scRNA-seq reveals that origin recognition complex subunit 6 regulates mouse spermatogonial cell proliferation and apoptosis via activation of Wnt/ß-catenin signaling.

ABSTRACT: The regulation of spermatogonial proliferation and apoptosis is of great significance for maintaining spermatogenesis. The single-cell RNA sequencing (scRNA-seq) analysis of the testis was performed to identify genes upregulated in spermatogonia. Using scRNA-seq analysis, we identified the spermatogonia upregulated gene origin recognition complex subunit 6 (Orc6), which is involved in DNA replication and cell cycle regulation; its protein expression in the human and mouse testis was detected by western blot and immunofluorescence. To explore the potential function of Orc6 in spermatogonia, the C18-4 cell line was transfected with control or Orc6 siRNA. Subsequently, 5-ethynyl-2-deoxyuridine (EdU) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays, flow cytometry, and western blot were used to evaluate its effects on proliferation and apoptosis. It was revealed that ORC6 could promote proliferation and inhibit apoptosis of C18-4 cells. Bulk RNA sequencing and bioinformatics analysis indicated that Orc6 was involved in the activation of wingless/integrated (Wnt)/ ß-catenin signaling. Western blot revealed that the expression of ß-catenin protein and its phosphorylation (Ser675) were significantly decreased when silencing the expression of ORC6. Our findings indicated that Orc6 was upregulated in spermatogonia, whereby it regulated proliferation and apoptosis by activating Wnt/ß-catenin signaling.

Low XIST expression in Sertoli cells of Klinefelter syndrome patients causes high susceptibility of these cells to an extra X chromosome.

Klinefelter syndrome (KS) is the most common genetic cause of human male infertility. However, the effect of the extra X chromosome on different testicular cell types remains poorly understood. Here, we profiled testicular single-cell transcriptomes from three KS patients and normal karyotype control individuals. Among the different somatic cells, Sertoli cells showed the greatest transcriptome changes in KS patients. Further analysis showed that X-inactive-specific transcript ( XIST ), a key factor that inactivates one X chromosome in female mammals, was widely expressed in each testicular somatic cell type but not in Sertoli cells. The loss of XIST in Sertoli cells leads to an increased level of X chromosome genes, and further disrupts their transcription pattern and cellular function. This phenomenon was not detected in other somatic cells such as Leydig cells and vascular endothelial cells. These results proposed a new mechanism to explain why testicular atrophy in KS patients is heterogeneous with loss of seminiferous tubules but interstitial hyperplasia. Our study provides a theoretical basis for subsequent research and related treatment of KS by identifying Sertoli cell-specific X chromosome inactivation failure.

Ultrasonographic evaluation of the rete testis thickness: a promising approach to differentiate obstructive from nonobstructive azoospermia.

This study aimed to evaluate the ability of rete testis thickness (RTT) and testicular shear wave elastography (SWE) to differentiate obstructive azoospermia (OA) from nonobstructive azoospermia (NOA). We assessed 290 testes of 145 infertile males with azoospermia and 94 testes of 47 healthy volunteers at Shanghai General Hospital (Shanghai, China) between August 2019 and October 2021. The testicular volume (TV), SWE, and RTT were compared among patients with OA and NOA and healthy controls. The diagnostic performances of the three variables were evaluated using the receiver operating characteristic curve. The TV, SWE, and RTT in OA differed significantly from those in NOA (all P ≤ 0.001) but were similar to those in healthy controls. Males with OA and NOA were similar at TVs of 9-11 cm 3 ( P = 0.838), with sensitivity, specificity, Youden index, and area under the curve of 50.0%, 84.2%, 0.34, and 0.662 (95% confidence interval [CI]: 0.502-0.799), respectively, for SWE cut-off of 3.1 kPa; and 94.1%, 79.2%, 0.74, and 0.904 (95% CI: 0.811-0.996), respectively, for RTT cut-off of 1.6 mm. The results showed that RTT performed significantly better than SWE in differentiating OA from NOA in the TV overlap range. In conclusion, ultrasonographic RTT evaluation proved a promising diagnostic approach to differentiate OA from NOA, particularly in the TV overlap range.

Efficacy of stepwise mini-incision microdissection testicular sperm extraction for nonobstructive azoospermia with varied etiologies.

Stepwise mini-incision microdissection testicular sperm extraction (mTESE) is a procedure that attempts to minimize testicular damage. However, the mini-incision approach may vary in patients with different etiologies. Here, we performed a retrospective analysis of 665 men with nonobstructive azoospermia (NOA) who underwent stepwise mini-incision mTESE (Group 1) and 365 men who underwent standard mTESE (Group 2). The results showed that the operation time (mean ± standard deviation) for patients with successful sperm retrieval in Group 1 (64.0 ± 26.6 min) was significantly shorter than that in Group 2 (80.2 ± 31.3 min), with P <0.001. The total sperm retrieval rate (SRR) was 23.1% in our study, and there was no significant difference between Group 1 and Group 2 ( P >0.05), even when the etiologies of NOA were taken into consideration. The results of consecutive multivariate logistic regression analysis (odds ratio [OR]: 0.57; 95% confidence interval [CI]: 0.38-0.87; P =0.009) and receiver operating characteristic (ROC) analysis (area under the ROC curve [AUC]=0.628) showed that preoperative anti-Müllerian hormone (AMH) level in idiopathic NOA patients was a potential predictor for surgical outcomes after initial three small incisions made in the equatorial region without sperm examined under an operating microscope (Steps 2-4). In conclusion, stepwise mini-incision mTESE is a useful technique for NOA patients, with comparable SRR, less surgical invasiveness, and shorter operation time compared with the standard approach. Low AMH levels may predict successful sperm retrieval in idiopathic patients even after a failed initial mini-incision procedure.

