Novel INSL3 variants cause male infertility with cryptorchidism

Cryptorchidism is one of the most prevalent male congenital abnormalities, affecting 1.6%-9% of newborn males, and it poses substantial risks to male fertility. INSL3 and its receptor RXFP2 play a major role in the first phase of the biphasic testicular descent process. The genetic etiology of cryptorchidism has long remained controversial, and the association between INSL3 gene mutations and cryptorchidism still requires robust evidence to substantiate. This study aims to clarify that the novel homozygous frameshift variants of INSL3 are the genetic cause of cryptorchidism in patients.

Whole-exome sequencing (WES) and Sanger sequencing were performed on peripheral blood samples collected from two infertile patients with cryptorchidism. The AlphaFold database and PyMOL software were used to predict the 3D structure of INSL3 protein. In vitro analyses were performed to determine the effects of the identified INSL3 variants on protein function.

We identified two novel homozygous frameshift variants (NM_005543:c.176_182delCGACCGG:p.Ala59GlufsTer66 for F1-II-1; c.148dupC:p.Arg50ProfsTer16 for F2-II-1) in INSL3. Both variants were absent or rare from public databases. Prediction of 3D protein structure indicated that INSL3 variants caused alterations in the spatial conformation of the protein. In vitro experiments further confirmed that these variants led to the production of truncated proteins, which potentially disrupt the function of INSL3 and its interaction with RXFP2.

In this study, two novel homozygous frameshift variants in INSL3 were detected in two patients with cryptorchidism. These findings strengthen the link between INSL3 mutations and male infertility caused by cryptorchidism.