[Novel compound heterozygous LoF mutations in SRD5A2 may result in disorders of sex development].

Objective: To investigate the novel genetic cause associated with hypospadias and the strategy for preventing offspring genetic defects in these patients. Methods: In March 2019, a patient with gonadal dysplasia (hypospadias associated with cryptorchidism) was referred to Shanghai General Hospital. His secondary sex characters, level of sex hormones and the development of male reproductive system was assessed through physical examination, sex hormone examination, male reproductive system B-ultrasound and computed tomography (CT). Whole-exome sequencing (WES) was preformed to investigate the pathogenic genetic variations associated with hypospadias and cryptorchidism. Also, Sanger sequencing was conducted to verify the WES results in the pedigree. Semen analysis was used to assess the fertility of the proband and the SRD5A2 gene analysis of his spouse was performed to assess the risk of genetic defects in the offspring. Results: The patient suffered from gonadal dysplasia (hypospadias associated with cryptorchidism). Physical examination showed an inverted triangular distribution of pubic hair, small penis and the volume of the testis was 8 ml. Sex hormone examination revealed the level of FSH, LH, Pituitary prolactin (PRL), estrogen (E(2)), testosterone (T), and sex hormone-binding globulin (SHBG) was 25.81 U/L, 10.84 U/L, 21.09 µg/L, 153 pmol/L, 16.95 nmol/L, and 36.15 nmol/L respectively. B-ultrasound and computed tomography (CT) showed left inguinal testis. Also, semen analysis illustrated that the volume was 0.05 ml and sperm concentration<2×10(6)/ml, suggesting oligospermia in this case. WES sequencing and Sanger sequencing showed compound heterozygous LoF mutations in SRD5A2 [NM_000348.3:C.679C>T(p.Arg227Ter) and NM_000348.3:C.16C>T(p.Gln6Ter)] in this patient. And there were no pathogenic genetic variations of SRD5A2 in the spouse. Conclusion: Novel compound heterozygous LoF mutations in SRD5A2[NM_000348.3:C.679C>T(p.Arg227Ter) and NM_000348.3:C.16C>T(p.Gln6Ter)] may be the primary cause of disorders of sex development.

[Efficacy and safety of low-intensity pulsed ultrasound at different intervals by mechanical force in treating erectile dysfunction: a preliminary study].

Objective: To study the efficacy and safety of low-intensity pulsed ultrasound (LIPUS) at different intervals by mechanical force in treating erectile dysfunction (ED). Method: Forty patients with mild to moderate ED were randomized in a 1∶1 ratio to receive 16-treatment sessions of LIPUS in group A and group B, applied 3 times per week and 2 times per week, respectively. End-point assessments were made at 8th week after treatment. Efficacy were evaluated using International Index of Erectile Function-Erectile Function domain score (IIEF-EF), Erectile Hardness Score (EHS), Self-Esteem and Relationship Questionnaire (SEAR), Sexual Encounter Profile (SEP), Global Assessment Question (GAQ), and pain were assessed by Visual Analogue Score (VAS).Treatment response was confirmed by a minimal clinically importance difference (MCID) at 8th week. Results: Compared with baseline, IIEF-EF score [(17.1±5.48 vs 23.4±3.75, P<0.05) and (18.9±4.34 vs 24.1±4.32, P<0.05)], proportion of EHS 4 [(0 vs 40%, P<0.05) and (16.7% vs 55.6%, P<0.05)], and Overall Relationship score [(50.6 vs 67.5, P<0.05) and (44.4 vs 70.1, P<0.05)] were significantly improved at 8th week in two groups, respectively. Compared with baseline, the positive responses to SEP-3 increased significantly at 8th week in two groups (50.0% vs 80.0%,P<0.05) and (44.4% vs 88.9%, P<0.05), respectively. The positive responses to GAQ-2 were 90.0% and 88.9% at 8th week in two groups, respectively. There were no significant differences in IIEF-EF, EHS, SEAR, SEP and GAQ at 8th week between two groups. There was no significant difference in treatment response using MCID between two groups at end-point (80.5% vs 77.5%). The treatment duration for full sessions were 2.5 weeks less in group A than group B. No adverse effects were reported in all cases. Conclusion: LIPUS at two different intervals is effective and safe for mild to moderate ED, and the regimen at 3 times per week can achieve quite good effect in relatively short duration,while the long-term effects is still be clarified in further study.

