The Expression Of HOXA4 In Prepuce Of Hypospadias Patients And Application Of Single Layer Porcine Small Intestinal Submucosa In Urethroplasty On Beagle Models

Background:Hypospadias is one of the most common congenital malformations in the urogenital system of children.The incidence of hypospadias is between 1/300-1/125 boys.In recent years,the incidence of hypospadias has also increased significantly in China.Due to hypospadias patients cannot urinate and ejaculation,will seriously affect the lives of patients.Hypospadias is believed to related to endocrine factors,environmental factors and genetic factors.However,these can only explain about 30% of hypospadias.Most hypospadias cannot be explained.In recent years,with the development and wide application of genome sequencing technology.More gene loci associated with hypospadias have been screened.It has been reported that HoxA4 gene may be involved in the development of hypospadias by genome-wide association (GWAS).The HoxA4 gene is a member of the HOX gene family,which is located on chromosome 7.It encodes a DNA binding transcription factor that regulates gene expression and tissue differentiation.As a result of GWAS research design methods and genetic statistics methods can not fundamentally eliminate the mixed population,multiple comparisons caused by false positive.Whether HOXA4 gene is involved in the pathogenesis of hypospadias needs further study.Surgery is currently almost one of the only treatments for hypospadias.However,in the repair of hypospadias,a variety of complications are prone to occur,and urethral fistula is one of the most common complications in the repair of hypospadias.Many pediatric urologists have made unremitting efforts to reduce urethral fistula after hypospadias.A common way to reduce postoperative fistula in hypospadias is to add additional coverage to the newly formed urethra during surgery.A common covering material is the autologous fascia adjacent to the patient’s surgical area.However,the preparation of autologous fascia is a time-consuming process and may lead to autologous fascia deficiency in special cases such as multiple operations.The small intestinal submucosa is a biomaterial with mature clinical experience.It is widely used in tissue repair surgery in many specialties including urology.However,the experience of using this material as an additional covering material in hypospadias surgery is limited.This study was conducted to investigate the development and clinical treatment of hypospadias.This study is divided into two parts.Part one:The expression of HoxA4 in prepuce of hypospadias patientsObjective:To study the expression of HoxA4 gene and its protein in the prepuce of children with hypospadias and the penis of mice with hypospadias induced by hexyl phthalate.Methods:This study is divided into two parts,one is the basic research part,and the second is the clinical research aspect.In basic research,the prepuce of normal phimosis children and the prepuce of hypospadias of light,medium and heavy type were selected as control.The expression of HoxA4 mRNA and protein were measured by real-time quantitative PCR and immunohistochemical methods.Secondly,the animal model of hypospadias induced by DEHP was established.The expression of HoxA4 gene in penis tissues of fetal rats with hypospadias and normal controls was studied by RT-PCR and immunohistochemistry.Results:DEHP induced mouse hypospadias animal model was successfully replicated.HoxA4 gene was transcribed and expressed in the prepuce of hypospadias and the penis of mice.Compared with GAPDH,the relative transcription levels of the three groups were 1.501±0.063) in the prepuce group,1.101±0.147) in the mild hypospadias,1.099±0.140) in the moderate hypospadias and 0.539 ± 0.056) in the severe hypospadias.The transcription level of HoxA4 in the normal prepuce group was higher than that in the hypospadias group (p<0.001).The HoxA4 gene transcription level in the mild and moderate hypospadias group was higher than that in the severe hypospadias group (p<0.001).There was no significant difference in mRNA transcriptional level of HoxA4 gene between mild and moderate hypospadias(p=0.964).Conclusions DEHP-induced mouse model of hypospadias was stable and effective.HoxA4 gene expression may be involved in the occurrence of hypospadias,which is the cause of some hypospadias.Part two:The application of single layer porcine small intestinal submucosa in urethroplasty on beagle modelsObjective:to evaluate the role of porcine small intestinal submucosa (SIS) in urethroplasty on beagle models and to observe the degradation process of SIS.Methods and materials:22 healthy male beagles were tested.The animals were randomly divided into SIS group and control group, each animal in SIS group was paired with an animal in control group.An animal model of hypospadias was established by surgery with general anesthesia.In the both groups,the urethroplasty was performed routinely.In SIS group,the urethra was covered with a single layer SIS material.In the control group,no material was covered outside the urethra.Retrograde cystourethrography was performed at the 4w and 12w after surgery,and the occurrence of urethral fistula and was recorded.After 2w,4w and 12w of the animals were anesthetized and sacrificed on schedule,the penis specimens were collected and histological observation were performed with hematoxylin-eosin (HE)and Mallory staining.Results:surgical site inflammation was observed in the early stage and returned to normal within 4w.Urethrography indicated 1/3 of the SIS group and 3/3 of the control group had urethral fistula at the surgical site 4 weeks after surgery.At the 12 weeks after operation urethral fistula was found in 1/5 of the SIS group and 3/5 of the control group.In total,the incidence of urethral fistula was 25% (2/8) in the SIS group and 75% (6/8) in the control group,SIS material was effective in reduce the occurrence of urethral fistula (p=0.036).The inflammation reaction was observed in both groups;however,there is no significant difference between them (t=-1.936,p=0.082).The collagen fiber increased in both groups,but SIS group has much more gentle increase compared to control group (t=-9.037,p<0.001).Conclusion:SIS materials have good biocompatibility,can remodel collagen fiber attached to the SIS material and grow regularly,have the possibility of reducing urethral scar in beagle hypospadias model.The material can decrease the incidence of urethral fistula on animal model,when used as a coverage layer.The SIS degradation process started 2w-4w after the operation and finished before 12w on the animal model.