| Objective: One of the limitations in embryo implantation research is a lack of well-defined in vitro systems that faithfully replicate embryo- uterine interactions. Among the previous model studies, embryos were generally cultured on the monolayer of either uterine epithelial cells or extracellular matrix substrates such as fibronectin, laminin on which embryos could adhere and outgrow. However, those models failed to display an invasion process of embryos into the cells or substrates mainly due to the quite different components and structure of the supporters from that in vivo. Moreover, years of efforts to culture the uterine horn in vitro has unfortunately not been rewarded so far because of the inability to maintain functionally and structurally normal uterine horns in vitro. In the present study, we collected the intact mouse uterine endometrium consisting both of epithelium and stroma as well as stromal glands and capillaries on the day 4 of pregnancy, then co-cultured with the blastocysts derived from the same mouse on the surface of the endometrium at the gas-liquid interface. Next, we investigated the receptivity of the endometrium, which was cultured for 24 hours in vitro, to embryo. Finally we co-cultured the blastocysts derived from the same mouse on the surface of the endometrium, observing embryonic adherence and invasion to the endometrium and eventually developed the three-dimensional co-culture model of blastocyst and endometrium in mouse. Methods: (1) Cultural medium were repeatedly optimized for the growth of mouse endometrium in vitro. ①63.5 nmol/L progesterone (P4),7.14 nmol/L estradiol (E2) and 55.7 nmol/L P4,35nmol/L E2 were added respectively into designed basic culture media which contained F12/DMEM (1:1), 20% fetal bovine serum (FBS),100ug/ml insulin. Mouse endometrium about 2 mm at length was cultured at the gas-liquid interface between above-mentioned two culture media and 5% carbon dioxide (CO2) plus 95% air for 24 hours respectively, then taken out to be stained with hematoxylin-eosin (H.E), following the necrosis rate of a sample measured by using the image analysis system (IAS). ② To screen epidermal growth factor (EGF) concentration in tissue culture media, mouse endometrium was cultured at the interface between culture media supplemented with 0ng/ml, 5ng/ml, 20ng/ml EGF and 5% CO2 plus 95% air, respectively. The necrosis rate of a sample was measured with the same method as above. ③ To screen the oxygen concentration which was most advantageous to endometrium culture in vitro, mouse endometrium was cultured at the gas-liquid interface with air and mixed gas containing 50%, 75%, 95% oxygen respectively. ⑵ To determine whether the endometrium cultured for 24 hours was still receptive to the blastocyst, expressions of leukemia inhibitory factor (LIF),heparin-binding epidermal growth factor-like growth factor (HB-EGF) and cyclooxygenase-2 (COX-2) proteins in mouse endometrium were detected by immunohistochemistry. ⑶ To develop the three-dimensional model of embryo implantation in vitro, we co-cultured the blastocyst derived from the same mouse on the surface of the endometrium at the gas-liquid interface with air and the mixed gas containing 50%, 75%, 95% oxygen, respectively. After 24 hours of culture, the adherence rate of blastocysts was recorded to explore the optimal concentration of oxygen for the co-culture system in vitro. At the same time, the endometrium which had been adhered by blastocyst was fixed in paraformaldehyde and examined after H.E staining.Results: ⑴ Necrosis rate (%) of endometrium in two groups containing different concentrations of E2 and P4 was 16.81±3.75,21.01±5.59 respectively, displaying a significant difference (p < 0.05). Necrosis rate of endometrium in three groups containing different EGF concentration was 19.40±3.38,16.45±4.87,16.41±3.40 respectively, necrosis rate of two posterior groups were significantly lower than that in the first group (p < 0.05), but there was no dominan...
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