5th Joint Meeting of ESPU-SPU - Virtual

S1: BASIC RESEARCH 1

Moderators: Martin Kaefer (USA)

ESPU-SPU Meeting on Thursday 23, September 2021, 12:25 - 13:19


12:25 - 12:28
S1-1: Withdrawn (video presentation not uploaded)
 
12:28 - 12:31
S1-2: Withdrawn (video presentation not uploaded)
 

12:31 - 12:34
S1-3 (SO)

CAN PENTRAXIN-3 PREDICT APOPTOSIS IN EXPERIMENTAL TESTICULAR TORSION?

Ozkan CESUR 1, Levent DUMAN 2, Ilter ILHAN 3 and Kanat GULLE 4
1) Ankara Training and Research Hospital, Pediatric Surgery, Ankara, TURKEY - 2) Süleyman Demirel University Medical School, Department of Pediatric Surgery, Isparta, TURKEY - 3) Medical School, Suleyman Demirel University, Department of Medical Biochemistry, Isparta, TURKEY - 4) Süleyman Demirel University Medical School, Department of Histology and Embryology, Isparta, TURKEY

INTRODUCTION

Rapid diagnosis in testicular torsion provides protecting functions and preventing loss. Experimental study shows diagnosis using serum pentraxin-3(PTX3) and apoptotic pathway in germ cells-specific cell death following acute ischemia.

MATERIAL AND METHODS

16 male Wistar albino rats were divided as control group (non-operated group, n=8) and testicular torsion group(n=8). Testicular torsion group had rotating left testicle with its cord720 degrees clockwise and fixing testicle to scrotum’s internal surface. At 3 hours after torsion, both testes were harvested for histopathological, immunohistochemical and western-blotting (caspase-3, 8, 9) studies, and blood samples were collected for PTX-3 levels, antioxidant status(TAS), oxidant status(TOS) and oxygen saturation index(OSI).

RESULTS

Mean plasma PTX-3 level was higher in testicular torsion group (2.78±0,70 ng/ml vs. 1.89±0,84 ng/ml, p=0,032). Mean plasma TAS, TOS and OSI values were increased in testicular torsion group  (0,650±0,073vs0,779±0,075;6,609±3,861vs3,609±1,823;1,037±0,484vs0,458±0,199;p>0.05, respectively). Caspase-3 immune-reactivity level was observed higher in torsion group(p=0,001) and also found correlation with high Caspase-3 immune-reactivity between increased-PTX-3 level(p=0,001). Germ cell desquamation and disorganization were seen in seminiferous tubules in torsion group(p=0,001), and congestion was detected in interstitial capillary space. Johnsen score was found being decreased in torsion group(p=0,001).

CONCLUSIONS

Testicular torsion causes loss of spermatogenesis and significant increase in germ cell apoptosis due to increase in testicular oxidative stress arising with recruitment of neutrophils. Experimental studies might reveal testicular damage pathways. Present study proposed that PTX-3 can be used as early diagnostic marker predicting apoptosis in testicular torsion, and results obtained with apoptosis will help developing new treatment strategies for testicular salvage.


12:34 - 12:37
S1-4 (SO)

★ NEXT GENERATION SEQUENCING IN HYPOSPADIAS: A CASE-CONTROL STUDY OF 576 CHILDREN

Nicolas KALFA 1, Anne BERGOUGNOUX 1, Pascal PHILIBERT 1, Alice FAURE 2, Rachel REYNAUD 3, Kathy WAGNER 4, Jean BRÉAUD 5, Eric DOBREMEZ 6, Laura GASPARI 1, Cyril AMOUROUX 1, Nadège FAUCONNET-SERVANT 1, Charles SULTAN 1 and Françoise PARIS 1
1) Université de Montpellier, National Reference Center of Genital Development CRMR DEV-GEN Constitutif Sud, Montpellier, FRANCE - 2) Assistance Publique Hopitaux de Marseille, Service de Chirurgie et Urologie pédiatrique, Marseille, FRANCE - 3) Assistance Publique Hopitaux de Marseille, Unité d'Endocrinologie et de Diabétologie Pédiatrique, Marseille, FRANCE - 4) Hôpital Lenval, CHU Nice, Service Pédiatrie, Nice, FRANCE - 5) Hôpital Lenval, CHU Nice, Service de Chirurgie et Urologie pédiatrique, Nice, FRANCE - 6) Hopital Pellegrin Enfant, Service de Chirurgie Pédiatrique, Bordeaux, FRANCE

PURPOSE

The pathophysiology of hypospadias remains undetermined. Next-generation-sequencing offers a major breakthrough for exploring human disease but has yet to be applied to hypospadias. We aimed to explore a large panel of 336 candidate genes from hypospadiac patients and identify new, frequently associated variants.

