Rescue Potential of Supportive Embryo Culture Conditions on Bovine Embryos Derived from Metabolically Compromised Oocytes

Abstract

Elevated non-esterified fatty acid (NEFA), predominantly palmitic acid (PA), concentrations in blood and follicular fluid are a common feature in maternal metabolic disorders such as obesity. This has a direct negative impact on oocyte developmental competence and the resulting blastocyst quality. We use NEFA-exposure during bovine oocyte in vitro maturation (IVM) as a model to mimic oocyte maturation under maternal metabolic stress conditions. However, the impact of supportive embryo culture conditions on these metabolically compromised zygotes are not known yet. We investigated if the addition of anti-apoptotic, antioxidative and mitogenic factors (namely, Insulin-Transferrin-Selenium (ITS) or serum) to embryo culture media would rescue development and important embryo quality parameters (cell proliferation, apoptosis, cellular metabolism and gene expression patterns) of bovine embryos derived from high PA- or high NEFA-exposed oocytes when compared to controls (exposed to basal NEFA concentrations). ITS supplementation during in vitro culture of PA-exposed oocytes supported the development of lower quality embryos during earlier development. However, surviving blastocysts were of inferior quality. In contrast, addition of serum to the culture medium did not improve developmental competence of PA-exposed oocytes. Furthermore, surviving embryos displayed higher apoptotic cell indices and an aberrant cellular metabolism. We conclude that some supportive embryo culture supplements like ITS and serum may increase IVF success rates of metabolically compromised oocytes but this may increase the risk of reduced embryo quality and may thus have other long-term consequences.

Keywords: fertility; free fatty acids; in vitro culture supplementation.

Figure 1

Mean total cell count (A) or apoptotic cell index (B) of Day 8 blastocysts resulting from oocytes exposed to BASAL (physiological NEFA concentrations), PA and HCOMBI NEFAs during maturation and cultured in the presence or absence of bovine serum albumin (BSA), ITS or serum. Data are presented as mean percentage ± SEM. Vertical superscripts (a, b) indicate significant differences between maturation conditions within the same IVC condition (p < 0.05). Horizontal superscripts (xindicate significant difference between culture conditions within the same in vitro maturation (IVM) condition (p < 0.05). Values labeled with “§” tend to be different from each other at p < 0.1 and > 0.05.

Figure 2

The effect of BSA, ITS or serum supplementation during IVC on (A) average glucose consumption, (B) lactate production, (C) lactate:(2 glucose) ratio and (D) pyruvate consumption (pmol/embryo/hour) of blastocysts resulting from BASAL-, PA- or HCOMBI-exposed oocytes. Data are presented as mean percentage ± SEM. Vertical superscripts (a, b) indicate significant differences between maturation conditions within the same IVC condition (p < 0.05). Horizontal superscripts (x, y, z) indicate significant difference between culture conditions within the same IVM condition (p < 0.05). Values labeled with “§” tend to be different from each other at p < 0.1 and > 0.05.

Figure 3

mRNA expression (fold change) of genes related to cellular stress of blastocysts resulting from BASAL-, PA- or HCOMBI-exposed oocytes cultured in serum, BSA and/or ITS. Data are presented as fold change ± SEM. Vertical superscripts (a, b) indicate significant differences between maturation conditions within the same IVC condition (p < 0.05). Horizontal superscripts (x, y) indicate significant difference between culture conditions within the same IVM condition (p < 0.05). Values labeled with “$” tend to be different from each other at p < 0.1 and > 0.05.

Figure 4

Overview of the nine treatment groups: BASAL-BSA, BASAL-ITS, BASAL-serum, PA-BSA, PA-ITS, PA-serum, HCOMBI-BSA, HCOMBI-ITS, HCOMBI-serum.

Figure 5

Overview of the experimental design.

Figure 6

Grades of cumulus cell expansion in cumulus-oocyte complexes (COCs) after 24 h incubation in maturation media. (a) unexpanded COC (score 0), (b) poorly expanded COC (score 1), (c) partially expanded COC (score 2) and (d) and full y expanded COC (score 3). Scale bar = 100 µm.