Abstract Study question Do different culture conditions and practices, regarding oxygen levels, temperature, and culture interruption for evaluation purposes, effect embryo developmental arrest? Summary answer Ultra-low oxygen levels at 2-3.5%, a temperature below 37oC, and more than one interruption during culture are associated with a higher developmental arrest rate. What is known already Literature indicates that embryo culture microenvironment differs among laboratories. According to data, oxygen levels range between 2%-20%, while the optimal temperature during embryo culture, ranging from 36oC-37oC, has not yet been validated. What is more, interrupting embryo culture to evaluate morphology disrupts the stable conditions of the incubator’s microenvironment, while various models of interruptive evaluation practices have been reported on time-points and assessment frequency. Data are lacking on the extent that oxygen levels, temperature and morphology evaluation practices affect the embryos’ developmental potential. Interestingly, reporting on optimal culture conditions and embryo evaluation practices still appears to be timely and essential. Study design, size, duration A systematic search of the literature was performed in Pubmed/Medline, Embase, and Cochrane Central Library up to October 2021. Seventeen published prospective studies were included in the three arms of this network meta-analysis. The outcome measure was developmental arrest rate prior to the blastocyst stage. To rank oxygen levels and interruption times, the P-Score was employed. Molecular pathways implicated in developmental arrest associated with each examined parameter were investigated as part of the systematic review. Participants/materials, setting, methods A network meta-analysis was performed regarding oxygen levels, temperature, and culture interruption practices for evaluation purposes. The population consisted of preimplantation embryos. Atmospheric conditions (20% O2), low-oxygen levels (5% O2) and ultra-low oxygen levels (2-3.5% O2), along with temperature below or at 37oC were compared. Finally, no interruptions (time-lapse microscopy), one interruption (fertilization evaluation), 2 and 3 interruptions for morphological evaluation were compared. The estimated network effect comprises of direct and indirect effects. Main results and the role of chance Eight studies reporting on the effect of different oxygen levels, presented with high heterogeneity I2=88%. No statistically significant difference was observed between atmospheric and low-oxygen level regarding arrest rate (RR:1.07; 95%CI:0.88-1.30). A significantly higher developmental arrest rate was observed in ultra-low oxygen levels versus low (RR:1.29; 95%CI:1.02-1.64). No statistically significant difference was observed between ultra-low oxygen levels and atmospheric conditions (RR:1.21; 95%CI:0.89-1.63). The optimal oxygen levels are low (P-Score=0.87), followed by atmospheric conditions (P-Score=0.57) and ultra-low (P-Score=0.06). Three studies reporting on the effect of different temperatures presented with low heterogeneity I2=30%. Temperature below 37oC presented with a statistically significant higher developmental arrest rate (RR:1.10; 95%CI:1.03-1.17). Six studies reporting on the effect of interruptive evaluation practices showed high heterogeneity I2=84%. Uninterrupted culture presented with no statistically significant difference with the other groups (1-interruption vs Uninterrupted: RR:0.93; 95%CI:0.78-1.12; 2-interruptions vs Uninterrupted: RR:1.08; 95%CI:0.91-1.28; 3-interruptions vs Uninterrupted: RR:1.11; 95%CI:0.92-1.34). A single-interruption presented with marginally no statistically significant difference when compared to two-interruptions (RR:0.87; 95%CI:0.74-1.02), however presented with a lower developmental arrest rate when compared to three-interruptions (RR:0.84; 95%CI:0.71-0.99). Two versus three-interruptions indicated no difference (RR:0.97; 95%CI:0.82-1.14). The optimal interruptive evaluation practice is one (P-Score=0.90) followed by uninterrupted (P-Score=0.63), two-interruptions (P-Score=0.30) and three-interruptions (P-Score=0.16). Limitations, reasons for caution The limited number of studies included and the heterogeneity are limitations of this study. The different number of studies and embryos included in each comparison group, along with the fact that randomization was not performed in all studies, present as reasons for caution when interpreting the results of this study. Wider implications of the findings Oxygen levels at 5%, 37oC temperature, and single or no-interruption embryo evaluation practices reduce developmental arrest rate. This systematic review indicates potential pathways implicated in developmental arrest as follows: oxygen tension influencing histone modifications, temperature influencing regulatory proteins’ activity, and stress-related cytokines’ differential expression when culture is interrupted multiple times. Trial registration number Not applicable