Effect of embryo in vitro culture on mESC transcriptome.
Ontology highlight
ABSTRACT: Transcriptome analysis of mouse embryonic stem cell lines derived from embryos cultured in optimal and suboptimal conditions compared to cell lines derived from control embryos. The use of assisted reproductive technologies (ART) such as in vitro fertilization (IVF) has resulted in the birth of more than 5 million children. While children conceived by these technologies are generally healthy, there is conflicting evidence suggesting an increase in adult-onset complications like glucose intolerance and high blood pressure in IVF children. Animal models indicate similar potential risks. It remains unclear what molecular mechanisms may be operating during in vitro culture to predispose the embryo to these diseases. One of the limitations faced by investigators is the paucity of the material in the preimplantation embryo to test for molecular analysis. To address this problem, we generated mouse embryonic stem cells (mESC) from blastocysts conceived after natural mating (mESCFB) or after IVF, using optimal (KSOM + 5% O2; mESCKAA) and suboptimal (Whitten’s Medium, + 20% O2, mESCWM) conditions.
Project description:Transcriptome analysis of mouse embryonic stem cell lines derived from embryos cultured in optimal and suboptimal conditions compared to cell lines derived from control embryos. The use of assisted reproductive technologies (ART) such as in vitro fertilization (IVF) has resulted in the birth of more than 5 million children. While children conceived by these technologies are generally healthy, there is conflicting evidence suggesting an increase in adult-onset complications like glucose intolerance and high blood pressure in IVF children. Animal models indicate similar potential risks. It remains unclear what molecular mechanisms may be operating during in vitro culture to predispose the embryo to these diseases. One of the limitations faced by investigators is the paucity of the material in the preimplantation embryo to test for molecular analysis. To address this problem, we generated mouse embryonic stem cells (mESC) from blastocysts conceived after natural mating (mESCFB) or after IVF, using optimal (KSOM + 5% O2; mESCKAA) and suboptimal (Whitten’s Medium, + 20% O2, mESCWM) conditions. We analyzed three female cell lines per group for a total of nine mouse embryonic stem cells on Affymetrix MoGene 1.0 ST Arrays.
Project description:Although chromosomal instability (CIN) is a common phenomenon in cleavage-stage embryogenesis following in vitro fertilization (IVF), its rate in naturally conceived human embryos is unknown. CIN leads to mosaic embryos that contain a combination of genetically normal and abnormal cells, and is significantly higher in in vitro-produced preimplantation embryos as compared to in vivo-conceived preimplantation embryos. Even though embryos with CIN-derived complex aneuploidies may arrest between the cleavage and blastocyst stages of embryogenesis, a high number of embryos containing abnormal cells can pass this strong selection barrier. However, neither the prevalence nor extent of CIN during prenatal development and at birth, following IVF treatment, is well understood. Here we profiled the genomic landscape of fetal and placental tissues postpartum from both IVF- and naturally conceived children, to investigate the prevalence and persistence of large genetic aberrations that probably arose from IVF-related CIN. We demonstrate that CIN is not preserved at later stages of prenatal development, and that de novo numerical aberrations or large structural DNA imbalances occur at similar rates in IVF- and naturally conceived live-born neonates. Our findings affirm that human IVF treatment has no detrimental effect on the chromosomal constitution of fetal and placental lineages.
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)
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.
Project description:Our study reported an increased risk of metabolic disorders in in-vitro Fertilization and Frozen-thawed Embryo Transfer (IVF-FET) conceived offspring compared with those born after IVF-ET. To explore the underlying mechanisms of the aberrant metabolism in male offspring conceived by IVF and FET compared with natural conceived (NC), we performed the RNA-sequencing of livers from three groups. We performed GSEA analysis. The results suggested more severe alterations in the expression of genes involved in insulin resistance pathway in the FET-chow group than that in the IVF-chow group.
Project description:Although in vitro fertilization (IVF) is associated with adverse perinatal outcomes, an increasing concern is the long-term health implications. We augmented our IVF mouse model to longitudinally investigate cardiometabolic outcomes in offspring from optimal neonatal litter sizes. We found that IVF-conceived females had higher body weight and cholesterol levels compared to naturally-conceived females, whereas IVF-conceived males had higher levels of triglycerides and insulin, and increased body fat composition. Through transcriptomics and proteomics of adult liver, we identified sexually-dimorphic dysregulation of the sterol regulatory element binding protein (SREBP) pathways that contribute to the sex-specfic phenotypes. We also found that global loss of DNA methylation in placenta was linked to higher cholesterol levels in IVF-conceived females. Our findings indicate that IVF procedures have long-lasting sex-specific effects on metabolic health of offspring and lay the foundation to utilize the placenta as a predictor of long-term outcomes.
