Sequential co-culture of preimplantation embryos with oviductal epithelial cells first is more effective than that with endometrial epithelial cells first in human

Abstract

To address the physiological relevance of the process that fertilized embryos gradually move from the oviduct to the uterine cavity during their development into the blastocyst. We studied preimplantation embryo development in vitro using two sequential co-culture conditions. One hundred and two surplus frozen embryos (G2 or G3 based on the Veeck's classification) in the IVF program were used for the co-culture. Such embryos at 4 cell stage were randomly allocated into two groups as follows: the oviductal (2 days)-endometrial (2 days) sequential co-culture group (OEG) and the endometrial (2 days)-oviductal (2 days) sequential co-culture group (EOG). SV40TAg-transfected oviductal or endometrial epithelial cell lines were used for co-culture. Cells were cultured in RPMI1640 supplemented with 10% FCS. The medium was replaced 24 h before co-culture was started. Only cells at 80% confluence in 6-well plates (Falcon) were used for embryo co-culture. Conditioned medium for 48 h cell culture was assayed for bFGF, HGF (hepatocyte growth factor), TGF-β1, IGF-I, IL-1β, IL-4, IL-6 and IL-8 with EIA (for bFGF, TGF-β1, IL-1β, IL-6) or IRMA (for IGF-I) or ELISA (for IL-4, IL-8 and HGF). Differences in embryonic development between OEG and EOG were compared with Fisher's exact test. Differences in concentrations between culture conditions were compared with two-tailed t-test. Of 46 and 43 frozen-thawed embryos with good quality (3 or 4 blastomeres were viable after thawing) in OEG and EOG, 31 (67.4%) and 26 (60.5%) embryos reached the morula stage (N.S.), 23 (50.0%) and 12 (27.9%) embryos reached the early blastocyst stage (P<0.05), and 8 (17.4%) and 6 (14.0%) reached the expanded blastocyst stage (N.S.), respectively. IL-1β and HGF concentrations were significantly higher in the conditioned medium of the oviductal epithelial cell culture. IL-6 and IL-8 concentrations were significantly higher in the conditioned medium of the endometrial epithelial cell culture. IGF-I and IL-4 were not detected in each assay. Although both oviduct and uterus are developed from the Müllerian tubercle, each tissue might play differentiated roles in the preimplantation embryo growth. We could not show clear difference in the development into expanded blastocyst stage possibly due to the limitation of our co-culture conditions that did not include hormonal regulation or stromal cells.