Protein supplementation and the incidence of apoptosis and oxidative stress in mouse embryos.
OBJECTIVE
To estimate the effect of protein supplementation of culture media on reactive oxygen species production and incidence of apoptosis in preimplantation mouse embryos.


METHODS
A total of 72 two-cell mouse embryos were cultured in human tubal fluid (HTF) alone (HTF-alone, control) and 71 embryos in HTF with protein supplementation (10% serum substitute supplement; HTF-SSS) for 72 hours. Total cell number per embryo was determined by staining with Hoechst 33258. Allocation of inner cell mass and trophectoderm in blastocysts and incidence of apoptosis were determined by confocal microscopy. Levels of reactive oxygen species in culture media were measured by chemiluminescence assay using luminol as probe.


RESULTS
Blastocyst development, total cell number, and the inner cell mass/trophectoderm ratio were similar between the 2 groups. The blastocyst hatching rate was significantly higher in the HTF-SSS group than in the HTF-alone group (20% compared with 4%, P = .007). Level of reactive oxygen species was significantly higher in HTF-alone compared with HTF-SSS at 24 hours (median and interquartile range 28 [13, 43] compared with 0 [0, 1], P = .02), 48 hours (24 [21, 26] compared with 2 [1, 2], P = .02), and 72 hours (26 [9, 32] compared with 2 [2, 3], P = .03). The incidence of apoptosis in blastocysts cultured in HTF-SSS was significantly lower than those in HTF-alone group (mean +/- standard deviation 2.38 +/- 0.68 and 5.81 +/- 1.11, respectively, P = .001).


CONCLUSIONS
Protein supplementation of culture media improves the hatching rate and reduces reactive oxygen species levels and the incidence of apoptosis in mouse preimplantation embryos.
Esfandiari
Navid
N
Department of Obstetrics and Gynecology, Glickman Urological Institute, Cleveland, Ohio 44195, USA.
Falcone
Tommaso
T
Agarwal
Ashok
A
Attaran
Marjan
M
Nelson
David R
DR
Sharma
Rakesh K
RK
D000818:Animals; D017209:Apoptosis; D001755:Blastocyst / Q000166:cytology / Q000378:metabolism; D001798:Blood Proteins / Q000494:pharmacology; D001826:Body Fluids; D002452:Cell Count; D002478:Cells, Cultured; D003470:Culture Media; D046149:Embryo Culture Techniques; D004622:Embryo, Mammalian / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D047108:Embryonic Development / Q000187:drug effects; D005187:Fallopian Tubes; D005260:Female; D020287:In Situ Nick-End Labeling; D051379:Mice; D008815:Mice, Inbred Strains; D018384:Oxidative Stress; D010952:Plasma Substitutes; D011506:Proteins / Q000494:pharmacology*; D017382:Reactive Oxygen Species / Q000378:metabolism
D001798:Blood Proteins; D003470:Culture Media; D010952:Plasma Substitutes; D011506:Proteins; D017382:Reactive Oxygen Species