MATERIAL AND METHODS

Prospective multicenter case-control study with 576 participants. Cases were children with hypospadias without pathogenic variants in the main candidate genes. Controls were matched for ethnicity. After targeted- exome-sequencing of 336 independent loci, variant filtering and calling were performed using validated algorithms. Comparison of distribution of frequent variants (minor allele frequency >0.01) in cases and controls using the PLINK reference software in different transmission model hypotheses.

RESULTS

Nineteen frequent variants on 16 loci showed a p-value below 10-2. Seven of them were previously reported to be associated with other human conditions (cleft palate, myopia, cardiac defects and Hirschsprung disease). In addition to confirming two previously associated DGKK variants, we identified new variants possibly involved in hypospadias, especially in TGFBR3 and other genes implicated in the TGFβ pathway (TGIF1, ZEB1, NTF3 and FGFR2), which regulates testis development and spermatogenesis. Other SNPs showed a significant trend in PPARGC1b, which is expressed in Leydig cells and germ cells; GLI4 and SUFU, two genes implicated in the Shh pathway in the urethral plate; and IRF6, which has been reported in midline defects.

CONCLUSIONS

Next-generation-sequencing applied to unexplained hypospadias identified at-risk SNPs compared to controls and highlighted original candidate genes and pathways. Future studies should test whether this genetic background indicates a susceptibility to endocrine-disrupting chemicals.


12:37 - 12:40
S1-5: Withdrawn (video presentation not uploaded)
 

12:40 - 12:43
S1-6 (SO)

CREATION OF TISSUE ENGINEERED URETHRAL CONSTRUCTS FOR PROXIMAL HYPOSPADIAS REPAIR IN A RABBIT EXPERIMENTAL MODEL

Maria Virginia AMESTY 1, Beatriz SANZ 2, Roberto LOBATO 1, Susana RIVAS 1, Maria Jose MARTINEZ URRUTIA 1 and Pedro LOPEZ PEREIRA 1
1) Hospital Universitario La Paz, Pediatric Urology, Madrid, SPAIN - 2) Hospital Universitario La Paz, Research, Madrid, SPAIN

PURPOSE

Tissue engineering is a potential treatment for urethral defects. Our aim is to create tissue-engineered urethral constructs from cells obtained with a non-invasive method, and test them in a proximal hypospadias rabbit model.

MATERIAL AND METHODS

An experimental study was conducted. Hypospadias model was created in male New-Zealand rabbits resecting the ventral penile urethra. Urethral constructs were created from bladder washing cells, seeded in a decellularized intestinal submucosa matrix (SIS-Cook-Biotech®). Constructs were tested in the model. Twenty-two rabbits were divided into 3 groups. Group-A (n=2) was control group (hypospadias unrepaired). Group-B (n=10) and group-C (n=10) underwent an Onlay urethroplasty, using the matrix unseeded in group-B; and the construct in group-C. Macroscopic, radiological and pathological results were assessed.

RESULTS

The model was successfully created. Stratified urothelial cultures were obtained from bladder washing and matrix cell adhesion was achieved. All group-A rabbits kept the urethral defect unchanged. All group-B rabbits presented urethroplasty dehiscence (median urethral repaired surface=15(5-20)%). In group-C, 5 presented complete urethral correction, and 5 almost total correction with small fistula, (median urethral repaired surface=97.5(70-100)%), demonstrating better results (p=7.85x10-5). Urethrography showed more fistulas in group-B (10/10, versus 5/10 in group-C)(p=0.04). No strictures were identified. Group-B pathology identified absence of ventral urethra in unrepaired areas, with dorsal urethra squamous metaplasia. In repaired areas of group-C, ventral multilayer urothelium was identified.

CONCLUSIONS

Bladder washing is a viable non-invasive cell source to create stratified urothelium. Urethroplasty with urethral constructs is feasible and presents superior results compared to using an isolated matrix in the rabbit model.


