman optics and motorized Eppendorf micromanipulators. No more than three embryos were manipulated outside of the incubator at any time. Embryos were held in place by gentle suction on an Eppendorf Sterile VacuTip holding pipette (15 μm diameter), and a dual-holder system was used to control the pipette containing acid Tyrodeâs (6 μm diameter) and the biopsy pipette (15 μm diameter). The zona pellucida was breached by gentle expulsion of a steady stream of acid Tyrodeâs solution (pH 1.6), and a single blastomere with a clear nucleus was removed by aspiration. Following each respective treatment, embryos were cultured individually in 10 μl drops of Potassium Simplex Optimization Medium supplemented with amino acids at 37oC in a humidified atmosphere of 5% CO2 in air until 96 h post-hCG injection. Four replicates of these five experimental groups, each containing 20 embryos, were generated for a total of 20 samples and 400 embryos. Experiment Overall Design: To determine the effect of the various aspects of the embryo biopsy procedure on global patterns of gene expression, total RNA was extracted from each set of 20 embryos in each treatment group at 96 h post-hCG injection. This total RNA was then used to prepare target cRNA by linear two-round amplification as described previously. The final yield of biotinylated cRNA/20 embryos ranged from 34 μg -78 μg, of which 20 μg was fragmented and submitted to the University of Pennsylvania Microarray Facility where the samples were serially hybridized to the MOE430v2 GeneChip. Quality control parameters for all samples were within the following ranges: scale factor: 0.27-0.47, percent of genes detected: 44-51%, actin 3â/5â signal ratio: 1.5-4.2, and GAPDH 3â/5â signal ratio: 3.1-4.3. The GC-RMA algorithm was used to quantify and normalize microarray signals using Stratagene Array Assist Lite, Version 3.4. The resulting .chp files were imported to GeneSpring (v7.31, Agilent Technologies) where probe sets were filtered to retain those that were flagged as P (present) in at least 3 of the 20 samples. The data was log2-transformed and subjected to analysis using Partek Genomics Suite (v6.3, Partek, Inc).
2008-06-11 | E-GEOD-11206 | biostudies-arrayexpress
Project description:BACKGROUND:
Preimplantation genetic diagnosis (PGD) enables profiling of embryos for genetic disorders prior to implantation. The majority of PGD testing is restricted in the scope of variants assayed or by the availability of extended family members. While recent advances in single cell sequencing show promise, they remain limited by bias in DNA amplification and the rapid turnaround time (<36 h) required for fresh embryo transfer. Here, we describe and validate a method for inferring the inherited whole genome sequence of an embryo for preimplantation genetic diagnosis (PGD).
METHODS:
We combine haplotype-resolved, parental genome sequencing with rapid embryo genotyping to predict the whole genome sequence of a day-5 human embryo in a couple at risk of transmitting alpha-thalassemia.
RESULTS:
Inheritance was predicted at approximately 3 million paternally and/or maternally heterozygous sites with greater than 99% accuracy. Furthermore, we successfully phase and predict the transmission of an HBA1/HBA2 deletion from each parent.
CONCLUSIONS:
Our results suggest that preimplantation whole genome prediction may facilitate the comprehensive diagnosis of diseases with a known genetic basis in embryos.
| EGAS00001001020 | EGA
Project description:Study question: Could extracellular vesicles (EVs) secreted by aneuploid embryos a) serve for development of RNA biomarkers for preimplantation genetic testing for aneuploidy (PGT-A) and b) elicit a relevant transcriptomic response in decidualised primary endometrial stromal cells (dESCs)? Summary answer: Aneuploid embryo EVs a) contain genes PPM1J, LINC00561, ANKRD34C and TMED10 in differential abundance from euploid EVs and b) induce up-regulation of MUC1 transcript in dESCs. What is known already: PGT-A is popular as it is thought to facilitate transfer of euploid embryos to increase implantation probability but the technology is controversial, as it requires an invasive embryo biopsy with an elusive long-term biosafety. The development of non-invasive methods to screen out aneuploid embryos is paramount. It is also critical to decode the embryo-endometrial dialog underlying implantation failure. We have previously reported that IVF embryos secrete EVs that can be internalised by ESCs, conceptualising that successful implantation to the endometrium is facilitated by EVs, which may additionally serve as biomarkers of ploidy status. Study design, size, duration: Embryos destined for biopsy on days 5-7 for PGT-A were grown under standard conditions. Spent media (30μl) were collected from euploid (n=175) and aneuploid (n=140)embryos at cleavage (days 1-3) stage and from euploid (n=187) and aneuploid (n=142) embryos at blastocyst (days 3-5) stage. Media