Objective: Coculture of embryos with monolayers of AECC has been shown to improve outcome for patients with multiple implantation failures after In Vitro Fertilization (IVF). Presumably, this effect is mediated through the production of trophic factors expressed by these helper cell lines. TGF-2 and EGF are known to be produced by the endometrium (Giudice LC. Trends Endocrinol Metab, 1995; 6:60), and receptors have been found on embryos (Lim H. Dev Biol, 1998;204:97–110). The specific aim of this study was to determine if TGF-2 and EGF levels as measured in the supernatants of conditioned media (CM) of embryos cocultured in AECC is predictive of IVF outcome.Design: CM from the ECC cells exposed or non-exposed to human embryos was collected in 27 patients and assayed for the presence of TGF-2 and EGF and correlated with outcome.Materials/Methods: 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 conditioned medium (CM) was changed every 2 days. Embryos were randomly grown on ECC or conventional media if more than 6 oocytes normally fertilized. Otherwise, all embryos were grown on AECC. TGF-2 and EGF levels were measured utilizing an immunoenzymetric assay. Background levels of TGF-2 and EGF were also determined from media alone (Hams F-10 supplemented with 15% patient serum). Statistics included Wilcoxon signed rank test, Mann-Whitney U test and Chi-square.Results: The average age of the patients studied was 36.5 ± 3.5 years. Mean numbers of mature oocytes, fertilized embryos, and embryos transferred were 8.3 ± 4.1, 5.9 ± 3.5, and 3.5 ± 1.4 respectively. Clinical pregnancy rate was 37% and implantation rate was 14%. Exposure or non-exposure to an embryo did not result in differing levels of TGF-2 and EGF in the CM. Embryos grown on ECC demonstrated a significant improvement in number of blastomeres and fragmentation (frag) when compared to embryos grown in conventional media without ECC (6.7 ± 1.3 vs. 5.6 ± 1.2 blastomeres and 17.6% ± 9.3 vs. 26.4% ± 9.8 frag; p <0.05). TGF-2 and EGF levels in the CM were not associated with embryo quality or pregnancy outcome.Conclusions: We have demonstrated a significant improvement in blastomere number and fragmentation with ECC. TGF-2 and EGF are expressed by the cells in the AECC. The presence of TGF-2 and EGF in the CM was not associated with embryonic development or outcome.Supported by: None. Objective: Coculture of embryos with monolayers of AECC has been shown to improve outcome for patients with multiple implantation failures after In Vitro Fertilization (IVF). Presumably, this effect is mediated through the production of trophic factors expressed by these helper cell lines. TGF-2 and EGF are known to be produced by the endometrium (Giudice LC. Trends Endocrinol Metab, 1995; 6:60), and receptors have been found on embryos (Lim H. Dev Biol, 1998;204:97–110). The specific aim of this study was to determine if TGF-2 and EGF levels as measured in the supernatants of conditioned media (CM) of embryos cocultured in AECC is predictive of IVF outcome. Design: CM from the ECC cells exposed or non-exposed to human embryos was collected in 27 patients and assayed for the presence of TGF-2 and EGF and correlated with outcome. Materials/Methods: 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 conditioned medium (CM) was changed every 2 days. Embryos were randomly grown on ECC or conventional media if more than 6 oocytes normally fertilized. Otherwise, all embryos were grown on AECC. TGF-2 and EGF levels were measured utilizing an immunoenzymetric assay. Background levels of TGF-2 and EGF were also determined from media alone (Hams F-10 supplemented with 15% patient serum). Statistics included Wilcoxon signed rank test, Mann-Whitney U test and Chi-square. Results: The average age of the patients studied was 36.5 ± 3.5 years. Mean numbers of mature oocytes, fertilized embryos, and embryos transferred were 8.3 ± 4.1, 5.9 ± 3.5, and 3.5 ± 1.4 respectively. Clinical pregnancy rate was 37% and implantation rate was 14%. Exposure or non-exposure to an embryo did not result in differing levels of TGF-2 and EGF in the CM. Embryos grown on ECC demonstrated a significant improvement in number of blastomeres and fragmentation (frag) when compared to embryos grown in conventional media without ECC (6.7 ± 1.3 vs. 5.6 ± 1.2 blastomeres and 17.6% ± 9.3 vs. 26.4% ± 9.8 frag; p <0.05). TGF-2 and EGF levels in the CM were not associated with embryo quality or pregnancy outcome. Conclusions: We have demonstrated a significant improvement in blastomere number and fragmentation with ECC. TGF-2 and EGF are expressed by the cells in the AECC. The presence of TGF-2 and EGF in the CM was not associated with embryonic development or outcome. Supported by: None.