Aim <p>To investigate the effects of two different gas blending methods on the microenvironment of in vitro embryo culture.</p> Methods <p>Using real-time monitoring systems, we tracked temperature, CO₂, and pressure in both incubator types. Blood gas analyzers measured media parameters (pCO₂, pO₂, pH). Retrospective analysis included 3658 frozen transfer cycles from women ≤ 35 without genetic/uterine anomalies. The participants were divided into two groups: the lumen blending group (n = 2746) and the preblending group (n = 912). The outcomes of frozen embryo transfer at cleavage stage and blastocyst stage were compared between the two groups.</p> Results <p>The concentration of CO₂ in the preblending incubator was significantly lower (4.40%vs. 5.82%, p &lt; 0.001). The results showed that the pH value (7.418 vs. 7.351, p &lt; 0.05) and O₂ pressure (90 vs. 81 mmHg, p &lt; 0.05) of the preblending incubator were significantly higher than that of the lumen blending incubator, while the CO₂ pressure was lower (31 vs. 38 mmHg, p &lt; 0.01). The clinical pregnancy rate (56.77% vs. 45.90%, p &lt; 0.05), implantation rate (40.64% vs. 32.52%, p &lt; 0.05) and live birth rate (45.81% vs. 37.70%, p &lt; 0.05) of cleavage embryos in the lumen blending group were significantly higher than those in the preblending group, though blastocyst results showed no intergroup differences. Binary logistic regression analysis revealed that the lumen blending group had a significantly higher clinical pregnancy rate compared to the preblending group (aOR = 1.431, 95% CI 1.024 ~ 2.001, p = 0.036). There was also a trend towards a higher live birth rate in the preblending group (aOR = 1.332, 95% CI 0.947 ~ 1.874, p = 0.100).</p> Conclusion <p>The lumen blending incubators may ensures superior gas concentration stability and faster CO₂ recovery. This method proves more beneficial for cleavage-stage embryos, whereas blastocyst development remains unaffected by gas blending methods.</p>