Hypospadias is a common congenital abnormality. In the Netherlands 1:200/300 boys is born with hypospadias. During hypospadias surgery there may be not enough local tissue. Oral mucosa is widely used in adults for urethroplasties and repair after failed hypospadias. Little is known about how oral mucosa reacts on the hormonal effect of puberty. To predict whether oral mucosa can safely be used in prepubertal hypospadias patients, we investigated the androgen receptor (AR) localization in oral mucosa, and tested testosterone sensitivity of cells isolated from oral mucosa.

Surgical waste (oral mucosa) from onlay urethroplasty, was collected under local biobank protocol. Part of the tissue was embedded in paraffin and tissue sections were prepared. Tissue sections were used in hemotoxilin and eosin (HE) staining, but also in immunohistochemistry for proliferation marker Ki-67 and AR. We collected tissue from 15 different patients. Human male urethra and oral mucosa as well as urethra from fertile male rats were used as controls for AR staining.
From five of the fifteen patients, we isolated cells. Using keratinocyte and fibroblast markers we optimized the efficiency of keratocytes isolation.

AR staining of rat oral mucosa showed nuclear localization in the basal layer, and cytoplasmic localization in the apical layer. The basal layer was Ki-67 positive, associated with cell proliferation. In oral mucosa from patients a diverse pattern was seen: in hypospadias patients cytoplasmic AR localization was seen throughout all layers of the epithelium, in other patients localization was similar to the rat mucosa: nuclear localization in the basal layer. In all oral mucosa samples the basal layer was Ki-67 positive. The human and rat urethra both showed nuclear localized AR expression, with less cells positive in the human sample. Human oral keratinocytes could be isolated from four of the five patients, from one patients cells stopped dividing after two passages and we were unable to test the cells for androgen sensitivity. One patient cell line exposed to testosterone showed switch from cytoplasmic to nuclear localized AR after 30 minutes, indicating active testosterone signaling. We are currently performing testosterone treatment for the other three patients cell lines.

In most oral mucosa AR is localized in the nucleus in the basal layer, indicating that the testosterone signaling is active in these cells. Active testosterone signaling overlaps with Ki-67 staining, which is a marker for proliferation. This finding suggests that graft taken from oral mucosa is sensitive to androgens and might grow during puberty like other hormone sensitive tissue. We found that in oral mucosa from hypospadias patients, androgen receptors were localized in the cytoplasm throughout the whole thickness of the epithelium. As we do not know the genetic background of these patients, we can only speculate that these patients have an aberrant testosterone signaling. However, in  isolated cells from a hypospadias  patient, testosterone treatment induced nuclear translocation of the androgen receptor, indicating active testosterone signaling.

Preliminary observations indicate that human oral keratinocytes are sensitive to testosterone, predicting that oral mucosa used for urethral reconstruction will keep up with penile growth during puberty.

Continence 2S2 (2022) 100457
DOI: 10.1016/j.cont.2022.100457