Project description:A multitude of factors encompassing assisted reproductive technologies present compounding stresses and cause synergistic damage to cell function and embryo development in vitro. We have previously reported that antioxidant supplementation of culture and vitrification media ameliorate some of the detrimental effects of in vitro culture and cryopreservation. The aim of this study was to identify the molecular mechanisms by which antioxidants improve embryo and fetal development by comparing mRNA profiles of E14.5 post-implantation fetal (liver) and placental tissue following supplementation of either in vitro culture or vitrificiation media with or without antioxidants. In vitro samples are compared with tissues derived from in vivo derived fetuses and placentae.
Project description:Ketogenic diet consumption elevates circulating levels of the ketones β-hydroxybutyrate (βOHB) and acetoacetate (AcAc). In vitro ketone exposure perturbs preimplantation mouse embryo viability and female-specific fetal development post-transfer. Here we assessed whether transient exposure of preimplantation embryos to ketones impacts post-implantation fetal and placental gene expression. Blastocysts cultured in vitro with or without 2 mmol/L βOHB alone (‘βOHB’) or combined with 0.8 mmol/L AcAc (‘Keto’) underwent embryo transfer. Transcriptional profiles of sexed E14.5 placentae, liver, and brain were examined via RNA-Seq and DAVID functional analysis, revealing a sexually dimorphic transcriptomic response. βOHB and Keto exposure both downregulated genes related to oxidative phosphorylation specifically in female liver. βOHB downregulated female placental steroid biosynthetic processes, while Keto treatment upregulated genes relevant to blood vessel formation and cell migration in male placentae. Brain transcriptomes were minimally affected. X-linked genes and chromatin modifiers were identified as differentially expressed, alluding to a sex-specific regulatory mechanism. Transient preimplantation ketone exposure therefore perturbs sex-specific fetal liver and placental gene expression demonstrating a developmental programming effect that warrants future investigation of male and female offspring postnatal metabolic health.
Project description:In vitro produced (IVP) bovine embryos are used to enhance herd genetics and support advanced reproductive technologies, however, less than 40% survive the first month of gestation after transfer to the surrogate. While the biochemical consequences of in vitro culture on the embryo before transfer have been well documented, less is known after transfer when the embryo interacts with the endometrium and pregnancy failure occurs. To investigate this, we utilized a Day 16 trophectoderm-endometrial mono-culture and co-culture system, integrating transcriptomic and proteomic analyses, to compare crosstalk between the IVP or in vivo derived (IVD) bovine conceptus and endometrium. We found that the IVP conceptus transcriptome (differentially expressed genes; DEG), diverged from the IVD conceptus but only after co-culture with endometrium (377 DEG; FDR < 0.05). Further, of the IVP conceptus DEG, 81 genes (21%) were associated with abnormal embryonic or fetal development. Significant biological processes associated with abundant endometrial DEG induced by IVP conceptuses were related to GTPase activity and the inflammatory response (FDR < 0.01), the latter of which may be associated with increased MHC class II expression, which was specific to IVP conceptuses. Proteomic analysis of the culture media identified 1,031 and 604 differentially abundant proteins (FDR < 0.05) associated with the IVD and IVP conceptus-endometrial co-cultures, respectively, compared to endometrium alone, indicating the proteomic environment surrounding the IVP conceptus may be suboptimal. Collectively, compared to the IVD conceptus, it appears the endometrium drives and responds to divergence of IVP conceptus biochemistry, likely contributing to pregnancy failure.
Project description:We used microarrays to detail the global gene expression in human placental tissues in first trimester from patients subject to in vitro fertilization and embryo transfer and normal pregnancy
Project description:Assisted Reproductive Technologies (ART) employ gamete/embryo handling and culture in vitro to produce offspring. ART pregnancies have increased risk of low birth weight, abnormal placentation, pregnancy complications, and imprinting disorders. We and others have previously shown that embryo culture induces low birth weight, abnormal placental morphology, and lower levels of DNA methylation in placentas in a mouse model of ART. We hypothesized that these adverse effects are linked to a subtle disruption of specific biological processes during preimplantation development. To test this hypothesis, we performed embryo culture for several discrete periods of preimplantation development and assessed fetal and placental outcomes at term. We observed a reduction in fetal:placental ratio in two distinct windows of preimplantation embryo development, while placental morphological abnormalities and reduced imprinting control region methylation were associated with culture prior to the morula stage. We also provide evidence that extended culture to the blastocyst stage induces additional placental DNA methylation changes compared to embryos transferred at the morula stage, and that female concepti exhibited a higher loss of DNA methylation than males. Altogether, this study identifies specific developmental windows of susceptibility and potential targets for embryo culture optimization.
