To assess the effect of reducing the oxygen concentration from 5% to 2% on Intracytoplasmic sperm injection (ICSI) outcome.

Oxygen level plays a critical role in reducing the high level of detrimental reactive oxygen species within cells, influences embryonic gene expression, helps with embryo metabolism of glucose, and enhances embryo development from 2PN to the blastocyst stage, the oxygen concentration in the incubator is an important determinant of the in vitro environment that an embryo encounters. The preimplantation embryo consumes oxygen at a relatively constant level from the one-cell through the morula stage, while at the blastocyst stage the consumption is considerably increased. Oxygen plays a role in energy production, but in too high concentrations it may have a toxic effect by the formation of reactive oxygen species. While in vivo oxygen levels range from 1.5 to 8.7% in the fallopian tubes and uterus of several species, in most laboratories in vitro culture of human embryos was traditionally performed under atmospheric oxygen concentrations (~20%) . In mammals, the oxygen (O2) tension in the uterus is lower (2%) than the oviduct (5-7%). This fits with a shift in the metabolic strategy of the embryo after compaction and may have implications for EC in clinical IVF. Indeed, in a recent study utilizing discarded human embryos, blastulation rates were superior when cultured in 2% compared to 5% after day day 3. However, it is exceedingly difficult to control for the myriad factors impacting blastulation when employing discarded material . The technologies available in the early days of IVF, and most likely also for financial reasons, led to the widespread implementation of incubators that used atmospheric air (i.e. 20% oxygen) . Around the turn of the last century, more advanced incubators capable of creating an environment of 5% oxygen, which better resembles the natural

environment for oocytes and embryos, were developed. Currently, in around 60% of the IVF cycles performed worldwide, the embryos are cultured under 5% oxygen . Kaser et al. add to the long history of experimental data evaluating different oxygen levels in embryo culture. They evaluated the impact of a sequential oxygen tension system (5% oxygen from day 1 to day 3; 2% oxygen from day 3 to day 5) in extended culture . The effect of oxygen level during embryo culture on pregnancy and live birth rates is less straightforward. Although most studies found an improvement in embryo quality when culturing embryos under 5% oxygen . This discrepancy is partly explained by lack of power due to small sample sizes in some studies. When data are pooled in meta-analyses, the overall effect favours culture under 5% oxygen . Another explanation for the inconsistent results among the studies could be due to the differences in duration of exposure, until cleavage or blastocyst stage. When data in the meta-analyses were stratified for duration of culture, there was no effect of oxygen on pregnancy or live birth rates after 2-3 days of culture. After culture to the blastocyst stage, however, culture under 5% oxygen resulted in improved ongoing pregnancy. In the previous studies reported that culture under 5% O2 results in significantly more human surplus embryos reaching the blastocyst stage, as well as a higher proportion of blastocysts consisting of a normal number of cells.

in the human, indirect evidence exists that a low O2 tension of 5% appears to enhance the blastulation rate of surplus embryos .

A reduction in oxygen tension from atmospheric to more physiologic levels is beneficial for the mammalian blastocyst, in terms of a higher cell number and decreased apoptosis, less DNA fragmentation and less oxidative stress. During this highly metabolically active period of growth of the embryo and differentiation into trophectoderm (TE) and inner cell mass (ICM), this decrease in oxygen may protect against oxidative stress .

Inclusion Criteria:

- Female age 20-35 years. Oocytes with normal morphology.

Exclusion Criteria:

• Embryo number less than 5. Male factor.