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01.12.2014 | Embryo Biology | Ausgabe 12/2014

Journal of Assisted Reproduction and Genetics 12/2014

Composition of protein supplements used for human embryo culture

Zeitschrift:
Journal of Assisted Reproduction and Genetics > Ausgabe 12/2014
Autoren:
Dean E. Morbeck, Melissa Paczkowski, Jolene R. Fredrickson, Rebecca L. Krisher, Heather S. Hoff, Nikola A. Baumann, Thomas Moyer, Dietrich Matern
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1007/​s10815-014-0349-2) contains supplementary material, which is available to authorized users.

Capsule

Protein supplements for IVF and embryo culture are highly heterogeneous and poorly defined. Some components of supplements adversely affect mouse embryo development and these effects are dependent on oxygen concentration used for culture.

Abstract

Purpose

To determine the composition of commercially available protein supplements for embryo culture media and test if differences in protein supplement composition are biologically relevant in a murine model.

Methods

Amino acid, organic acid, ion and metal content were determined for 6 protein supplements: recombinant human albumin (AlbIX), human serum albumin (HSA and Buminate), and three complex protein supplements (SSS, SPS, LGPS). To determine if differences in the composition of these supplements are biologically relevant, mouse one-cell embryos were collected and cultured for 120 hours in each protein supplement in Global media at 5 and 20 % oxygen in an EmbryoScope time-lapse incubator. The compositions of six protein supplements were analyzed for concentrations of 39 individual amino acids, organic acids, ions and elements. Blastocyst development and cell cycle timings were calculated at 96-hours of culture and the experiments were repeated in triplicate. Blastocyst gene expression was analyzed.

Results

Recombinant albumin had the fewest undefined components , the lowest concentration of elements detected, and resulted in high blastocyst development in both 5 and 20 % oxygen. Buminate, LGPS and SPS had high levels of transition metals whereas SSS had high concentrations of amino acids. Pre-compaction mouse embryo development was delayed relative to embryos in AlbIX for all supplements and blastocyst formation was reduced in Buminate, SPS and SSS.

Conclusions

The composition of protein supplements are variable, consisting of previously undescribed components. High concentrations of pro-oxidant transition metals were most notable. Blastocyst development was protein dependent and showed an interaction with oxygen concentration and pro-oxidant supplements.

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Zusatzmaterial
ESM 1 (DOC 38 kb)
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ESM 2 (DOC 39 kb)
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ESM 3 (DOC 34 kb)
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ESM 7 (DOC 35 kb)
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Supplemental Figure 1 Specific cell division timings (hours; mean±se) from the 2-cell stage to 3-, 4-, 5- and 8 cells for embryos cultured with different protein supplements. Time to the 3-cell stage relative to AlbIX was longer for SPS (p<0.01) and Buminate and SSS (p<0.001). Time to 8-cell was delayed relative to AlbIX for Buminate, SSS, SPS and LGPS (p<0.01). (PPT 201 kb)
10815_2014_349_MOESM8_ESM.ppt
Supplemental Figure 2 Gene expression analyses, relative to 18s rRNA, for hatching mouse blastocysts cultured in 5 mg/mL AlbIX,10% Buminate, 5 mg/mL HSA, 10% LGPS, 10% SPS, or 10% SSS at 5% O2 compared to 20% O2. Positive fold changes indicate an increase in transcript abundance in embryos cultured at 20% O2 as compared to 5% O2, whereas a negative fold change indicates a decrease in transcript abundance in embryos cultured at 20% O2 as compared to 5% O2. *, P < 0.05; ‡, P < 0.1. (DOC 190 kb)
10815_2014_349_MOESM9_ESM.doc
Literatur
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