Objective To investigate the real correlation between embryo density and developmental outcome of cleavage embryos on day 3 by adjusting covariates.   Methods  Data of 1196 embryos from 206 couples undergoing in vitro fertilization and embryo transfer(IVF-ET) treatment were retrospectively analyzed. Embryos were hypoxia-cultivated in a fixed 30 μl microdrop in culture dishes. Embryo quality on day 3 was evaluated and proportion of good quality cleavage embryos on day 3 was used as the endpoint. Maternal age, paternal age, antral follicles, level of anti-mullerian hormone (AMH), type of infertility, controlled ovarian stimulation(COS) protocol, length of stimulation, number of retrieved oocytes, and type of insemination were chosen as covariates.   Results  A total of 1196 embryos were included and analyzed. Three embryo densities were routinely used: 30 μl/embryo ［1 embryo/(30 μl·drop)］, 15 μl/embryo ［2 embryos /(30 μl·drop)］ and 10 μl/embryo ［3 embryos/(30 μl·drop)］. The number of embryos assigned into these three groups were 434, 646 and 116 embryos separately. The average proportion of good quality embryos on day 3 in 10 μl/embryo group were lower than that in both 15 and 30 μl/embryo group (29.31% vs 40.25%，P<0.05；29.31% vs 42.17%，P<0.05), and it was comparable between groups of 15 and 30 μl/embryo (40.25% vs 42.17%，P>0.05). In the regression analysis, without adjusting any covariables, the good quality embryos rate on day 3 of the 10 μl/embryo group was 43% lower than that of the 30 μl/embryo group (0.57, 95% CI 0.32, 1.03), and there was no statistical difference (P>0.05). In the minimum-adjusted model 2 (adjusted the level of AMH and type of insemination), proportion of good quality embryos on day 3 in group of 10 μl/embryo significantly decreased by 51% (0.49, 95%CI 0.27，0.90, P<0.05) compared with that in group of 30 μl/embryo. 

  ConclusionIn a 30 μl microdrop, compared with individual cul turing, group culturing with the density of 10 μl/embryo did not benefit the development of the cleavage embryos and 30 μl/embryo was the optimal embryo density.