INFLUENCE OF GLUCOSE AND OXYGEN ON HUMAN EMBRYOS AND EMBRYONIC STEM CELLS

  • Maribel Montufar Martinez

Student thesis: Phd

Abstract

Despite the significant improvements in Assisted Reproductive Technologies (ART) there is an extensive area that remains unknown in regards to the effect of embryo culture on the long-term health of the offspring. The preimplantation embryo has specific requirements depending on the developmental stage; however, it remains unclear how the different compositions of commercial culture media can affect embryo development. The in vitro factors that most influence embryo health include cryopreservation methods, culture media components, and oxygen levels. Several studies have reported alterations in birth weight, and a higher risk of congenital anomalies when comparing between assisted and naturally conceived babies. Others have reported effects on epigenetic regulation, gene expression and imprinting, suggesting that embryo culture medium may have a potential impact on the further development of the newborn. Nevertheless, the most commonly reported alterations in offspring health outcomes include foetal growth, birth weight, childhood growth and long-term diseases such as metabolic, cardiovascular, and neurological. Due to the number of reports indicating the possible effect of the environment on embryo development, it is crucial to be aware of the mechanisms involved in order to prevent embryo alterations and long-term diseases. Therefore, the objective of this thesis was to determine whether specific components of embryo culture such as glucose and oxygen had any effect on the epigenetic patterns, at transcript levels, as well as on mitochondrial function. The analysis of transcripts was assessed to determine differential expression of genes related to pluripotency, mitochondrial function, apoptosis, oxidative stress, DNA methylation, glucose transporters, among others. The epigenome analysis (Bisulfite Sequencing) was carried out to determine differential DNA methylation levels on human blastocyst cultured in high or low glucose (0.9mM or 3.5mM). Finally, mitochondrial function was evaluated through the measurement of mitochondrial membrane potential (MitoProbe™ JC-1 assay) and through the analysis of mitochondrial respiration (Seahorse XF Cell Mito Stress Test) by directly measuring the oxygen consumption rate (OCR). In order to accomplish the objectives of this project and to obtain the most relevant clinical data, this research was conducted using preimplantation human embryos at blastocyst stage (day 6) and a model of human embryonic stem cells (HESCs). HESCs are pluripotent stem cells derived from the inner cell mass (ICM) of human blastocysts. The combination of the analysis on human embryos and HESCs allowed the validation of initial protocols, set up experimental conditions and the evaluation of different culture conditions. Firstly, I evaluated combined oxygen levels (5% and 20%) and glucose concentration (5mM and 17.5mM) on HESCs to determine their effect on transcript levels and mitochondrial function. Findings revealed altered transcript levels and mitochondrial function when cells were exposed to different concentrations of either oxygen or glucose, as well as to the combinations of both. However, the most significant effects were observed with changes in glucose concentration. Secondly, oxygen levels (5% and 20%), glucose concentration (0.9mM and 3.5mM) and the effect of cryoprotectants were evaluated on human blastocysts to determine their effect on transcript levels, mitochondrial function and DNA methylation. Findings revealed altered gene expression due to changes in oxygen but more significantly due to changes in glucose. Also, the analysis of DNA methylation showed significant differences as a result of glucose concentration. Furthermore, for the evaluation of mitochondrial function, the analysis of membrane potential was only carried out on blastocysts, treated/untreated with cryoprotectant. On the other hand, the measurement of OCR for mitochondrial respiration was only possible to determine the basel
Date of Award31 Dec 2021
Original languageEnglish
Awarding Institution
  • The University of Manchester
SupervisorSusan Kimber (Supervisor) & Daniel Brison (Supervisor)

Keywords

  • EMBRYO DEVELOPMENT
  • OCR
  • MITOCHONDRIA
  • GENE EXPRESSION
  • DNA METHYLATION
  • HESC
  • HUMAN EMBRYO
  • OXYGEN
  • GLUCOSE
  • DOHAD
  • ART

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