[Screening of candidate proteins related to maturation arrest based on iTRAQ technique].

Objective: To screen candidate proteins related to maturation arrest of human spermatogenesis. Methods: From March 2015 to September 2016, 3 cases of obstructive azoospermia and 3 cases of patients with maturation arrest, who were admitted to the Shanghai General Hospital, were selected for proteomic sequencing, and the sequencing results were identified and analyzed. Results: A total of 125 differentially expressed proteins were identified, of which 21 were up-regulated and 104 were down-regulated. Analysis of biological process shows that differentially expressed proteins participate in reproductive and developmental processes. Participation in reproductive processes accounts for 29.00% of the difference proteins. Participation in the development process accounted for 41.00% of the difference proteins. Cell component(CC) analysis showed that 68.00% different proteins were attached to organelles. Molecular function analysis showed that the binding function accounted for 85.00% of the differential proteins. The proportion of proteins involved in transportation, regulation of enzyme activity and anti-oxidation were 11.05%, 4.62% and 8.34% respectively. Signal pathways involved in sperm deformation included MAPK pathway, Ca(2+)/calmodulin pathway. Conclusions: The candidate proteins and its associated signaling pathways were selected. The abnormal structuration and function of mature arrest could lead to severe male infertility.

[Efficacy and safety study of microsurgical varicocelectomy in the treatment of non-obstructive azoospermia with varicocele].

Objective: To discuss the efficacy and safety of subinguinal microsurgical varicocelectomy in the treatment of non-obstructive azoospermia (NOA) with varicocele. Methods: The clinical data of 141 patients with NOA and varicocele who underwent subinguinal microsurgical varicocelectomy from March 2015 to June 2017 in Shanghai General Hospital was collected.One hundred and ten patients suffered from varicocele on the left side, 1 on the right side, and the rest (30 cases) were bilateral varicocele. Grade Ⅰ varicocele were found on 7 sides (the right and left side was count respectively), grade Ⅱ on 121 sides, and grade Ⅲ on 43 sides. Sperm analysis, pregnancy rate and complications were recorded after at least 6 months since operation. Results: Eleven cases were lost during the follow-up. Eighteen of the remaining 130 NOA patients processed successful sperm retrieval in post-operative semen analysis (18/130, 13.8%). Six couples(6/130, 4.6%) succeeded in natural pregnancy. Five couples (5/130, 3.8%)underwent successful pregnancy following with intracytoplasmic sperm injection(ICSI). Twenty-six out of the remaining 112 patients underwent the micro dissection testicular sperm extraction (micro-TESE), and 4 patients got a successful sperm retrieval (4/26, 15.4%). Among them, 2 couples had successful pregnancy with ICSI. Totally 2 cases of postoperative infection of incision were found. Conclusions: Microsurgical varicocelectomy had a beneficial effect on sperm quality of patients suffered from NOA with varicocele to some extent, even leading to unassisted pregnancy or avoiding micro-TESE before ICSI. Microsurgical varicocelectomy could be applied in the treatment of NOA with varicocele.

[Correlation between anatomical factors of spermatic vessels and varicocele].