12:43 - 12:46
S1-7 (SO)

A HISTOLOGICAL AND RNA-SEQ STUDY OF THE DIVERGENT CORPUS SPONGIOSUM IN HYPOSPADIAS

Yichen HUANG 1, Hua XIE 1, Yiqing LYU 1, Zhengjun XI 2, Lujie SONG 3 and Fang CHEN 3
1) Shanghai Children's Hospital, Urology, Shanghai, CHINA - 2) Shanghai Children's Hospital, Pathology, Shanghai, CHINA - 3) Shanghai 6th People's Hospital, Urology, Shanghai, CHINA

PURPOSE

To investigate the histological and RNA-seq changes of the divergent corpus spongiosum in hypospadias

MATERIAL AND METHODS

Seventy hypospadias boys from 9 months to 4 years old were included and divided into the distal (n=44) and the proximal (n=26) groups according to the location of the ectopic meatus. Another 7 cases with the urethral rupture were taken as the controls. The histopathological features of the corpus spongiosum were evaluated by HE staining, masson staining and immunohistochemical study of α-SMA, CD34. RNA-seq was conducted for 7 proximal hypospadias and 5 controls.

RESULTS

Histological evaluation suggested an abnormal structure of the cavernous sinus in the hypospadias group, which is characterized by a wider vascular lumen (control 7.20 ± 1.12μm, hypospadias 13.75 ± 8.08μm,p=0.0068), a thicker vascular wall (control 5.40 ± 1.28μm, hypospadias 14.11 ± 7.59μm,p=0.0006), decreased vascular density (control 7.24 ± 4.19, hypospadias 15.66 ± 1.17, p<0.0001), decreased trabecula density (control 3.68 ± 2.87, hypospadias 9.80 ± 1.92, p<0.0001). The severity of the structural abnormity was more obvious in the proximal group. The results of RNA-seq suggested there were 334 significantly overexpressing genes and 1233 down-regulated genes in hypospadias group. KEGG analysis showed significant pathway of DEGs were enriched in complement and coagulation cascades, cell adhesion molecules and  cytokine−cytokine receptor interactions.

CONCLUSIONS

The histological variation of the structure is positively correlated with the severity of hypospadias. RNA-seq provideds the potential pathways for studying the etiology of hypospadias.


12:46 - 12:49
S1-8 (SO)

GENETIC INTERACTIONS CAUSE DIVERSE DEVELOPMENTAL PHENOTYPES OF HYPOSPADIAS

Zhongzhong CHEN, Hua XIE and Fang CHEN
Shanghai Children's Hospital, Department of Urology, Shanghai, CHINA

PURPOSE

Common variants have been identified in hypospadias using genome-wide association studies (GWASs). However, the GWASs of hypospadias cohort only explained 9.5% of the genetic variance. The rare coding variants with larger effects on risk of hypospadias are poorly understood, and the genetic etiology of hypospadias is still unclear.

MATERIAL AND METHODS

To identify rare coding variants with large effects on hypospadias risk, we carried out whole exome sequencing (WES) in five severe patients with the variable outcomes in a large hypospadias family with an X-linked recessive inheritance. SNVs and Indels variants were annotated based on the Variant Effect Predictor (VEP) and Ensembl canonical and APPRIS transcripts. Each variant was classified into groups of LoF (loss of function), missense, synonymous and others using the Sequence Ontology.

RESULTS

Rare damaging variant p.R841H in AR gene was identified in three patient and p.T353M in HSD3B1 gene was found in two patients. Additionally, two rare damaging variants (p.E293* and p.G121C) in one gene SLC25A5, located on the X chromosome, were identified in all patients. All of these two damaging mutants are very rare in the ExAC database and 1000 Genomes Project, with minor allele frequency (MAF) < 0.0001. Patients with different rare damaging variants combinations were observed to have different hypospadias phenotypes.

CONCLUSIONS

In conclusion, previous knowledge of genetic variants that affect hypospadias risk is primarily based on GWASs of common variants. This study indicates that these genetic interactions of rare damaging variants rather single mutation yielded hypospadias with the variable outcomes. These mutations provide new insight into etiology of genetic contribution of hypospadias.


