We have investigated the effect of protein- and amino acid-free simple human tubal fluid (HTF) medium conditioned with bovine granulosa cells (BGC) or Vero cells on the development of early bovine embryos to the blastocyst stage. Serum-containing medium (SCM) and serum-free medium (CM) conditioned by BGC (BGC-SCM, BGC-CM) and by Vero cells (VC-SCM, VC-CM) were prepared. Early embryos (1-cell stage and 5- to 8-cell stage) were obtained by in vitro maturation and fertilization of oocytes from slaughtered cows. Embryos were randomly divided into 7 culture groups as follows: culture with BGC-SCM, BGC-CM, VC-SCM, or VC-CM; coculture with BGC or Vero cells; or culture with fresh HTF medium without serum. The proportion of 5- to 8-cell embryos developing to the blastocyst stage in BGC-CM (16%) and VC-CM (12%) was significantly lower (p < 0.05) than in BGC-SCM (41%) and in VC-SCM (29%) and after coculture with BGC (48%) and Vero cells (30%). Similarly, the percentages of 1-cell embryos developing to blastocyst in BGC-CM and VC-CM were significantly lower than in BGC-SCM and VC-SCM and after coculture. Cell numbers per blastocyst developed from 5- to 8-cell embryos in BGC-CM (96.8 cells) and in VC-CM (94.0 cells) were somewhat lower than those in BGC-SCM (128.5 cells) and VC-SCM (117.1 cells) and after coculture with BGC (124.2 cells) and Vero cells (115.3 cells). These results suggest that BGC and Vero cells cultured in a protein- and amino acid-free simple HTF medium synthesize and secrete factor(s) promoting blastocyst formation in vitro. Physiochemical analysis indicated that the embryotrophic substances in BGC-CM were distributed in two molecular size ranges, one between 10 kDa and 30 kDa and another greater than 30 kDa.

As is often the case with cell culture, normal human endometrial epithelial cells show a low, squamous shape with flattened nuclei and lack three-dimensional morphology in in-vitro culture systems. Here we report the first well-differentiated epithelial culture system using basement membrane extracts (BME) from an Engelbreth-Holm-Swarm tumour (EHS tumour). In this model, BME regulated the reconstruction of glandular formation and subsequent reformation of the epithelium by epithelial cells. An electron microscopic study clearly indicated that there were two distinct types of cells grown on the substrate. The first, having a high columnar shape, formed the glandular epithelium, and the second, having a cuboidal shape, covered the surface of the BME. Study of epithelial reformation indicated that the regeneration of superficial epithelium could occur, following the development of the glandular formation, in a helix-like pattern. Total protein secretion was greater when the cells were grown on the BME than on plastic. Thus, normal human endometrial epithelial cells cultured on the BME assumed a phenotype and morphology characteristic of those in vivo.

Serum concentrations of human hepatocyte growth factor (hHGF) were measured in normal menstrual cycles and during uncomplicated pregnancies. In the normal menstrual cycle, the concentrations of hHGF increased through the mid and the late luteal phases to reach the highest peak during the menstrual phases, followed by a gradual decline during the follicular phase toward the trough levels seen in the ovulatory and the very early luteal phases. During pregnancy, serum hHGF concentrations increased continuously from the late luteal levels and constituted 4 distinct peaks. By immunostaining, c-Met protein, a receptor for hHGF, was localized not in the stromal but in the epithelial layer of the endometrium. In cultured isolated endometrial cells, hHGF stimulated the proliferation of both the epithelial and the stromal cells. It is likely that hHGF is involved in the repair or reconstruction process of the endometrium after menstrual shedding and implantation.

We report the effects of PRL on cultured human endometrial cells (epithelial cells and stromal cells) and discuss the role of PRL in implantation. Essentially similar responses to PRL in cell attachment and growth were obtained both in epithelial and stromal cells: a low concentration of PRL (3–30 ng/ml of PRL) enhanced cell attachment and growth. In contrast, the presence of a high concentration of PRL (over 100 ng/ml) resulted in a decrease in cell attachment and growth in normal culture conditions. Morphological analysis of epithelial glandular reformation on basement membrane extracts indicated that PRL had dual effects on endometrial epithelial cells: as a stimulator in low concentrations and an inhibitor in high concentrations. These data suggest that PRL may influence the functions of human endometrial cells in a dual way depending on its level and plays an important role in the implantation process.

The simplified SART embryo scoring system is highly correlated to implantation and live birth in single blastocyst transfers. Journal of assisted reproduction and genetics, 30(4), 563-567.