Objective To detect the gene expression changes between urethra plates from hypospadias patients and foreskins from non-hypospadias patients by microarray and to investigate the underlying mechanisms of hypospadias.Methods Twelve hypospadias patients,aged 6-12 months (mean,8 months),were enrolled as the test group,including 5 moderate and 7 severe hypospadias patients.Six age-matched patients underwent circumcision were enrolled as controls.Samples from hypospaidas patient's urethra plates during hypospadias repair and samples from the foreskins during circumcision were obtained and processed into Tri-Reagent immediately for RNA extraction.Oligonucleotide expression microarrays were used to detect genes expression changes in tissues from patients with and without hypospadias.This microarray analysis incorporated 22 000 genes.The intensity of all genes present was analyzed by one-way ANOVA (P＜0.01) and Tukey's test.Four estrogen responsive genes,CYR61,CTGF,ATF3 and GADD45β,were tested by RT-PCR in 8 controls,8 moderate hypospadias and 8 severe hypospadias as well.Results Ninty-four genes were detected differentially expressed in hypospadias patients compared with phimosis patients.There were 47 genes upregulated in moderate hypospadias compared with controls(P＜0.01),68 genes up-regulated in severe hypospadias compared with controls(P＜0.001),17 genes up-regulated in severe hypospadias compared with moderate hypospadias(P＞0.05).These genes were involved in different cell functions such as growth regulation and signal transduction.CYR61,CTGF,ATF3 and GADD45β,known to be estrogen responsive or to interact with estrogen receptor were found up-regulated in microarray and the up-regulations were confirmed by RT-PCR.Conclusions The up-regulated genes contribute to the development of hypospadias.Up-regulation of estrogen responsive genes may play important roles in the development of hypospadias.