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 (PP)

MECHANISM OF ROS MEDIATED MAPK SIGNALING PATHWAY IN IMPAIRMENT OF BLOOD-TESTIS BARRIER CAUSED BY PM2.5

Guanghui WEI, Xing LIU, Deying ZHANG and Dawei HE
Children's Hospital of Chongqing Medical University, Urology, Chongqing, CHINA

PURPOSE

To explore the reason and molecular mechanism of spermatogenesis dysfunction caused by PM2.5, and to investigate whether combined vitamin intervention can reconstruct the blood-testis barrier and rescue the spermatogenesis dysfunction.

MATERIAL AND METHODS

8-week-old male SD rats and primary Sertoli cell were randomly divided into normal saline control group, PM2.5 exposure group, PM2.5 exposure group plus vitamin C and E intervention group. Then we observed the changes of testicular histomorphology, sperm quality and quantity, Western blot and IF examined expression and localization of the key junctional proteins (including ZO-1, occludin, β-catenin and Cx43) that constitute the blood-testis barrier, Western blot detected expressions of SOD, MDA, NRF-2 expression and MAPK signal pathway activity and apoptosis related proteins.

RESULTS

Compared with the control group, the sperm quality and quantity of rats exposed to PM2.5 decreased significantly, the seminiferous tubules atrophied, the spermatogenic cells decreased significantly, the expression of blood-testis barrier associated junctional proteins decreased significantly, SOD and NRF-2 expressions in testis and Sertoli cell decreased dramatically, MDA increased significantly, Bax/Bcl-2 ratio decreased significantly, cleaved Caspase-3, p-ERK1/2, p-p38 and p-JNK were significantly increased. On the contrary, these indicators were recovered and close to normal after administering vitamin C and E intervention.

CONCLUSIONS

PM2.5, the source of automobile exhaust, causes oxidative stress injury in Sertoli cells, then destroys blood-testis barrier and causes spermatogenesis dysfunction. Combined use of vitamin C and E can reduce oxidative stress induced by PM2.5, reconstructs blood-testis barrier integrity and rescues spermatogenesis by inhibiting MAPK pathway.


12:28 - 12:31
S1-2 (PP)

RETINOIC ACID IS EFFICACIOUS FOR PROMOTING SPERMATOGENESIS VIA KEEPING THE INTEGRALITY OF BLOOD-TESTIS BARRIER IN CRYPTORCHIDISM RATS

Deying ZHANG, Xing LIU, Dawei HE and Guanghui WEI
Children's Hospital of Chongqing Medical University, Urology, Chongqing, CHINA

PURPOSE

To investigate the effect and mechanism of retinoic acid on cryptorchidism rats.

MATERIAL AND METHODS

30 pregnant rats were divided into 3 groups randomly. a: Normal control group(n=10), corn oil was hypodermic between gestation 12-20d. b: Flutamide-induced cryptorchidism group (n=10), flutamide carried with corn oil was hypodermic between gestation 12-20d. c: Retinoic acid (RA) group (n=10), the method of modeling cryptorchid offspring was same with b group, pups male rats were hypodermic injected with retinoic acid between 1-10d. At postnatal 30d and 60d, offspring male rats testicular histopathology, sperm quality test were performed. The proteins level of blood-testis barrier and spermatogenesis markers protein were conducted with immunofluorescence and Western-blot. Biotin tracer was conducted for evaluating BTB permeability.

RESULTS

In group b, testicular weight and volume were significant decreased compared with group a and c (P<0.05). Sperm count was also significant decreased while sperm deformity rate significantly increased in group b. After treating with RA, sperm count and sperm quality was ameliorated, and had no significant difference compared with group a. Western-blot results shown that the expression of BTB protein (ZO-1, CX43 and Claudin 11) was decreased sharply in cryptorchidism testis (P<0.05). Additional, the quantitative analysis of spermatogenesis markers (SCP3, Stra8, c-Kit) also lower in cyrptorhidism testicles. After RA treatment, the expression of BTB related proteins and spermatogenesis markers were partially recovery. Immunofluorescence change were keep concert with the trend of Western-blot results. Biotin tracer indicated that the BTB permeability damage in cryptorchidism could be ameliorated after RA treatment.

CONCLUSIONS

Retinoic acid could restore the spermatogenic process in cryptorchid through regulating BTB function, which could provide a novel clue to clinic practice for avoiding cryptorchidism- induced infertility.


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

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 - 12:43
Discussion
 

12:43 - 12:46
S1-4 (PP)

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.


12:46 - 12:49
S1-5 (PP)

FABRICATION, CHARACTERIZATION AND DEGRADABILITY ASSESSMENT OF A NOVEL, TRI-LAYER ELECTROSPUN, 3D SILK FIBROIN/PLA/SILK FIBROIN NANOCOMPOSITE SCAFFOLDS FOR URETHRAL REPLACEMENT

Tariq Osman ABBAS 1, Huseyin YALCIN 2, Hemalatha PARANGUSAN 3, Mohamed HASSAN 3, Lubna ZAKRIF 4 and Cristian PENNISI 5
1) Sidra Medicine, Urology, Doha, QATAR - 2) Qatar University, Biomedical Research Centre, Doha, QATAR - 3) Qatar Univeristy, Centre for Advanced Materials, Doha, QATAR - 4) Qatar Univeristy, Biomedical Research Centre, Doha, QATAR - 5) Aalborg University, Laboratory for Stem Cell Research, Aalborg, DENMARK

PURPOSE

Hybrid tissue-engineered scaffolds combining various synthetic and natural biodegradable materials have potential applications in regenerative medicine, particularly reconstructive urology. In this study, silk fibroin (SF) and PHBV hybrid composites were produced, analyzed, and a suitable design was optimized. This study generated a tri-layer, electrospun, silk fibroin/PHBV membrane, for the spatially segregated culture of smooth muscle cells (SMCs).

MATERIAL AND METHODS

The SF/PHBV/SF scaffolds were made using electrospinning. Porosity, water absorption, mechanical properties and pore size of different scaffold was assessed. SMCs were seeded onto scaffolds, cultured 14 days, proliferation and metabolism of cells were detected in different timings using the thiazolyl blue tetrazolium bromide (MTT) assay method, and cell morphology and distribution were observed by histological analysis and scanning electron microscopy (SEM).

RESULTS

SF/PHBV/SF films showed good porosity, water absorption expansion rate, elasticity modulus and pore size, cells grow well inside the scaffolds, and was very suitable for tissue engineering. The MTT results showed that the metabolism of cells in the SF/PHBV/SF was better than PHBV or small intestinal submucosa (SIS) alone.

CONCLUSIONS

This novel tri-layer membrane is capable of supporting the tissue engineered regenerative capacity of urethral replacement. In addition, the materials were examined for their ability to support SMCs adhesion, proliferation, and multilayered formation mimicking the spongiosum. We provided evidence that these unique composite scaffolds provide significant benefit over commonly used acellular materials in vitro and suggest that they be further examined in vivo.


12:49 - 12:52
S1-6 (PP)

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.


12:52 - 13:01
Discussion
 

13:01 - 13:04
S1-7 (PP)

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.


13:04 - 13:07
S1-8 (PP)

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.


13:07 - 13:10
S1-9 (PP)

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.


13:10 - 13:19
Discussion