samples from n=35 cleavage embryos were pooled in order to obtain five euploid and four aneuploid pools. Similarly, media samples from blastocyst were pooled to create one euploid and one aneuploid pool. ESCs were obtained from five women undergoing diagnostic laparoscopy. Participants/materials, setting, methods: EVs were isolated from pools of media derived from euploid and aneuploid day 3 embryos with differential centrifugation and EV-RNA sequencing was performed following the SMARTer Stranded Total RNA-Seq approach. ESCs were decidualised (E2:10nM, P4:1uM, cAMP:0.5 mM twice every 48 hours) and treated for 24 hours with 50 ng/ml euploid or aneuploid EVs extracted from blastocyst media. RNA sequencing was performed on ESCs following the Illumina Truseq RNAseq protocol. Main results and the role of chance: Aneuploid cleavage stage embryos secreted EVs that were less abundant in RNA fragments originating from the genes PPM1J (log2fc=-5.13, p=0.011), LINC00561 (log2fc=-7.87, p=0.010) and ANKRD34C (log2fc=-7.30, p=0.017) and more abundant in TMED10 (log2fc=1.63 p=0.025) compared to EVs from euploid embryos. Decidualisation per se induced downregulation of MUC1 (log2FC=-0.54, p=0.0028) in ESCs as prerequisite for the establishment of receptive endometrium. The expression of MUC1 transcript in decidualised ESCs was significantly increased following treatment with aneuploid compared to euploid embryo-secreted EVs (log2FC=0.85, p=0.0201). Limitations, reasons for caution: The findings of the study may require validation utilising a second cohort of EVs samples. Wider implications of the findings: The discovery that the RNA cargo of EVs secreted from aneuploid cleavage stage embryos is diverse from that of euploid embryos potentiates the development of non-invasive methodology for PGT-A. The upregulation of MUC1 in dESCs following aneuploid embryo EV treatment proposes a new mechanism underlying implantation failure. Funding: The study was supported by a MSCA fellowship awarded to SM by the European Commission (CERVINO grant agreement ID: 79620) and by a BIRTH research grant from Theramex HQ UK Ltd
2024-04-03 | GSE234338 | GEO
Project description:In total, 240 single blastomeres from nine top-quality day-4 embryos frozen at day 3 of development and four fresh top-quality day-4 embryos that had one-cell biopsy on day 3 for preimplantation genetic diagnosis (PGD) were collected. Blastomeres' DNA was amplified using SurePlex DNA Amplification System (BlueGnome, Cambridge, UK) . Array-CGH was carried out using 24Sure Cytochip microarrays following the standard protocol (BlueGnome, www.cytochip.com).
2013-04-05 | GSE39518 | GEO
Project description:In total, 240 single blastomeres from nine top-quality day-4 embryos frozen at day 3 of development and four fresh top-quality day-4 embryos that had one-cell biopsy on day 3 for preimplantation genetic diagnosis (PGD) were collected. Blastomeres' DNA was amplified using SurePlex DNA Amplification System (BlueGnome, Cambridge, UK) . Array-CGH was carried out using 24Sure Cytochip microarrays following the standard protocol (BlueGnome, www.cytochip.com). BAC array-CGH on single blastomeres amplified by SurePlex amplification Kit.
2013-04-05 | E-GEOD-39518 | biostudies-arrayexpress
Project description:Preimplantation Genetic Testing (PGT) of in vitro fertilized (IVF) embryos has been proposed as a method to reduce transmission of common disease, although comprehensive genetic assessment remains unavailable. Here we use a combination of molecular and statistical techniques to reliably and accurately infer inherited genome sequence in 110 embryos. We combine polygenic risk scores (PRS) and rare variation to model susceptibility across 12 common conditions.
| EGAS00001005619 | EGA
Project description:Objective: To prove the ability to distinguish between balanced and normal chromosomes in embryos from a translocation carrier. Design: Case report. Setting: Academic center for reproductive medicine. Patient: A female with a balanced translocation causing Alagille Syndrome seeking preimplantation genetic diagnosis (PGD). Interventions: Blastocyst biopsy for PGD. Main outcome measures: Consistency of 3 methods of embryo genetic analysis (real-time PCR, SNP microarray, and FISH) and normalcy in the newborn derived from PGD. Results: PGD was applied to 48 embryos. Real-time PCR, SNP microarray, and FISH demonstrated 100% consistency, although FISH failed to detect aneuploidies observed by comprehensive SNP microarray based analyses. Two blastocysts were identified to be normal for all 3 factors using SNP microarray technology alone. The two normal embryos were transferred back to the patient resulting in the delivery of a healthy baby boy with a normal karyotype. Conclusions: This is the first report of validation and successful clinical application of microarray based PGD to distinguish between balanced and normal chromosomes in embryos from a translocation carrier.
