Analysis of gene expression in uniparental mouse blastocysts produced in different culture media
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE111112 | GEO | 2022/02/01
REPOSITORIES: GEO
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Project description:Following intracytoplasmic sperm injection (ICSI), in vitro culture (IVC) in ART media causes imbalance in the blastocyst's inner cell mass (ICM)with reduction of the primitive endoderm compartment compared to non-IVC blastocystsFollowing embryo transfer (ET), these imbalances do not influence birth rates, and the postnatal effects are inconspicuous. ICSI alters gene expression in mouse embryos. Choice of embryo culture media has been correlated with birth and body growth rates in human studies. No analysis has yet been made combining ICSI and IVC in the mouse. We produced mouse concepti by ICSI followed by IVC and ET so as to mimic,at least in part, the way human embryos are produced and handled in the clinical practice. Fertilized oocytes were cultured in two sets of sequential ART media, compared with an optimized mouse medium and with the mouse oviduct. Embryos were analyzed or implanted to produce newborns. In total, 3706 fertilized mouse oocytes, 1672 blastocysts and 78 newborns were examined during the course of the study. Mice of the B6C3F1, C57Bl/6 and CD1 strains were used as oocyte donors, sperm donors and embryo recipients, respectively. B6C3F1 oocytes were fertilized by intracytoplasmic C57Bl/6 sperm injection and were allowed to cleave in HTF/MultiBlast and in ISM1/ISM2 sequential culture protocols as well as in optimized mouse medium (KSOM(aa)) and in vivo in the CD1 mouse oviduct. Cleavage and blastocyst rates were analyzed by Wilcoxon / Kruskal-Wallis test and the blastocysts were split in two subgroups:one subgroup was analyzed for cell lineage allocation and for transcriptome, the other subgroup was transferred to genital tract, allowed to develop to term and characterized for birth rate, litter size, neonatal bodyweight and behavior using ANOVA F-test and t-test. The effects of ART media on blastocyst rates exceed those of ICSI. Protein and mRNA analysis reveal that IVC has a marked effect on the primitive endoderm lineage of ICSI blastocysts. In contrast to preimplantation, birth rates after ET are indistinguishable not only among ICSI but also in comparison to non-injected controls. At birth, ICSI-IVC-ET concepti are outwardly normal and weigh the same as non-ICSI controls. Confounding effects of subfertility, lifestyle and genetic heterogeneity are reduced to a minimum in the hybrid inbred mouse model compared to human patients. Confounding effects of polyspermy and parthenogenetic activation are prevented by ICSI. There is more to ART than ICSI, IVC and ET, and only a small number of ART media were examined. Preimplantation effects of ART culture media can effectively be modeled in the mouse because the in vitro environment is suboptimal for both mouse and human. However, in the optimal uterine environment of their own species, mouse and human ART blastocysts may be supported and selected for differently, depending on species-specific features such as the kinetics of implantation and the endometrium. 14 samples were analyzed. HTF/MultiBlast: Mouse multiblastocysts in HTF medium, 3 biological rep ISM1/ISM2: Mouse blastocysts in ISM1 and ISM2 medium, 3 biological rep KSOM(aa): Mouse blastocysts in KSOM(aa) medium, 2 biological rep oviduct: Mouse oviduct, 3 biological rep micromanipulation control: Mouse micromanipulation control, 3 biological rep
2016-08-25 | E-GEOD-50193 | biostudies-arrayexpress
