Objective: Apoptosis is a common mode of programmed cell death occurring during embryonic development as well as in many pathological conditions. In the mouse blastocyst, apoptosis eliminates redundant cells from the inner cell mass (ICM) to maintain appropriate ratio of ICM/trophectoderm (TE) and plays an important role in normal development. However, cell death is increased under sub-optimal culture and stressful conditions. In vitro culture of embryos results in production of higher amounts of reactive oxygen species (ROS) which can influence the in vitro development of early mammalian embryos. The objective of this study was to determine the role of ROS in induction of apoptosis in early mouse embryos. Design: A prospective study in a reproductive research laboratory. Materials/Methods: Two-cell mouse embryos (n = 72) were cultured for 72h in HTF medium. Blastocyst development rate (%BDR) and total cell number in blastocyst were recorded. Total blastomere count per embryo was determined by staining with bisbenzimide (Hoechst 33258). The number of nuclei in ICM and TE in blastocyst were determined using confocal microscopy. Control group (n = 71) consisted of the embryos cultured in HTF alone and HTF medium supplemented with 10% serum supplement as a ROS scavenger. The levels of ROS produced by embryos in culture medium were measured at 24h, 48h, and 72h by chemiluminescence assay using luminol as the probe and the results were expressed as × 106 counted photons per minute (cpm). The incidence of apoptosis was detected by terminal transferase-mediated DNA end labeling (TUNEL) using confocal microscopy. Results: The median (25th, 75th percentile) value of total cell number (TCN) of blastocysts in HTF + serum group <53.64 (45.20, 60.75)>was similar to blastocysts cultured in HTF-alone <55.38 (53.71, 57.6)>(p = 0.31). Levels of ROS in HTF and HTF + serum media after 24h was 0.28 (0.13, 0.43) vs. 0.0 (0.0, 0.01) (p = 0.02), at 48h was 0.24 (0.21, 0.26) vs. 0.02 (0.01, 0.02) (p = 0.02), and was 0.26 (0.09. 0.32) vs. 0.02 (0.02, 0.03) (p = 0.01) at 72h, respectively. There was a significant positive correlation between the percentage of apoptosis and the levels of ROS after 24h (r = 0.76, p = 0.01), 48h (r = 0.87, p = 0.001), and 72h (r = 0.53, p = 0.05). Results of BDR and apoptosis in both groups are provided in the table. TableGroupBDR (%)Apoptotic blastocyst (%)Apoptosis per blastocystApoptosis in ICM (%)Apoptosis in TE (%)HTF-alone92985.84 (5.19, 6.60)∗Values are median and interquartile range.25.13.5HTF + serum92942.27 (1.89, 3.30)15.21.2P-value†P < 0.05 was considered significant using repeated measures ANOVA.0.80.3<0.001<0.001<0.001∗ Values are median and interquartile range.† P < 0.05 was considered significant using repeated measures ANOVA. Open table in a new tab Conclusions: Our results indicate that higher amounts of reactive oxygen species induce apoptosis in cultured mouse pre-implantation embryos. Addition of serum may help scavenge the excessive amounts of ROS in culture media and decrease the incidence of apoptosis. Supported by: None.