2011-05-18 | GSE21732 | GEO
Project description:Single-cell whole-genome haplotyping allows simultaneous detection of haplotypes associated with monogenic diseases, chromosome copy-numbering and subsequently, has revealed mosaicism in embryos and embryonic stem cells. Methods, such as karyomapping and haplarithmisis, were deployed as a generic and genome-wide approach for preimplantation genetic testing (PGT) and are replacing traditional PGT methods. While current methods primarily rely on SNP array, we envision sequencing-based methods to become more accessible and cost-efficient. Here, we developed a novel sequencing-based methodology to haplotype and copy-number profile single cells. Following DNA amplification, genomic size and complexity is reduced through restriction enzyme digestion and DNA is genotyped through sequencing. This single-cell genotyping-by-sequencing (scGBS) is the input for haplarithmisis, an algorithm we previously developed for SNP array-based single-cell haplotyping. We established technical parameters and developed an analysis pipeline enabling accurate concurrent haplotyping and copy-number profiling of single cells. We demonstrate its value in human blastomere and trophectoderm samples as application for PGT for monogenic disorders. Furthermore, we demonstrate the method to work in other species through analyzing blastomeres of bovine embryos. Our scGBS method opens up the path for single-cell haplotyping of any species with diploid genomes and could make its way into the clinic as a PGT application.
2021-08-18 | GSE178156 | GEO
Project description:This was a retrospective comparison study of SNP-based preimplantation genetic screening (SNP-PGS) and FISH-based preimplantation genetic diagnosis (FISH-PGD) for 575 couples in total with chromosome translocations, including 169 couples treated by SNP-PGS between October 2011 and August 2012, and 406 couples treated by FISH- PGD between January 2005 and October 2011. In total, 773 blastocysts obtained from 169 couples were biopsied and frozen, embryo transfer was carried out on the balanced embryos. The PGS results and pregnancy outcomes were compared with those of FISH-PGD for 406 translocation carriers with 3,968 embryos biopsied on day 3. Of the 773 biopsied blastocysts, reliable SNP-PGS results were obtained for 717 (92.76%). For Robertsonian translocation carriers, the rate of normal/balanced embryos, embryos with translocation-related abnormalities, and embryos with abnormalities unrelated to a translocation were 57.80%, 23.39% and 18.81%, respectively. In reciprocal translocation carriers, the rate of normal/balanced embryos, embryos with translocation-related abnormalities and embryos with abnormalities unrelated to translocation were 35.47%, 52.10% and 12.42%, respectively. There was no significant differences in patient age, basal endocrine level and the average number of retrieved oocytes and good quality day 3 embryos before biopsy in the SNP-PGS group compared with the FISH-PGD group. The number of embryos biopsied in the FISH-PGD group was higher than in the SNP-PGS group. However, the pregnancy rate with successful delivery per oocyte retrieval and the implantation rate were both lower in the FISH-PGD group than in the SNP-PGS group. The spontaneous abortion rate was higher in the FISH-PGD group than in the SNP-PGS group. Affymetrix SNP arrays were performed according to the manufacturer's directions on DNA extracted from trophectoderm cells.
2013-03-11 | E-GEOD-44994 | biostudies-arrayexpress
Project description:This was a retrospective comparison study of SNP-based preimplantation genetic screening (SNP-PGS) and FISH-based preimplantation genetic diagnosis (FISH-PGD) for 575 couples in total with chromosome translocations, including 169 couples treated by SNP-PGS between October 2011 and August 2012, and 406 couples treated by FISH- PGD between January 2005 and October 2011. In total, 773 blastocysts obtained from 169 couples were biopsied and frozen, embryo transfer was carried out on the balanced embryos. The PGS results and pregnancy outcomes were compared with those of FISH-PGD for 406 translocation carriers with 3,968 embryos biopsied on day 3. Of the 773 biopsied blastocysts, reliable SNP-PGS results were obtained for 717 (92.76%). For Robertsonian translocation carriers, the rate of normal/balanced embryos, embryos with translocation-related abnormalities, and embryos with abnormalities unrelated to a translocation were 57.80%, 23.39% and 18.81%, respectively. In reciprocal translocation carriers, the rate of normal/balanced embryos, embryos with translocation-related abnormalities and embryos with abnormalities unrelated to translocation were 35.47%, 52.10% and 12.42%, respectively. There was no significant differences in patient age, basal endocrine level and the average number of retrieved oocytes and good quality day 3 embryos before biopsy in the SNP-PGS group compared with the FISH-PGD group. The number of embryos biopsied in the FISH-PGD group was higher than in the SNP-PGS group. However, the pregnancy rate with successful delivery per oocyte retrieval and the implantation rate were both lower in the FISH-PGD group than in the SNP-PGS group. The spontaneous abortion rate was higher in the FISH-PGD group than in the SNP-PGS group.
2013-03-11 | GSE44994 | GEO