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Secretion of 17β-estradiol in the multi-layered ovarian cell spheroids was higher than in the non-layered constructs. Increased secretion of progesterone was detected in the multi-layered ovarian cell spheroids at day 5 of culture and was sustained during the culture period. The initial secretion level of progesterone in the non-layered ovarian cell spheroids was similar to those from the controls and increased significantly from days 21 to 30. An in vitro rat model of engineered ovarian cell spheroids was developed that was capable of s