Project description:Following intracytoplasmic sperm injection (ICSI), in vitro culture (IVC) in ART media causes imbalance in the blastocyst's inner cell mass (ICM)with reduction of the primitive endoderm compartment compared to non-IVC blastocystsFollowing embryo transfer (ET), these imbalances do not influence birth rates, and the postnatal effects are inconspicuous. ICSI alters gene expression in mouse embryos. Choice of embryo culture media has been correlated with birth and body growth rates in human studies. No analysis has yet been made combining ICSI and IVC in the mouse. We produced mouse concepti by ICSI followed by IVC and ET so as to mimic,at least in part, the way human embryos are produced and handled in the clinical practice. Fertilized oocytes were cultured in two sets of sequential ART media, compared with an optimized mouse medium and with the mouse oviduct. Embryos were analyzed or implanted to produce newborns. In total, 3706 fertilized mouse oocytes, 1672 blastocysts and 78 newborns were examined during the course of the study. Mice of the B6C3F1, C57Bl/6 and CD1 strains were used as oocyte donors, sperm donors and embryo recipients, respectively. B6C3F1 oocytes were fertilized by intracytoplasmic C57Bl/6 sperm injection and were allowed to cleave in HTF/MultiBlast and in ISM1/ISM2 sequential culture protocols as well as in optimized mouse medium (KSOM(aa)) and in vivo in the CD1 mouse oviduct. Cleavage and blastocyst rates were analyzed by Wilcoxon / Kruskal-Wallis test and the blastocysts were split in two subgroups:one subgroup was analyzed for cell lineage allocation and for transcriptome, the other subgroup was transferred to genital tract, allowed to develop to term and characterized for birth rate, litter size, neonatal bodyweight and behavior using ANOVA F-test and t-test. The effects of ART media on blastocyst rates exceed those of ICSI. Protein and mRNA analysis reveal that IVC has a marked effect on the primitive endoderm lineage of ICSI blastocysts. In contrast to preimplantation, birth rates after ET are indistinguishable not only among ICSI but also in comparison to non-injected controls. At birth, ICSI-IVC-ET concepti are outwardly normal and weigh the same as non-ICSI controls. Confounding effects of subfertility, lifestyle and genetic heterogeneity are reduced to a minimum in the hybrid inbred mouse model compared to human patients. Confounding effects of polyspermy and parthenogenetic activation are prevented by ICSI. There is more to ART than ICSI, IVC and ET, and only a small number of ART media were examined. Preimplantation effects of ART culture media can effectively be modeled in the mouse because the in vitro environment is suboptimal for both mouse and human. However, in the optimal uterine environment of their own species, mouse and human ART blastocysts may be supported and selected for differently, depending on species-specific features such as the kinetics of implantation and the endometrium.
2016-08-25 | GSE50193 | GEO
Project description:Two major factors contributing to reduced fertility is use of exogenous hormones and old age. We use mouse model to study transcriptional and cell-cell communication changes upon superovulation and ageing in female reproductive cells - oocytes (OC) - and somatic cells - granulosa (GC) - surrounding them. In this experiment, we are testing how different GC transcriptional profiles correlate with embryo quality. 3M old C57BL6/J mice were naturally or superovulated and COCs singularized as described above. Granulosa cells were immediately flash frozen and matched oocytes were divided into individual drops of CARD media under mineral oil for fertilization. The oocytes were incubated in 37oC, 5% CO2 incubator for 30-40 min prior to fertilization. Frozen Mus musculus (C57BL6/J) male sperm straws were thawed and capacitated in Fertiup media for 30 min. Fertilization was achieved by combining 2.5 µl of activated sperm with 20 µl CARD media drop containing a single oocyte and incubated at 37oC, 5% CO2 for 3 hours. Then, oocytes were individually washed in Human Tubal Fluid (HTF) media drops and transferred to 20 µl HFT drops for overnight culture. After 24 hours, fertilization rate was evaluated by 2-cell stage and embryos transferred to G1+ media for further development. Early morula stage embryos were continuously collected based on observation 62-64 h post-fertilization. For late blastocysts, fertilization media was additionally changed to G2+ after 48h post- fertilization and blastocysts collected at 108h post-fertilization. For collection, the embryos were washed in DPBS and flash frozen. Granulosa cells and embryo samples were further processed by Smart-seq2, the cDNA amplification PCR cycle number for embryos was 16-17.
