Maternal diet impacts in vitro culture requirements for optimal embryo development

Abstract

To determine the effect of maternal high fat diet and exogenous lipid provision in vitro on mouse embryo development and subsequent embryo outgrowth in an extended culture system. Research study IVF zygotes from mice fed either a High Fat (HFD) or Normal Diet (ND) for 8 wk were allocated into medium with two albumin supplements; 2.5 mg/mL fatty acid free (FAF) or lipid rich (AlbuMAX (ALBMX)) BSA. In vivo produced blastocysts (VIVO) were also obtained from ND and HFD mice. Embryos were moved to fibronectin coated chambers (Ibidi) and cultured in IVC1 medium (Cell Guidance Systems) for 72 h, then media was replaced with IVC2 and embryos cultured for an additional 48 h (D8.5). Embryos (ND ALBMX, 8; ND FAF, 10; ND VIVO, 6; HFD ALBMX, 8; HFD FAF, 8; HFD VIVO, 6) were fixed and assessed for outgrowth cell number (DAPI) and lipid volume (Nile Red). Lipid volume was normalized to total actin staining. Data are reported as mean ± SEM; significance p<0.05. Body weight was not different (ND, 25.6 ± 0.4g; HFD, 27.2 ± 0.9g). Abdominal fat pad weight was greater in high fat fed females (ND, 0.75 ± 0.10g; HFD, 1.27 ± 0.20g). There was no difference in embryo production between treatments, but there tended to be a higher percentage (P=0.10) of morulae produced in HFD FAF (77% ± 5.8) than ND FAF (58% ± 4.9). There was a higher percentage of hatched blastocysts in ND FAF (19% ± 4.6) than in HFD ALBMX (4% ± 2.2) and HFD FAF (5% ± 2.5%). Embryos from HFD females cultured in a low lipid environment were more successful in forming embryo outgrowths in extended culture (100%) than HFD embryos cultured with high lipid (62%). The total cell number of embryo outgrowths was significantly higher in ND in vivo produced embryos; there were no other differences between treatments (ND VIVO, 453 ± 56; ND FAF, 219 ± 46; ND ALBMX, 202 ± 56; HFD VIVO, 177 ± 61; HFD FAF 88 ± 56; HFD ALBMX,147 ± 52). There were no differences in the total amount of endogenous lipid (in relative fluorescence units) per embryo outgrowth (ND VIVO, 2.2 ± 0.9; ND FAF, 2.0 ± 0.8; ND ALBMX, 0.65 ± 1.1; HFD VIVO, 2.4 ± 1.1; HFD FAF 1.6 ± 1.2; HFD ALBMX, 1.1 ± 1.0). Embryos from mice fed a high fat diet, regardless of lipid provision in culture media, developed slower than embryos from mice fed a normal diet in lipid free conditions, suggesting reduced embryo quality. Interestingly, in vivo produced embryos and ND embryos cultured in lipid free conditions had the highest levels of endogenous lipid in outgrowth culture, suggesting embryos from obese females and those cultured under suboptimal lipid conditions may excessively metabolize endogenous lipids leading to reduced embryo quality. Additional experimentation will investigate this hypothesis. Manipulation of lipids in culture may optimize embryo quality, particularly from obese females.