Project description:Underdeveloped lungs are the primary cause of death in premature infants, however, little is known about stem and progenitor cell maintenance during human lung development. In this study, we have identified that FGF7, Retinoic Acid and CHIR-99021, a small molecule that inhibits GSK3 to activate Wnt signaling, support in vitro maintenance of primary human fetal lung bud tip progenitor cells in a progenitor state. Furthermore, these factors are sufficient to derive a population of human bud tip-like progenitor cells in 3D organoid structures from human pluripotent stem cells (hPSC). Functional studies showed that hPSC-derived bud tip progenitor organoids do not contain any mesenchymal cell types, maintain multilineage potential in vitro and are able to engraft into the airways of injured mice and respond to systemic factors. We performed RNA-sequencing to assess the degree of similarity in global gene expression profiles between the full human fetal lung (59-127 days gestation), isolated human fetal bud tip progenitors, organoids grown from primary fetal bud tip progenitors, and hPSC-derived bud tip organoids. Results showed that hPSC-derived organoids have molecular profiles similar to organoids generated from primary human fetal lung tissue. Gene expression differences between hPSC-derived bud tip organoids and fetal progenitor organoids may be related to the presence of contaminating mesenchymal cells in primary cultures. hPSC-derived bud tip organoids are generated from a well-defined human cell sources, offering a distinct advantage over rare primary tissue as a means to study human specific lung development, homeostasis and disease.<br>Sample Nomenclature - Description<br> -------------------------------------------------------------------------<br> Peripheral fetal lung the distal/peripheral portion of the fetal lung (i.e., distal 0.5 cm) was excised from the rest of the lung using a scalpel. This includes all components of the lung (e.g., epithelial, mesenchymal, vascular). <br>Isolated fetal bud tip the bud peripheral portion of the fetal lung was excised with a scalpel and subjected to enzymatic digestion and microdissection. The epithelium was dissected and separated from the mesenchyme, but a small amount of associated mesenchyme likely remained. <br>Fetal progenitor organoid 3D organoid structures that arose from culturing isolated fetal epithelial bud tips. <br>Foregut spheroid 3D foregut endoderm structure as described in Dye et al. (2015). Gives rise to patterned lung organoid (PLO) when grown in 3F medium. <br> Patterned lung organoid (PLO) lung organoids that were generated by differentiating hPSCs, as described throughout the manuscript. <br> Bud tip organoid organoids derived from PLOs, enriched for SOX2/SOX9 co-expressing cells, and grown/passaged in 3F medium.
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
Project description:Pluripotency, the capacity of embryo-derived stem cells to generate all tissues in the organism, can be induced in somatic cells by nuclear transfer into oocyte, fusion with embryonic stem cells, and for male germ cells by cell culture alone. Recently, murine fibroblasts have been reprogrammed directly to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4, and Myc) to yield induced Pluripotent Stem (iPS) cells. Using the same four factors, we have derived iPS cells from human embryonic stem cell-derived fibroblasts, primary human fetal cells, and diverse cells of neonatal and adult human origin. The human iPS cells manifest the colony morphology, gene expression patterns, and epigenetic characteristics of human Embryonic Stem (hES) cells, and form well-differentiated teratomas in immune-deficient mice. These data demonstrate that defined factors can reprogram human cells to pluripotency, and establish a method whereby patient-specific cells might be established in culture. Biological replicates: GSM248201 and GSM248202; GSM248205 and GSM248206; GSM248207 and GSM248208; GSM248209 and GSM248210; GSM248211 and GSM248212; GSM248213 and GSM248214. Sample descriptions: H1-OGN: ES cells expressing GFP-NEO marker under OCT4 promoter dH1f: differentiated H1-OGN fibroblasts dHcf16: differentiated H1-OGN cloned fibroblasts MRC5: fetal lung fibroblasts BJ1: neonatal fibroblasts Keywords: cellular reprogramming
Project description:whole genome sequencing data of parent blood samples. Single cell full-length RNA-seq and PBAT-Seq data of in vitro culture D6 to D14 human embryo.