Gene Expression Differences Between Ruptured Anterior Cruciate Ligaments in Young Male and Female Subjects

Background: The incidence of anterior cruciate ligament (ACL) injuries is two to eightfold greater in female compared with male athletes. Anatomic, hormonal, and neuromuscular factors have been associated with this disparity. This study compared gene expression and structural features in ruptured but otherwise normal ACL tissue from young female and male athletes. Methods: A biopsy sample of ruptured ACL tissue (which would normally have been discarded) was obtained intraoperatively from seven female and seven male athletes (12.7 to 22.6 years old). Each sample was divided into portions for histological and gene expression analyses. Specimens for gene analysis were frozen and ground, and RNA was extracted and purified. Microarray analysis was performed on RNA isolated from four female and three male study participants (13.9 to 18.5 years old) who had a noncontact injury. Genes with an expression level that differed significantly between these female and male athletes were grouped into functionally associated networks with use of IPA software (Qiagen). Three genes of interest were chosen for further validation by RT-qPCR (reverse transcription-quantitative polymerase chain reaction) analysis of the samples from all fourteen patients. Several statistical methods were used to examine sex-related differences. Results: Microarray analysis of the RNA isolated from the ruptured ACL tissue from the female and male athletes identified thirty-two genes with significant differential expression. Fourteen of these genes were not linked to the X or Y chromosome. IPA analysis grouped these genes into pathways involving development and function of skeletal muscle and growth, maintenance, and proliferation of cells. RT-qPCR confirmed significant differences in expression of three selected genes: ACAN (aggrecan) and FMOD (fibromodulin) were upregulated in female compared with male study participants, and WISP2 (WNT1 inducible signaling pathway protein 2) was downregulated. No morphological differences among the ruptured tissue from the various participants were apparent on histological examination. Conclusions: The genes identified in this study as differing distinctly according to sex produce major molecules in the ACL extracellularmatrix. Significant upregulation of ACAN and FMOD (which regulate the matrix) and downregulation of WISP2 (which is involved in collagen turnover and production) may account for the weaker ACLs in female compared with male individuals.