Co-culture of embryos with monolayers of AECC has been shown to improve outcome for patients with multiple implantation failures. Presumably, this effect is mediated through the production of trophic factors expressed by these helper cell lines. The endometrial cells elaborate multiple cytokines including IL-4 soluble Receptor (IL4-sR), IL-6, IL-10, IL-15. The specific aim of this study was to determine if IL-4sR, IL-6, IL-10, IL-15 levels as measured in the supernatants of conditioned media (CM) of embryos co-cultured in AECC is predictive of IVF outcome. CM from the AECC cells exposed or non-exposed to human embryos was collected in 118 consecutive patients and assayed for the presence of these cytokines and correlated with outcome. During a luteal phase biopsy (5–10 days after LH surge) made prior to the treatment cycle, glandular (G) and stromal (S) endometrial cells were isolated by enzymatic digestion and separated based on differential sedimentation rates. These cells were cryopreserved, then plated as a 50%/50% combination of G and S cells prior to embryo exposure. The CM was changed every 2 days. Embryos were randomly grown on ECC or conventional media. IL-4sR, IL-6, IL-10, IL-15 levels were measured utilizing an immunoenzymetric assay. Background levels of these cytokines were also determined from media alone (C-2 media supplemented with 15% patient’s serum). Statistics included Wilcoxon signed rank test, Mann-Whitney U test and Chi-square. Not all cytokines could be tested in all CM supernatants because of the limitations of the volumes of the samples. The pregnancy rate was 49.1% (58/118). The clinical pregnancy rate (fetal heart activity) was 42.3% (50/118). The mean age of the patients was 37.4 (3.8) years. The patients had had a mean number of 2.9 (1.5) previous failed cycles and 3.8 (1.8) years of infertility. A total of 10.4 (5.0) mean oocytes were obtained at the retrieval. On average, 3.5 (1.1) embryos were replaced. Exposure or non-exposure to an embryo did not result in differing levels of IL-4sR, IL-6, IL-10, and IL-15 in the CM. The endometrial coculture cells produced these cytokines. Embryos grown on AECC demonstrated a significant improvement in number of blastomeres and fragmentation (frag) when compared to embryos grown in conventional media without AECC (5.8 + 1.2 vs. 4.8 + 1.2 blastomeres and 12.6 % + 6.3 vs. 22.6% + 8.4 frag; P < 0.001). When IL-4sR was greater than 62.8 ng/mL (1 std dev above mean) in the CM, the embryos grown in ECC were of improved quality as compared to the embryos grown only in conventional media (7.4 + 1.1 vs. 5.2 + 1.2 blastomeres and 3.6 % + 2.3 vs. 16.6% + 11.4 frag; P < 0.01). A trend towards a higher level of IL-4sR was found in the CM from embryos that resulted in a clinical pregnancy. When IL-15 was less than 5.8 ng/mL (1 std dev below mean) in the CM, the embryos grown in ECC were of improved quality as compared to the embryos grown only in conventional media (5.8 + 1.3 vs. 4.9 + 1.0 blastomeres and 10.0 % + 2.3 vs. 13.6% + 3.4 frag; P < 0.03). A trend towards a lower level of IL-15 was found in the CM from embryos that resulted in a clinical pregnancy. No obvious correlations were found with IL-6 and IL-10. We have demonstrated a significant improvement in blastomere number and fragmentation with AECC. The cells in the AECC express IL-4sR, IL-6, IL-10, and IL-15. IL-4sR and IL-15 appear to play an important role in the AECC system.