W.F.J. Feitz

Aetiology of hypospadias: a systematic review of genes and environment

BACKGROUND Hypospadias is a common congenital malformation of the male external genitalia. Most cases have an unknown aetiology, which is probably a mix of monogenic and multifactorial forms, implicating both genes and environmental factors. This review summarizes current knowledge about the aetiology of hypospadias. METHODS Pubmed was used to identify studies on hypospadias aetiology published between January 1995 and February 2011. Reference lists of the selected manuscripts were also searched to identify additional studies, including those published before 1995. RESULTS The search provided 922 articles and 169 articles were selected for this review. Studies screening groups of patients with hypospadias for single gene defects found mutations in WT1, SF1, BMP4, BMP7, HOXA4, HOXB6, FGF8, FGFR2, AR, HSD3B2, SRD5A2, ATF3, MAMLD1, MID1 and BNC2. However, most investigators are convinced that single mutations do not cause the majority of isolated hypospadias cases. Indeed, associations were found with polymorphisms in FGF8, FGFR2, AR, HSD17B3, SRD5A2, ESR1, ESR2, ATF3, MAMLD1, DGKK, MID1, CYP1A1, GSTM1 and GSTT1. In addition, gene expression studies indentified CTGF, CYR61 and EGF as candidate genes. Environmental factors consistently implicated in hypospadias are low birthweight, maternal hypertension and pre-eclampsia, suggesting that placental insufficiency may play an important role in hypospadias aetiology. Exogenous endocrine-disrupting chemicals have the potential to induce hypospadias but it is unclear whether human exposure is high enough to exert this effect. Other environmental factors have also been associated with hypospadias but, for most, the results are inconsistent. CONCLUSIONS Although a number of contributors to the aetiology of hypospadias have been identified, the majority of risk factors remain unknown.

Common variants in DGKK are strongly associated with risk of hypospadias

Hypospadias is a common congenital malformation of the male external genitalia. We performed a genome-wide association study using pooled DNA from 436 individuals with hypospadias (cases) and 494 controls of European descent and selected the highest ranked SNPs for individual genotyping in the discovery sample, an additional Dutch sample of 133 cases and their parents, and a Swedish series of 266 cases and 402 controls. Individual genotyping of two SNPs (rs1934179 and rs7063116) in DGKK, encoding diacylglycerol kinase κ, produced compelling evidence for association with hypospadias in the discovery sample (allele-specific odds ratio (OR) = 2.5, P = 2.5 × 10−11 and OR = 2.3, P = 2.9 × 10−9, respectively) and in the Dutch (OR = 3.9, P = 2.4 × 10−5 and OR = 3.8, P = 3.4 × 10−5) and Swedish (OR = 2.5, P = 2.6 × 10−8 and OR = 2.2, P = 2.7 × 10−6) replication samples. Expression studies showed expression of DGKK in preputial tissue of cases and controls, which was lower in carriers of the risk allele of rs1934179 (P = 0.047). We propose DGKK as a major risk gene for hypospadias.

Genome-wide association analyses identify variants in developmental genes associated with hypospadias

Hypospadias is a common congenital condition in boys in which the urethra opens on the underside of the penis. We performed a genome-wide association study on 1,006 surgery-confirmed hypospadias cases and 5,486 controls from Denmark. After replication genotyping of an additional 1,972 cases and 1,812 controls from Denmark, the Netherlands and Sweden, 18 genomic regions showed independent association with P < 5 × 10−8. Together, these loci explain 9% of the liability to developing this condition. Several of the identified regions harbor genes with key roles in embryonic development (including HOXA4, IRX5, IRX6 and EYA1). Subsequent pathway analysis with GRAIL and DEPICT provided additional insight into possible genetic mechanisms causing hypospadias.

In utero Repair of an Experimental Neural Tube Defect in a Chronic Sheep Model Using Biomatrices

Objective: Persistent exposure of the unprotected spinal cord to amniotic fluid and the uterine wall can lead to progressive damage of neural tissue in case of a myelomeningocele (two-hit hypothesis). The aim of this study was to evaluate whether in utero repair of an experimental neural tube defect in a fetal lamb could protect neural tissue from secondary injury and save neurologic functions after birth. Methods:In 19 fetal lambs, a neural tube defect was created at 79 days’ gestation. In 12 lambs the defect was covered either with a novel, molecular defined collagen-based biocompatible and biodegradable matrix (UMC) or with a small intestinal submucosa (SIS) biomatrix (Cook®) or by closing the skin over the defect. Results: All lambs with the defect covered showed no or minor neurologic morbidity in contrast to the lambs with the defect uncovered in which major neurologic morbidity was seen. Conclusions:These results demonstrate that long-term exposure of the open spinal cord to the intrauterine environment can lead to damage of neural tissue and, consequently loss of neurologic functions and that coverage of the defect can lead to a better neurologic outcome. Furthermore, we could show that a UMC biomatrix and an SIS biomatrix are useful for in utero coverage of a surgically created neural tube defect in our model.

