HUMAN TUBAL CELL COCULTURE : IN VITRO CELL BEHAVIOUR AND USE OF CONDITIONED MEDIUM FOR EMBRYONIC SUPPORT

Abstract

The growth of human embryos on human fallopian tubal monolayers has offered much promise in improving embryo quality for in vitro fertilization programs. To study the mode of action of such 'helper' cells and decide which cell generation would be most ideal, the in vitro behaviour of tubal epithelial cells from 20 premenopausal women aged 30 to 42 years was investigated. Large patches of epithelioid-like cells with vigorously beating cilia were observed under light microscopy (LM) in primary cultures after 4 to 7 days. Scanning electron micrographs (SEM) confirmed the presence of ciliated and secretory cells in primary cultures. The ciliated cells gradually shed their cilia as the days advanced in primary culture and transformed to the secretory type with numerous microvilli on their surfaces. At the first subculture, confluent epithelioid-like monolayers were formed in 4 to 6 days. These cells changed to spindle-shaped fibroblasts in the 2nd and 3rd subcultures in all patients. In some patients in the 4th to 7th subcultures, the fibroblast-like cells changed to broad band-like cells which had a prominent cytoskeleton and reduced mitotic index. The cells remained fibroblast-like or band-like until the 20th subculture in all 20 patients irrespective of the stage of the menstrual cycle. SEM of all cells from the 1st until the 15th subculture showed numerous varieties of microvilli. The surfaces of cells from the 15th to 20th subculture were very smooth with minimal microvilli. Chromosome analysis of the epithelioid-like cells (primary and 1st subculture), and fibroblast and band-like cells (2nd to 20th subculture) in 5 different patients showed a 46XX karyotype. Human tubal epithelial cells can thus be maintained through at least 20 generations without a change in karyotype. The significance of these morphological and genetic changes with respect to coculture systems is discussed. A 48 hour preconditioned medium prepared from early tubal subcultures was evaluated for fertilization rates and embryonic behaviour in vitro. Eighteen patients provided 195 mature oocytes, 97 of which were cultured in conditioned medium and 98 in conventional medium. Sixty-nine percent of oocytes fertilized in conditioned medium compared to 62% in controls. A significantly higher percentage of good quality 2 to 4- cell embryos were produced in conditioned medium compared to controls (88% vs 68%; p< 0.01) and a significantly greater number of embryos cavitated at the blastocyst stage at 110 to 115 hours postinsemination in conditioned medium (47% vs 29%; p<0.05). The percentage of expanded blastocysts at 120 hours postinsemination was, however, not significantly different between the two groups. The ionic constituents and pyruvate levels in a 48 hour medium prepared from 1 x 105 cells/ml were not significantly different from controls. However, glucose levels in conditioned medium were reduced and lactate levels increased. The improved embryo quality in conditioned medium may be due to metabolic redesigning of the medium, removal of undesirable metabolites by the cells and the release of putative embryotrophic factors by the cells into the medium.