2024-09-22 | E-MTAB-13480 | biostudies-arrayexpress
Project description:Background: In vitro culture of preimplantation mouse embryos is associated with changes in gene expression. It is however not known if the method of fertilization affects the global pattern of gene expression. Method: We compared gene expression and development of mouse blastocysts produced by intra-cytoplasmic sperm injection and cultured in Whitten’s medium (ICSIWM) or KSOM medium with amino acids (ICSIKSOMaa) with control blastocysts flushed out of the uterus on post coital day 3.5 (In vivo). Global pattern of gene expression was assessed using the Affymetrix 430 2.0 chip. In addition we compared gene expression in embryos generated in IVF or ICSI using WM. Results: Blastocysts resulting from ICSI fertilization have a reduction in the number of trophoblastic and inner cell mass cells compared to in vivo generated embryos. Approximately 1000 genes are different between ICSI blastocyst and in vivo blastocysts; proliferation, apoptosis and morphogenetic pathways are the most common pathways altered after in vitro culture. Unexpectedly, only 41 genes were statistically different between embryo cultured in suboptimal conditions (WM) or optimal conditions (KSOMaa). Conclusion: The method of fertilization plays a more important role in shaping the transcriptome of the developing mouse embryo than the culture media used.
2010-11-10 | GSE23009 | GEO
Project description:Background: In vitro culture of preimplantation mouse embryos is associated with changes in gene expression. It is however not known if the method of fertilization affects the global pattern of gene expression. Method: We compared gene expression and development of mouse blastocysts produced by intra-cytoplasmic sperm injection and cultured in Whitten’s medium (ICSIWM) or KSOM medium with amino acids (ICSIKSOMaa) with control blastocysts flushed out of the uterus on post coital day 3.5 (In vivo). Global pattern of gene expression was assessed using the Affymetrix 430 2.0 chip. In addition we compared gene expression in embryos generated in IVF or ICSI using WM. Results: Blastocysts resulting from ICSI fertilization have a reduction in the number of trophoblastic and inner cell mass cells compared to in vivo generated embryos. Approximately 1000 genes are different between ICSI blastocyst and in vivo blastocysts; proliferation, apoptosis and morphogenetic pathways are the most common pathways altered after in vitro culture. Unexpectedly, only 41 genes were statistically different between embryo cultured in suboptimal conditions (WM) or optimal conditions (KSOMaa). Conclusion: The method of fertilization plays a more important role in shaping the transcriptome of the developing mouse embryo than the culture media used. 16 samples were used in 4 treatment groups (4 replicate samples per treatment)
2010-11-10 | E-GEOD-23009 | biostudies-arrayexpress
Project description:With the increasing use of Assisted Reproductive Technologies (ART) for treatment of human infertility, there is an increasing requirement for embryo culture conditions that perform as similar to nature as possible. How good the match, however, cannot be tested experimentally in human. We solved the central question of how well ART culture protocols prepare embryos for postimplantation development, under the provisions of the 'mouse embryo assay' (MEA). Our side-by-side comparison of 8 conditions [i.e., 3 culture conditions (KSOM, HTF and ISDM1) plus the in vivo system in two different mouse strains (B6 and CD1)] shows that mouse embryos cultured under ART conditions are differentially primed for postimplantation development, and that certain ART protocols outperform the oviduct. The distinct performances of blastocysts formed in ART vs. oviduct do not correlate with any significant transcriptome changes, whereas protein analysis by immunoconfocal microscopy reveals differences in the allocation of embryonic cells to the three germ layers of blastocysts. We conclude that in vitro technology is not always a defective copy of nature, and that the choice of ART protocol primes the embryos for subsequent development. 22 samples were analyzed. B6KSOM: Mouse B6 background, E3.5 blastocysts in KSOM medium, 3 biological rep B6HTF: Mouse B6 background, E3.5 blastocysts in HTF medium, 3 biological rep B6ISM1: Mouse B6 background, E3.5 blastocysts in ISM1 medium, 3 biological rep B6vivo: Mouse B6 background, E3.5 blastocysts in vivo, 3 biological rep CD1KSOM: Mouse CD1 background, E3.5 blastocysts in KSOM medium, 1 biological rep CD1HTF: Mouse CD1 background, E3.5 blastocysts in HTF medium, 3 biological rep CD1ISM1: Mouse CD1 background, E3.5 blastocysts in ISM1 medium, 3 biological rep CD1vivo: Mouse CD1 background, E3.5 blastocysts in vivo, 3 biological rep
2012-05-02 | E-GEOD-31414 | biostudies-arrayexpress
Project description:Our data provided a genome-wide DNA methylation landscape of human early development embryos, including human MII oocytes, sperm, zygotes, 2-cell to 8-cell embryos, morula, blastocyst and postimplantation embryos at single base resolution. In total, 44 samples including biological and technical replicates, from 12 different human embryo development stages were analyzed, including two metaphase II oocytes, two zygotes, three first polar bodies, two second polar bodies, four sperm samples, two 2-cell-stage embryos, two 4-cell-stage embryos, three 8-cell-stage embryos, three morulae, three inner cell masses (ICMs) and three trophectoderms (TEs) seperated from late blastocysts, and three post-implantation embryos. In addition, 12 different human embryo development stages were analyzed, including metaphase II oocytes, zygotes, first polar bodies, second polar bodies, sperm samples, 2-cell-stage embryos, 4-cell-stage embryos, 8-cell-stage embryos, morulae, inner cell masses (ICMs) and trophectoderms (TEs) seperated from late blastocysts, and post-implantation embryos.