12:49 - 12:52
S1-9 (SO)

GENETICS OF HYPOSPADIAS IN CONSANGUINOUS CASES AND FAMILIAL HISTORY: WHOLE EXOME SEQUECNING IS AN INTERESTING TOOL

Hélène MOREL 1, Alexandre ROUEN 1, Nicolas KALFA 2, Valeska BIDAULT 3, Geneviève QUENUM-MIRAILLET 1, Anne BERGOUGNOUX 4, Sandra CHANTOT-BASTARAUD 1, Christine GRAPIN 3, Muriel HOUANG 5, Laetitia MARTINERIE 6, Georges AUDRY 7, Marie LEGENDRE 1, M. Francesca MONN 8, Agathe VEILLET-LAVALLEE 1, Annabel PAYE-JAOUEN 3, Serge AMSELEM 1, Alaa EL GHONEIMI 3, Pascal PHILIBERT 4, Jean-Pierre SIFFROI 9, Matthieu PEYCELON 10, Matthieu PEYCELON 1 and Matthieu PEYCELON 3
1) Sorbonne Université, INSERM UMRS_933, Maladies génétiques d'expression pédiatrique, APHP, Hôpital d'Enfants Armand Trousseau, Paris, FRANCE - 2) Pediatric Surgery and Urology, CHU de Montpellier; Université de Montpellier; National Reference Network DSD DevGen, Centre Constitutif Sud, Montpellier, FRANCE - 3) Pediatric Urology, Robert-Debré Hospital, AP-HP; University of Paris; National Reference Center DSD MERC, Paris, FRANCE - 4) Molecular Genetics, CHU de Montpellier; Université de Montpellier; National Reference Network DSD DevGen, Centre Constitutif Sud, Montpellier, FRANCE - 5) Sorbonne Université, Pediatric Endocrinology, APHP, Hôpital d'Enfants Armand Trousseau; National Reference Center DSD MERC, Paris, FRANCE - 6) Pediatric Endocrinology, Robert-Debré Hospital, AP-HP; University of Paris; National Reference Center DSD MERC, Paris, FRANCE - 7) Sorbonne Université, Pediatric Surgery, APHP, Hôpital d'Enfants Armand Trousseau; National Reference Center DSD MERC, Paris, FRANCE - 8) Pediatric Urology of Riley Children Hospital; Indiana University, School of Medicine, Indianapolis, USA - 9) Sorbonne Université, INSERM UMRS_933, Maladies génétiques d'expression pédiatrique, APHP, Hôpital d'Enfants Armand Trousseau; National Reference Center DSD MERC, Paris, FRANCE - 10) Pediatric Urology, Riley Children Hospital at Indiana University Health; Indiana University School of Medicine, Indianapolis, USA

PURPOSE

Hypospadias is the most common malformation affecting male genitalia and its incidence is increasing. Beside a strong environmental contribution to this phenotype, the heritability has been estimated at 54-77%, with familial clustering in about 10%. Most cases remain undiagnosed at the molecular level. The aim of this study was to identify new genes and new variants involved in hypospadias using Whole Exome Sequencing (WES), on consanguineous patients or with a relevant familial history.

MATERIAL AND METHODS

Genomic DNA was extracted from blood lymphocytes using standard techniques. Constitutional WES using NovaSeq and Hiseq4000 (Illumina) was performed on 30 patients born with hypospadias (57% distal, 43% proximal): 10 patients were consanguineous, and 20 patients had an affected relative that was also included in the cohort. Statistical analysis: Fisher’s exact test.

RESULTS

15 variants were identified in genes previously associated with the phenotype and reported in the literature (SOX8, CYR61, ZFHX3, PDGFC, SAMD9, SCARB1, EXOC3, ATF3, NR5A1, CYP11A1, APOE, NR2F1, NR5A2, RTN4) in these 30 patients: 13 in case of familial history (5/10 families) and 2 in consanguineous patients (p<0.05). Moreover, in seven families, we also identified at least one variant in new genes that appeared relevant in hypospadias (steroid or developmental pathways). No significant variant was identified in 3 families (30%) and 8 consanguineous patients (80%) (p=0.02).

CONCLUSIONS

Whole exome sequencing is an interesting tool, especially in case of familial history. We identified several variants in genes already described to be implicated in the pathology and some other of unknown significance that will need to be further replicated and investigated with functional studies. According to our results, hypospadias could be associated with a large genetic heterogeneity, and maybe with an oligogenic inheritance.


12:52 - 13:19
Discussion