Immunofluorescent Analysis of Testicular Biopsies with Germ Cell and Sertoli Cell Markers Shows Significant MVH Negative Germ Cell Depletion with Older Age at Orchiopexy Ruili Li,* Jorgen Thorup, Cong Sun, Dina Cortes, Bridget Southwell and John Hutson From the Surgical Research Laboratory (RL, BS, JH), and Environmental and Genetic Epidemiology Research (CS), Murdoch Childrens Research Institute, Department of Pediatrics (RL, CS, BS, JH), University of Melbourne and Urology Department, Royal Children’s Hospital (JH), Parkville, Victoria, Australia, and Department of Pediatric Surgery, Rigshospitalet (JT) and Faculty of Health Science, University of Copenhagen (JT, DC), Copenhagen, Denmark Purpose: Undescended testis is the most common defect in male newborns. This condition is associated with increased risks of infertility and testicular malig- nancy due to abnormal germ cell development in the testes. Early surgery may limit such risks. We analyzed germ cell development vs age at orchiopexy using a germ cell marker and a Sertoli cell marker on testicular biopsies. Materials and Methods: A total of 22 testicular biopsies at orchiopexy in 20 patients 5 to 24.5 months old were fixed and embedded in paraffin. Sections were processed and labeled with AMH antibody for Sertoli cells and MVH antibody for germ cells for immunofluorescent histochemical analysis. Confocal images were counted using ImageJ (National Institutes of Health, Bethesda, Maryland) for germ cells and testicular tubules. The data were analyzed using linear regression. Results: Sertoli cells were clearly distinguished from MVH positive and negative germ cells located centrally or on basement membranes of tubules. Percentage of tubules with MVH negative germ cells significantly decreased with increasing age at orchiopexy (b ¼À0.03, p ¼ 0.03). Total tubular numbers and “empty” tu- bules without germ cells significantly increased with age at orchiopexy (b ¼ 1.15, p ¼ 0.02 and b ¼ 0.44, p ¼ 0.04, respectively). Conclusions: AMH antibody distinguished Sertoli cells from germ cells, and MVH antibody distinguished 2 types of germ cells at different developmental stages. Biopsy at orchiopexy in older patients showed significant germ cell depletion. These results lend support to early surgery to optimize germ cell number. Key Words: cryptorchidism, germ cells, orchiopexy, Sertoli cells, testis UNDESCENDED testis is a major prob- lem in male newborns, with 1% to 2% of boys requiring orchiopexy to pull the testis down into the scrotum. 1,2 Cryptorchidism is associated with germ cell loss leading to infertility. 3,4 Depletion of germ cells may be pri- mary but it may also be secondary to abnormally high temperature of the undescended testis. 5 Long-term fol- lowup studies of men with previous cryptorchidism reveal a fivefold to tenfold increase in testicular malig- nancy risk. 6,7 The timing of surgery for orchiopexy may be important to prevent these problems, and knowl- edge of early postnatal germ cell development might help to optimize Abbreviations and Acronyms AD-S ¼ adult dark spermatogonia AMH ¼ anti-Mullerian hormone GC ¼ germ cell GCs/T ¼ germ cells per tubule MVH ¼ mouse VASA homologue PLAP ¼ placental alkaline phosphatase UDT ¼ undescended testes Accepted for publication August 26, 2013. Supported by NH&MRC Grant 607365 and the Victorian Government Operational Infra- structure Support Program. Study received institutional ethics committee approval (No. KF-01299830). * Correspondence: ruili.li@mcri.edu.au; john. hutson@rch.org.au . 458 j www.jurology.com 0022-5347/14/1912-0458/0 THE JOURNAL OF UROLOGY ® © 2014 by AMERICAN UROLOGICAL ASSOCIATION EDUCATION AND RESEARCH,INC. http://dx.doi.org/10.1016/j.juro.2013.08.075 Vol. 191, 458-464, February 2014 Printed in U.S.A.