2014-07-22 | E-GEOD-49828 | biostudies-arrayexpress
Project description:Assisted reproductive technologies (ART) account for 1-6% of live births in developed countries. While most children conceived using ART are healthy, increases in birth and genomic imprinting defects have been reported; such abnormal outcomes have been attributed to underlying parental infertility and/or the ART used. Here, we assessed whether paternal genetic and lifestyle factors, that are associated with male infertility and affect the sperm epigenome, can influence ART outcomes. We examined how paternal factors, Dnmt3L haploinsufficiency and/or diet-induced obesity, in combination with ART (superovulation, in vitro fertilization, embryo culture and embryo transfer), could adversely influence embryo development and DNA methylation patterning in mice. While male mice fed high-fat diets (HFD) gained weight and showed perturbed metabolic health, their sperm DNA methylation was minimally affected by the diet. In contrast, Dnmt3L haploinsufficiency induced a marked loss of DNA methylation in sperm; notably, regions affected were associated with neurodevelopmental pathways and enriched in young retrotransposons, sequences that can have functional consequences in the next generation. Following ART, placental imprinted gene methylation and growth parameters were impacted by one or both paternal factors. For the embryos conceived by natural conception, the abnormality rates were similar for WT and Dnmt3L+/- fathers. In contrast, paternal Dnmt3L+/- genotype, as compared to WT fathers, resulted in a 3-fold increase in the incidence of morphological abnormalities in embryos generated by ART. Together, the results indicate that embryonic morphological and epigenetic defects associated with ART may be exacerbated in offspring conceived by fathers with sperm epimutations.
2023-12-05 | GSE234855 | GEO
Project description:Competent oocytes can be discriminated by BCB staining. Positive stained oocytes are considered more competent than BCB negative oocyte, and injection of BCB+ oocyte extracted mitochondria into BCB negative oocytse can increase fertilisation and blastocyst rate. Here we have analysed the impact of mitochondrial supplementation on subsequent blastocyst transcriptome using agilent one color microarray that is specificly design to study the porcine embryo preimplantation period. Blastocysts were produced by intra cytoplasmic sperm injection (ICSI) from BCB positive and BCB negative oocytes as well as BCB negative oocytes supplemented with mitochondrial extract during ICSI (mICSI), and 3-4 single blatocyst transcriptomes were analysed for each group. 3-4 single blastocysts were analysed at the RNA level after whole transcriptome amplification, and level of gene expression was compared between groups, i.e ICSI BCB+ blastocysts (4), ICSI BCB- blastocysts (3) and mICSI BCB- blastocysts (4).
2016-09-01 | E-GEOD-65310 | biostudies-arrayexpress
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 Tyrode’s, 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 Tyrode’s treatment, blastomere aspiration), performed individually or in combination, on global patterns of gene expression in the resulting blastocysts. Keywords: embryo biopsy, preimplantation genetic diagnosis, blastocyst
2008-04-22 | GSE11206 | GEO