Background:The aim of this study was to design and assess the effects of hydroalcoholic extract of Matricaria chamomilla (MC) on preantral follicle culture of mouse ovaries in a three-dimensional culture system.Methods:Isolated preantral follicles were randomly divided into three main groups:the control group containing 10％ fetal bovine serum without MC extract (G 1),the first experimental group supplemented with 25 μg/ml hydroalcoholic extract of chamomile (G2),and the second experimental group supplemented with 50 μg/ml hydroalcoholic extract of chamomile (G3).Results:After 12 days of culture,the survival rate (P ＜ 0.05),antrum formation (P ＜ 0.01),metaphase two oocytes (P ＜ 0.01),and the expression ofPCNA (P ＜ 0.05) and FSHR (P ＜ 0.05) genes significantly decreased in G3 as compared with G1.On the other hand,at the last day of culture (day 12),the mean diameter of follicles cultured in the medium which was supplemented with 50 μg/ml hydroalcoholic extract of chamomile significantly decreased as compared with the G1 (P ＜ 0.05).In addition,the levels of progesterone and dehydroepiandrosterone hormones significantly increased in the medium of G3 relative to G1 (P ＜ 0.01),while in the medium of G 1,the level of 17β-estradiol was significantly higher than that of other groups (P ＜ 0.01).Reactive oxygen species levels of metaphase Ⅱ oocytes were significantly decreased in G2 as compared with G1 (P ＜ 0.01).Conclusion:Adding chamomile extract to culture media appeared to decrease follicular function and development.