Urethral reconstruction of critical defects in rabbits using molecularly defined tubular type I collagen biomatrices: key issues in growth factor addition.

Tubular type I collagen biomatrices with and without growth factors (GFs) were constructed and evaluated in a rabbit model for critical urethral defects. Porous tubular biomatrices with an inner diameter of 3  mm were prepared using highly purified collagen fibrils and were crosslinked with or without heparin. Heparinized biomatrices were supplemented with the heparin-binding GFs vascular endothelial GF, fibroblast GF-2, and heparin-binding epidermal GF. Biomatrices with and without GFs were used to replace a critical 1 cm urethral segment in rabbits (n = 32). All animals showed normal urination without urinary retention. General histology and immunohistology of graft areas (2, 4, 12, and 24 weeks after implantation) indicated that all biomatrices were replaced by urethra-like structures with normal appearing cytokeratin-positive urothelium surrounded by vascularized tissue. The GF-containing biomatrices showed an increase in extracellular matrix deposition, neovascularization, urothelium, glands, granulocytes, and fibroblasts, compared with biomatrices without GF. GFs substantially improved molecular features of healing but failed to be superior in functional outcome. Retrograde urethrography indicated a normal urethral caliber in case of biomatrices without GF, but a relative narrowing of the urethra at 2 weeks postsurgery and diverticula after 4 weeks in case of biomatrices with GF. In conclusion, tubular acellular type I collagen biomatrices were successful in repairing urethral lesions in artificial urethral defects, and inclusion of GF has a profound effect on regenerative processes.

Rabbit urethra replacement with a defined biomatrix or small intestinal submucosa.

OBJECTIVE The evaluation of collagen-based biomatrix (SIS COOK((R))) in comparison to a biochemically reconstructed biomatrix for replacement of the urethra in a rabbit model as a preclinical model. MATERIAL AND METHODS Rabbits underwent partial urethra replacement (resection of 0.5 to 1.0 cm segment of the urethra), which was replaced with 1 or 4 layers Small Intestinal Submucosa (SIS COOK) patch grafts or with a biochemically defined collagen biomatrix, partly sutured with unresolvable sutures for future reference. Six animals underwent a sham control operation. The grafts of regenerated urethras were harvested at 1, 3 and 9 months after implantation. Urethrography was performed pre-operatively and before sacrificing. The animals were evaluated macroscopically and by routine histology and immunohistochemistry. RESULTS At 1 month after implantation, the biomatrices (1 layer, 4 layers and our biochemically defined biomatrix) were well distinguishable from the normal surrounding tissues and showed blood vessels at the periphery. Macroscopically, the unresolvable reference sutures were easy to find at all time points. At 3 months the graft was still distinguishable in the 4 layers SIS group. In the 1 layer and the defined biomatrix group a good regeneration of the urethra within the graft was seen with some central fibrosis. Histological and immunohistochemical evaluation showed urothelium regeneration on the 1 layer and on biochemically defined biomatrix with decreasing number of inflammatory cells from 1 month on. In the group treated with 4 layers SIS the urothelium was completely regenerated at 3 months. Histologically, the regeneration of muscle cells in the three biomatrices was comparable. The smooth muscle cells regenerated very slowly as 1 month after implantation no muscle cells were detectable within the grafts. At 3 months a few muscle cells were present in the graft, but cell density did not increase in the following 6 months. Strictures were not observed on control urethrography pre-operatively in the animals. In one case slight narrowing of the urethra on urethrography was seen, but apparently without causing voiding problems. One rabbit developed a fistula near the operation site. CONCLUSION The biomatrices investigated are feasible scaffolds to repair urethral lesions. The results with our biochemically defined biomatrix are comparable to one layer Small Intestinal Submucosa. Almost no smooth muscle cells population was observed after nine months for the three biomatrices. We conclude that an improved molecularly defined biomatrix focussed on stimulation of smooth muscle cell growth may be necessary to obtain optimal cellular grafting results.

Antibodies to cytokeratin and vimentin in testicular tumour diagnosis

Thirteen primary and metastatic testicular germ cell tumours, including classical and anaplastic seminomas, and non-seminomatous testicular tumours were examined for their intermediate filament protein (IFP) types. The seminomas were shown to react with a monoclonal and a polyclonal antibody to bovine lens vimentin, while non-seminomatous germ cell tumours were strongly positive for a polyclonal and a monoclonal antibody to cytokeratin. In one case of seminoma with elevated serum levels ofβHCG andαFP, cytokeratin positive tumour cells were found. In the case of teratocarcinoma, several components of the tumour could be distinguished using a combination of antisera in double-label immunofluorescence microscopy. The glandular component of this tumour was positive with the polyclonal antikeratin, but also with the monoclonal cytokeratin antibody specific for glandular epithelia (RGE 53). However, the squamous component was negative with this latter antibody. Strikingly, the spindle cell component showed focal positivity for vimentin, with coexpression of cytokeratin and vimentin in some cells. Our data show that antibodies to cytokeratin and vimentin can be helpful in the diagnosis of testicular germ cell tumours, especially in the differentiation between seminomas and non-seminomatous tumours.