Project description:Children conceived using Assisted Reproductive Technologies (ART) have a higher incidence of growth and birth defects, attributable in part to epigenetic perturbations. Both ART and germline defects associated with parental infertility could interfere with epigenetic reprogramming events in germ cells or early embryos. Mouse models indicate that the placenta is more susceptible to the induction of epigenetic abnormalities than the embryo, and thus the placental methylome may provide a sensitive indicator of ‘at risk’ conceptuses. Our goal was to use genome-wide profiling to examine the extent of epigenetic abnormalities in matched placentas from an ART/infertility group and control singleton pregnancies (n=44/group) from a human prospective longitudinal birth cohort, the 3D Study. Principal component analysis revealed a group of ART outliers. The ART outlier group was enriched for females and a subset of placentas showing loss of methylation of several imprinted genes including GNAS, SGCE, KCNQT1OT1 and BLCAP/NNAT. Within the ART group, placentas from pregnancies conceived with IVF/ICSI showed distinct epigenetic profiles as compared to those conceived with less invasive procedures (ovulation induction, intrauterine insemination). Male factor infertility and paternal age further differentiated the IVF/ICSI group, suggesting an interaction of infertility and techniques in perturbing the placental epigenome. Together, the results suggest that the human placenta is sensitive to the induction of epigenetic defects by ART and/or infertility, and we stress the importance of considering both sex and paternal factors and that some but not all ART conceptuses will be susceptible.
Project description:Since the first human conceived through in vitro fertilisation in 1978, over 8 million babies have been born by assisted reproductive technologies (ART). Although most ART babies and children seem healthy, in recent years, several human and animal model studies have evidenced a potential impact of ART on long-term development. However, the long-term follow-up data in this field is still limited. Till now, studies are mainly focused on techniques such as in vitro fertilisation or in vitro culture, being the information from gametes/embryos cryopreservation field practically missing. Herein, we have developed an animal model to determine whether vitrified-thawed embryo transfer procedure has long-term consequences over the offspring. The birth weight, growth performance and adult body weight of the rabbits derived from vitrified-thawed embryos was compared with that of the naturally-conceived animals. In adulthood, the liver, heart, kidneys, spleen, lungs, gonads and adrenal glands of the males were weighed and compared. Moreover, some haematological and biochemical parameters were assessed on peripheral blood. Besides, some liver samples were obtained to perform a comparative proteomic study. The embryo vitrification-transfer process modified the birth weight and the growth pattern of the offspring, reducing the growth performance in a sex-specific manner. In adulthood, animals derived from vitrified embryos showed a significantly lower body, liver and heart weight. Molecular analyses revealed that vitrified-thawed embryo transfer procedure triggers concordant reprogramming of the liver proteome. The most relevant metabolic alteration denoted by the protein profile was that related to the oxidative phosphorylation, suggesting an impaired oxidative metabolism in the mitochondria. Furthermore, hints of dysregulation in the zinc and lipid metabolism were identified. These results evidence long-term consequences in the offspring derived from cryopreserved-transferred embryos and represented an evident example of the phenotypic plasticity exhibited by the mammalian embryo.
Project description:Since the first human conceived through in vitro fertilisation in 1978, over 8 million babies have been born by assisted reproductive technologies (ART). Although most ART babies and children seem healthy, in recent years, several human and animal model studies have evidenced a potential impact of ART on long-term development. However, the long-term follow-up data in this field is still limited. Till now, studies are mainly focused on techniques such as in vitro fertilisation or in vitro culture, being the information from gametes/embryos cryopreservation field practically missing. Herein, we have developed an animal model to determine whether vitrified-thawed embryo transfer procedure has long-term consequences over the offspring. The birth weight, growth performance and adult body weight of the rabbits derived from vitrified-thawed embryos was compared with that of the naturally-conceived animals. In adulthood, the liver, heart, kidneys, spleen, lungs, gonads and adrenal glands of the males were weighed and compared. Moreover, some haematological and biochemical parameters were assessed on peripheral blood. Besides, some liver samples were obtained to perform a comparative proteomic study. The embryo vitrification-transfer process modified the birth weight and the growth pattern of the offspring, reducing the growth performance in a sex-specific manner. In adulthood, animals derived from vitrified embryos showed a significantly lower body, liver and heart weight. Molecular analyses revealed that vitrified-thawed embryo transfer procedure triggers concordant reprogramming of the liver proteome. The most relevant metabolic alteration denoted by the protein profile was that related to the oxidative phosphorylation, suggesting an impaired oxidative metabolism in the mitochondria. Furthermore, hints of dysregulation in the zinc and lipid metabolism were identified. These results evidence long-term consequences in the offspring derived from cryopreserved-transferred embryos and represented an evident example of the phenotypic plasticity exhibited by the mammalian embryo.
Project description:Chromosomal instability (CIN) occurs at high frequency during early in vitro embryogenesis and is known to be associated with early embryonic loss in humans. The chromosomal stability of in vivo-conceived cleavage stage embryos largely remains unknown. Here, we applied haplotyping and copy number profiling to investigate genomic architecture of 171 single bovine blastomeres and to compare the nature and frequency of CIN between in vivo embryos, in vitro embryos produced from ovum pick up with ovarian stimulation (OPU-IVF), and in vitro produced embryos from in vitro matured oocytes without ovarian stimulation (IVM-IVF). Our data shows that CIN is significantly lower in in vivo conceived cleavage stage embryos when compared to in vitro cultured embryos, as genomic stability of single blastomeres in both IVF embryos was severely compromised (P<0.0001)