Objective: To analyze the correlation between anatomy of spermatic vessels and varicocele, providing reference for the preoperative assessment and treatment of varicocele. Methods: A total of 156 patients who underwent microsurgical left subinguinal varicocelectomy at Shanghai General Hospital between May 2015 and July 2016 were included in this study. The severity of varicocele and number of spermatic vessels detected in operations were recorded. According to the number of internal spermatic arteries (ISAs), the patients were divided into three groups: single-ISA group (55 cases), double-ISAs group (63 cases) and multi-ISAs group (38 cases), to analyze the correlation among spermatic vessels and to compare varicocele grade, the volume of testes, the parameter of semen analysis, serum reproductive hormone, surgery time, and hospital stay among the three groups. Results: The number of ISAs was positively correlated with the ipsilateral internal spermatic veins (ISVs) (r=0.210; P=0.008)and lymphatic vessels (r=0.224; P=0.005); the number of lymphatic vessels was positively correlated with the ipsilateral gubernacular veins (r=0.172; P=0.032)and ISVs (r=0.296; P=0.000) . The number of ISVs in the multi-ISAs group (10.58±4.28) was significantly larger than that in the single-ISA group (8.22±3.10, P=0.003). The number of lymphatic vessels in the multi-ISAs group(4.11±1.90)was also significantly larger than that in the double-ISA group(3.76±1.40, P=0.020) and the single-ISA group(3.13±1.52, P=0.007). The number of ISVs in grade 2 varicocele patients (9.74±3.90) was significantly higher than that in grade 3 varicocele patients (8.33±3.10, P=0.013). No significant differences in varicocele grade, change of pre- and post-operative semen analysis, serum reproductive hormone, the volume of ipsilateral testes, surgery time, and hospital stay were observed among the three groups. Conclusions: There is a correlation among various kinds of spermatic vessels. Patients with grade 2 varicocele, especially who have multiple ISAs, are likely to have more ISVs and lymphatic vessels. For these patients, surgeons should pay more attention to protect spermatic arteries and lymphatics carefully while ligating varicose veins completely to prevent recurrence and complications.

Multicentre study of Y chromosome microdeletions in 1,808 Chinese infertile males using multiplex and real-time polymerase chain reaction.

Azoospermia factor (AZF) genes on the long arm of the human Y chromosome are involved in spermatogenesis, and microdeletions in the AZF region have been recognised to be the second major genetic cause of spermatogenetic failure resulting in male infertility. While screening for these microdeletions can avoid unnecessary medical and surgical treatments, current methods are generally time-consuming. Therefore, we established a new method to detect and analyse microdeletions in the AZF region quickly, safely and efficiently. In total, 1,808 patients with spermatogenetic failure were recruited from three hospitals in southern China, of which 600 patients were randomly selected for screening for Y chromosome microdeletions in AZF regions employing real-time polymerase chain reaction with a TaqMan probe. In our study, of 1,808 infertile patients, 150 (8.3%) were found to bear microdeletions in the Y chromosome using multiplex PCR, while no deletions were found in the controls. Among the AZF deletions detected, two were in AZFa, three in AZFb, 35 in AZFc, three in AZFb+c and two in AZFa+b+c. Our method is fast-it permits the scanning of DNA from a patient in one and a half hours-and reliable, minimising the risk of cross-contamination and false-positive and false-negative results.

[Effectiveness of microsurgical crossover anastomosis in treating complicated obstructive azoospermia].

Objective: To evaluate the efficacy and safety of microsurgical crossover vasovasostomy in treating complicated obstructive azoospermia. Methods: The data of 14 patients with complicated obstructive azoospermia treated with microsurgical crossover vasovasostomy were reviewed from October 2012 to March 2016.Ten of them underwent microsurgical crossover vasovasostomy. Intraoperative exploration revealed that 2 patients had vas deferens injury and contralateral testicular atrophy or epididymal obstruction due to previous hernia repair; 7 patients had obstruction of intracorporeal vas deferens on one side and epididymal obstruction on the other side; the other 1 patient had unilateral vasal obstruction with contralateral epididymal obstruction. Furthermore, 4 patients underwent microsurgical crossover vasoepididymostomy, including 3 patients who had obstruction at caput epididymis on one side, and obstruction at cauda epididymis and distal vas deferens on the other side; the other patient had absence of vas deferens in the scrotum on one side, and testicular atrophy on the other side. Regular follow-up visits were conducted after the surgery. Results: Two patients were lost to follow-up; the other 12 patients were follow-up for an average of 11 (range: 2-23) months. In the 10 cases receiving microsurgical crossover vasovasostomy (including 2 patients lost to follow-up), 1 has not undergone semen re-analysis, 6 were confirmed patent, including 3 reporting spontaneous pregnancy. The patency rate in the 4 patients receiving microsurgical crosseover vasoepididymostomy was 2/4, with 1 patient reporting spontaneous pregnancy. There was no complaint of discomfort or complications following the surgery. Conclusions: Microsurgical crossover anastomosis may be effective and safe for patients with complicated obstructive azoospermia, according to preoperative assessment and intraoperative exploration. It allows natural conception for patients with refractory infertility. The microsurgical crossover anastomosis could be an effective therapy to achieve satisfactory patency of vas deferens.