Denise Laskowski

Insulin exposure during in vitro bovine oocyte maturation changes blastocyst gene expression and developmental potential.

Metabolic imbalance impairs fertility, because changes in concentrations of metabolites and hormones in the blood and follicular fluid create an unfavourable environment for early embryonic development. Insulin is a key metabolic hormone known for its effects on fertility: insulin concentrations are increased during energy balance disturbances in diabetes or metabolic syndrome. Still, insulin is frequently used at supraphysiological concentrations for embryo in vitro culture with unknown consequences for the developmental potential of the offspring. In the present study we investigated the effects of insulin exposure during in vitro bovine oocyte maturation on developmental rates, embryo quality and gene expression. Supplementation of the maturation media with insulin at 10 or 0.1 µg mL-1 decreased blastocyst rates compared with an insulin-free control (19.8 ± 1.3% and 20.4 ± 1.3% vs 23.8 ± 1.3%, respectively; P < 0.05) and led to increased cell numbers (nearly 10% more cells on Day 8 compared with control; P < 0.05). Transcriptome analysis revealed significant upregulation of genes involved in lipid metabolism, nuclear factor (erythroid-derived 2)-like 2 (NRF2) stress response and cell differentiation, validated by quantitative polymerase chain reaction. To conclude, the results of the present study demonstrate that insulin exposure during in vitro oocyte maturation has a lasting effect on the embryo until the blastocyst stage, with a potential negative effect in the form of specific gene expression perturbations.

Hyperinsulinemia during in vitro oocyte maturation changes gene expression of insulin signaling in bovine Day-8 embryos

The obesity-metabolic-syndrome-complex is a growing problem in humans as in other species and known to be associated with decreased fertility. Insulin concentrations differ from physiological levels during periods of metabolic imbalance. The dairy cow suffers from metabolic disturbances due to a stressed metabolism caused by high milk-production.

A comparison study of insulin concentrations in follicular fluid, serum and in vitro-production of bovine embryos – risks of generating unfavourable metabolic conditions during early development

Insulin has frequently been used as a stimulatory factor in routine in vitro embryo production (IVP) and is added in supra-physiological concentrations to different media. Meanwhile, insulin as a key metabolic hormone is elevated in patients with metabolic syndrome or diabetes, syndromes known to impair fertility.

Lipid profile of bovine blastocysts exposed to insulin during in vitro oocyte maturation

Insulin is a key hormone with important functions in energy metabolism and is involved in the regulation of reproduction. Hyperinsulinaemia is known to impair fertility (for example, in obese mothers); therefore, we aimed to investigate the impact of elevated insulin concentrations during the sensitive period of oocyte maturation on gene expression and lipid profiles of the bovine Day-8 embryo. Two different insulin concentrations were used during in vitro oocyte maturation (INS10=10µgmL-1 and INS0.1=0.1µgmL-1) in order to observe possible dose-dependent effects or thresholds for hyperinsulinaemia in vitro. By investigating gene expression patterns by an mRNA microarray in combination with lipid profile analysis by desorption electrospray ionisation-mass spectrometry (DESI-MS) of embryos derived from insulin-treated oocytes, we gained further insights regarding molecular responses of embryos to insulin provocation during the first days of development. Lipid metabolism appeared to be influenced on multiple levels according to gene expression results but the profiles collected in positive-ion mode by DESI-MS (showing mostly ubiquinone, cholesteryl esters and triacylglycerols) did not differ significantly from controls. There are parallels in follicular development of ruminants and humans that make this bovine model relevant for comparative research on early human embryonic development during hyperinsulinaemia.

DNA methylation pattern of bovine blastocysts associated with hyperinsulinemia in vitro: LASKOWSKI et al.

Insulin functions as a regulator of metabolism and plays an important role in reproduction. Hyperinsulinemia is often observed in patients with obesity and diabetes type 2 and is known to impair fertility, but the underlying molecular mechanisms are only partly understood. Metabolic programming through epigenetic mechanisms such as DNA methylation during embryonic development can lead to health implications for the offspring later in life. Our aim was to study the potential effect of hyperinsulinemia on gene expression and DNA methylation of embryos by adding insulin (0.1 µg/ml = INS0.1 or 10 µg/ml = INS10) during in vitro oocyte maturation by using the EmbryoGENE DNA methylation array for a study of the bovine epigenome. Our results showed significant differences between blastocysts originating from insulin‐treated oocytes compared with untreated control blastocysts. In total, 13,658 and 12,418 probes were differentially methylated (DM) in INS0.1 and INS10, respectively, with an overlap of 3,233 probes in the DM regions (DMR) for both insulin groups. Genes related to pathways such as lipid metabolism, growth and proliferation, mitochondrial function, and oxidative stress responses were influenced at both the epigenetic and transcriptomic levels. In addition, imprinted genes and genes with functions in the epigenetic machinery were among the DMRs. This study identified DMRs correlated to differential expression of genes involved in metabolic regulation and should help to improve our knowledge of the underlying molecular mechanisms of metabolic imbalance.