The Effect Of Progesterone On Pinopode Appearance Of 3-dimensionally Cultured Human Glandular Cells

ObjectiveTo isolate and purify human endometrial epithelial and stromal cells and establish a system of 3-dimensionally cultured human endometrial cells which could be used as a tissue model for mechanism study of blastocyst implantation and endometrial receptivity in vitro, In order to study the correlation between progesterone and pinopode formation on 3-dimensionally cultured human endometrial epithelial cells.MethodsEndometrial tissues were obtained by surgical curette from normal ovulating women admitted to our department due to myoma other than endometrial diseases, aged 20-45 years old, whose menstral cycle was regularly, 24-35 days, not admitting hormonal therapy within prior three months. Proliferative endometrial biopsies were included in this experiment. Employing human endometrial glandular, luminal cells and stromal cells from hysterectomy biopsy by first layering a mixture of rat tail collagen I and endometrial sromal cells, then overlaying a thinner layer of matrigel, the upper layer is endometrial glandular and luminal cells, to establish a new system of human endometrial cells in vitro. Progesterone concentration was different in culture medium among study groups, 10^-8 mol/L, 10^-7 mol/L, but no progesterone in control ones. All of estrogen level was 10^-9 mol/L.ResultsEpithelial cells grew in whorls, stained positively by CK19 (cytokeratin 19) at 92%. Stromal cells were fibrospindle-shaped. They were stained positively by vimentin above 95%. Light microscope reveals monolayer columnar glandular cells, underlying was stromal cells, culture system grew 3-dimensinally, The ultrastructural examination of 4-6 day endometrial cells culture reveals pinopode structure with progesterone, but control ones. Where pinopodes covered 5% epithelial surface,and the appearance of tight junction and gap junction as well as denosome between glandular cells, endoplasmic reticulum, and secreted granules in plasma.ConclusionThis culture system represents a 3-dimensional system that closely resembles the native endometium morphologically, expression of pinopodes which was similar in study groups despite of different progesterone level. Our finding suggest that pinopodes formation may be dependent on progesterone, but no correlation was found between pinopoes and progesterone level.