OBJECTIVE:To investigate whether Zichong gran-ules(资冲颗粒,ZCKL),a very effective herbal for-mula for treating infertility,have an impact on the differentiation of ovarian granulosa cells from human embryonic stem cells(hESCs)in vitro,and to explore the cellular mechanisms of its clinical effects.METHODS:Serum from ZCKL-medicated rats was prepared and used to treat mesoderm cells derived from hESCs for 6 d.Normal rat serum and a set of growth factors were used as negative and positive controls,respectively.RESULTS:ZCKL-medicated rat serum,but not nor-mal rat serum,induced hESCs-derived mesoderm cells to differentiate into functional ovarian granu-losa-like cells(OGLCs)in a similar manner to de-fined growth factors.The induced OGLCs resem-bled the morphology of native human granulosa cells,expressed granulosa cell-specific markers at both the mRNA and protein levels,produced high levels of estradiol and strongly responded to folli-cle-stimulating hormone stimulation.Furthermore,mRNA levels of follistatin,mothers against decap-entaplegic homolog 8 and bone morphogenetic protein 6 were dynamically changed during the process.CONCLUSION:In the ZCKL treatment of infertility,one mechanism by which ZCKL may act is by influ-encing ovarian granulosa cell differentiation and development,possibly through the follistatin and BMP/SMAD signaling pathways.