Genetics of hypospadias: are single-nucleotide polymorphisms in SRD5A2, ESR1, ESR2, and ATF3 really associated with the malformation?

CONTEXT Hypospadias is a common congenital malformation of the male external genitalia with a multifactorial etiology. Little is known about the genes involved in hypospadias. A few genetic associations have been reported but mainly in studies of small sample size. Most of these associations have not been replicated. OBJECTIVE The aim of this study was to investigate whether previously reported associations for four single-nucleotide polymorphisms (SNPs) in genes involved in hormonal pathways could be replicated in a large Dutch hypospadias sample. The SNPs investigated are rs523349 in steroid-5 alpha-reductase (SRD5A2), rs6932902 in estrogen receptor 1 (ESR1), rs2987983 in ESR2, and rs11119982 in activating transcription factor 3 (ATF3). DESIGN, PARTICIPANTS, AND METHODS We genotyped 620 Caucasian hypospadias cases and 596 controls for these SNPs using TaqMan-based genotyping. RESULTS We did not replicate the associations of the SNPs in SRD5A2 and ESR1 with hypospadias. The SNPs in ESR2 and ATF3 were borderline associated with hypospadias [odds ratios 0.9 (95% confidence interval 0.7-1.0) and 1.2 (95% confidence interval 1.0-1.4), respectively] but in the opposite direction compared with earlier publications. Stratification according to localization of the urethral opening produced comparable results in the subgroups. CONCLUSIONS The lack of consistency between our and previously performed studies might represent spurious results or chance findings in our or the earlier studies, differences in criteria used to select the study populations, or a real difference between populations, i.e. different genes contributing to disease risk. These results once again confirm the importance of replication in genetic association approaches.

Urotherapy in children: quantitative measurements of daytime urinary incontinence before and after treatment according to the new definitions of the International Children's Continence Society

OBJECTIVE To assess the effectiveness of urotherapy in children with lower urinary tract dysfunction, according to the new definitions of the International Childrens Continence Society. MATERIAL AND METHODS We performed a retrospective review of 122 children (aged 8.8 ± 2.0 years) treated in an outpatient program for lower urinary tract dysfunction. Exclusion criteria included all neurologic abnormalities. In 98 children (80%) daytime urinary incontinence was a predominant symptom. Therapy consisted of an individually adapted drinking and voiding schedule, pelvic floor relaxation, instructions on toilet behavior, biofeedback uroflowmetry and if necessary recommendations for regulation of defecation. Before and at the end of training, patients were evaluated for number and severity of daytime wet accidents per week, using a scoring system to grade the severity of incontinence. Secondary measurements of accompanying voiding symptoms were performed. RESULTS Of the 90 children with daytime urinary incontinence for whom sufficient objective data were collected, 42% became completely dry during the daytime and 36% showed a 50% or greater level of response. Secondary measurements showed a significant reduction in daily voiding frequency (mean 7.0 ± .3, P<0.0001) and mean post-void residual (P<0.003), and an improvement in flow pattern (P<0.05). CONCLUSIONS Urotherapy is successful for the treatment of daytime urinary incontinence in children. Additional benefit was evident in improvement of accompanying voiding symptoms. A combination of the definitions of the International Childrens Continence Society and a scoring system to grade severity improved the evaluation method. Further research into long-term efficacy will be performed.

Tissue Engineered Tubular Construct for Urinary Diversion in a Preclinical Porcine Model

PURPOSE The ileal conduit has been considered the gold standard urinary diversion for patients with bladder cancer and pediatric patients. Complications are mainly related to the use of gastrointestinal tissue. Tissue engineering may be the technical platform on which to develop alternatives to gastrointestinal tissue. We developed a collagen-polymer conduit and evaluated its applicability for urinary diversion in pigs. MATERIALS AND METHODS Tubular constructs 12 cm long and 15 mm in diameter were prepared from bovine type I collagen and Vypro® II synthetic polymer mesh. Characterized tubes were sterilized, seeded with and without primary porcine bladder urothelial cells, and implanted as an incontinent urostomy using the right ureter in 10 female Landrace pigs. At 1 month the newly formed tissue structure was functionally and microscopically evaluated by loopogram and immunohistochemistry, respectively. RESULTS The survival rate was 80% with 1 related and 1 unrelated death. By 1 month the collagen was resorbed and a retroperitoneal tunnel had formed that withstood 40 cm H(2)O water pressure. In 5 cases the tunnel functioned as a urostomy. Histological analysis revealed a moderate immune response, neovascularization and urothelial cells in the construct lumen. The polymer mesh provoked fibroblast deposition and tissue contraction. No major differences were observed between cellular and acellular constructs. CONCLUSIONS After implanting the tubular constructs a retroperitoneal tunnel was formed that functioned as a urinary conduit in most cases. Improved large tubular scaffolds may generate alternatives to gastrointestinal tissue for urinary diversion.