idea. In particular, INSL3 (Leydig insulin-like 3 hormone) has been proposed as putative gene for cryptorchidism, since male mice mutant for Insl3 (Insl3 -/- ) exhibit bilateral abdominal cryptorchidism (1, 2). Insl3 null mutant mice showed no abnormalities in the urogenital tract with the exception of an alteration in gubernaculum development, strongly supporting the role of this gene in testicular de- scent. INSL3 is a member of the insulin-like hormone su- perfamily (3) and is predominantly expressed in Leydig cells (4). The active INSL3 peptide is structurally characterized by a B chain and an A chain connected by a C-peptide (3, 5). The gene maps to chromosome 19p13.2-p12 (5), and con- sists of two exons: exon 1 encodes for a signal peptide (that is present in the preproprotein), the B chain and the first eight aminoacids of the C-peptide; exon 2 encodes for the rest of the C-peptide and the A chain. Preliminary studies of INSL3 in cryptorchid patients showed the presence only of sequence variants probably repre- senting polymorphisms unrelated to the phenotype, as they were found also in the control groups (6, 7). However, the sample groups were relatively small and heterogeneous. Furthermore, ethnic differences may be present. Therefore, in this study we looked for INSL3 mutations in a highly se- lected group of ex-cryptorchid patients, classified on the basis of their history (unilateral vs bilateral maldescent, as- sociated malformations, etc.), sperm count (normo-, oligo- azoospermia), and testicular histology. INTRODUCTION Cryptorchidism (impaired testicular descent) occurs in 1-2% of full-term boys and is associated with a high risk of inferti- lity and testicular cancer. Testicular descent from abdomen to scrotum during embryonic development requires the interaction of hormonal and anatomical factors, and it oc- curs in two different phases: the transabdominal and the inguinoscrotal phases. The outgrowth of gubernaculum and regression of the cranial suspensory ligament (CSL) result in the transabdominal descent into the inguinal region. During the second phase, the testes descend to the scro- tum by regression of the gubernaculum and this phase is androgen-dependent. Disruption of any of these processes results in cryptorchidism. However, the specific etiology remains for the most part unknown. Familial cases of cryptorchidism in humans suggest that it may have genetic causes and animal models support this RAPID COMMUNICATION Different insulin-like 3 ( INSL3 ) gene mutations not associated with human cryptorchidism P. Marin, A. Ferlin, E. Moro, A. Garolla, and C. Foresta Department of Medical and Surgical Sciences, Clinical Medicine III, University of Padova, Padova, Italy Key-words: Cryptorchidism, INSL3, Leydig insulin-like hormone, polymor- phism. Correspondence: Prof. Carlo Foresta, Dipartimento di Scienze Mediche e Chirurgiche, Clinica Medica III, Università di Padova, Via Ospedale 105, 35128 Padova, Italy. E-mail: forestac@protec.it Accepted January 4, 2001. RC13 ABSTRACT. Cryptorchidism is the most frequent congenital anomaly of the urogenital tract in the male, but its etiology is for the most part unknown. Evidence suggests that a possible genetic cause may be in- volved. Animal models support this hypothesis, and in particular INSL3 (Leydig insulin-like 3 hormone) has been proposed as putative gene for cryptorchidism, since male mice mutant for Insl3 exhibit bilateral ab- dominal cryptorchidism due to alteration of gubernaculum development. In this study, we analyzed whether mutations in INSL3 could be associated with human cryptorchidism. Heteroduplex analysis and sequencing of both exons of INSL3 in 65 ex-cryptorchid patients and a group of control subjects allowed us to find four nucleotide changes in the sequence of exon 1. These mutations are all single base substi- tutions from G to A at position 27, 96, 126 and 178. Only the 178G→A substitution changes codon 60 from alanine to threonine (A60T). All mutations were found in comparable distribution in ex-cryptorchid patients and non-cryptorchid men. Therefore, all mutations represent neutral polymorphisms not asso- ciated with phenotype. This study confirms previous observations and demonstrates a novel polymor- phism in the INSL3 gene. In contrast to that described for the mutant mouse, these data indicate that mutations of INSL3 do not seem to represent a frequent cause of cryptorchidism. (J. Endocrinol. Invest. 24: RC13-RC15, 2001) © 2001, Editrice Kurtis J. Endocrinol. Invest. 24: RC13-RC15, 2001