Clinical features and microsurgical reconstruction of congenital unilateral absence of the vas deferens with obstructive azoospermia: a tertiary care center experience.

Patients with congenital unilateral absence of the vas deferens (CUAVD) manifest diverse symptoms from normospermia to azoospermia. Treatment for CUAVD patients with obstructive azoospermia (OA) is complicated, and there is a lack of relevant reports. In this study, we describe the clinical features and evaluate the treatments and outcomes of CUAVD patients with OA. From December 2015 to December 2020, 33 patients were diagnosed as CUAVD with OA in Shanghai General Hospital (Shanghai, China). Patient information, ultrasound findings, semen analysis, hormone profiles, and treatment information were collected, and the clinical outcomes were evaluated. Of 33 patients, 29 patients were retrospectively analyzed. Vasoepididymostomy (VE) or cross VE was performed in 12 patients, the patency rate was 41.7% (5/12), and natural pregnancy was achieved in one of the patients. The other 17 patients underwent testicular sperm extraction as the distal vas deferens (contralateral side) was obstructed. These findings showed that VE or cross VE remains an alternative treatment for CUAVD patients with OA, even with a relatively low rate of patency and natural pregnancy.

Novel mutation in ODF2 causes multiple morphological abnormalities of the sperm flagella in an infertile male.

Numerous genes have been associated with multiple morphological abnormalities of the sperm flagella (MMAF), which cause severe asthenozoospermia and lead to male infertility, while the causes of approximately 50% of MMAF cases remain unclear. To reveal the genetic causes of MMAF in an infertile patient, whole-exome sequencing was performed to screen for pathogenic genes, and electron microscope was used to reveal the sperm flagellar ultrastructure. A novel heterozygous missense mutation in the outer dense fiber protein 2 (ODF2) gene was detected, which was inherited from the patient's mother and predicted to be potentially damaging. Transmission electron microscopy revealed that the outer dense fibers were defective in the patient's sperm tail, which was similar to that of the reported heterozygous Odf2 mutation mouse. Immunostaining of ODF2 showed severe ODF2 expression defects in the patient's sperm. Therefore, it was concluded that the heterozygous mutation in ODF2 caused MMAF in this case. To evaluate the possibility of assisted reproductive technology (ART) treatment for this patient, intracytoplasmic sperm injection (ICSI) was performed, with the help of a hypo-osmotic swelling test and laser-assisted immotile sperm selection (LAISS) for available sperm screening, and artificial oocyte activation with ionomycin was applied to improve the fertilization rate. Four ICSI cycles were performed, and live birth was achieved in the LAISS-applied cycle, suggesting that LAISS would be valuable in ART treatment for MMAF.

[Imaging quality of the robotic visualization 4K3D system and its application in microscopic vasectomy reversal in rats].

OBJECTIVE: To quantitatively assess the performance of the new robotic visualization system (Zeiss, KINEVO 900) in terms of visual imaging effect and evaluate its potential application in microscopic vasectomy reversal. METHODS: We made a parallel comparison between the effects of the plane and stereo visual images of KINEVO 900 and optical surgical microscopy (Zeiss, S7), and performed microscopic vasectomy reversal on the rat model using KINEVO 900. RESULTS: Compared with S7, KINEVO 900 provided an even longer working distance (200－625 mm), a 3－4 times larger effective field area and a 1.5－2 times deeper front depth of field with the same working distance of 200 mm. No statistically significant difference was observed in the average anastomosis time and immediate patency rate between the two platforms (P > 0.05). CONCLUSION: The 4K3D video image stream outputted by KINEVO 900 is not inferior to that of the optical surgical microscope represented by S7 and is sufficient for microsurgeries in urology and andrology. More prospective randomized clinical animal experiments are needed to further evaluate its application value in andrology.

3D digital image microscope system-assisted vasovasostomy and vasoepididymostomy in rats.

Optimal vision and ergonomics are essential factors contributing to the achievement of good results during microsurgery. The three-dimensional (3D) digital image microscope system with a better 3D depth of field can release strain on the surgeon's neck and back, which can improve outcomes in microsurgery. We report a randomized prospective study of vasoepididymostomy and vasovasostomy using a 3D digital image microscope system (3D-DIM) in rats. A total of 16 adult male rats were randomly divided into two groups of 8 each: the standard operating microscope (SOM) group and the 3D-DIM group. The outcomes measured included the operative time, real-time postoperative mechanical patency, and anastomosis leakage. Furthermore, a user-friendly microscope score was designed to evaluate the ergonomic design and equipment characteristics of the microscope. There were no differences in operative time between the two groups. The real-time postoperative mechanical patency rates were 100.0% for both groups. The percentage of vasoepididymostomy anastomosis leakage was 16.7% in the SOM group and 25.0% in the 3D-DIM group; however, no vasovasostomy anastomosis leakage was found in either group. In terms of the ergonomic design, the 3D-DIM group obtained better scores based on the surgeon's feelings; in terms of the equipment characteristics, the 3D-DIM group had lower scores for clarity and higher scores for flexibility and adaptivity. Based on our randomized prospective study in a rat model, we believe that the 3D-DIM can improve surgeon comfort without compromising outcomes in male infertility reconstructive microsurgery, so the 3D-DIM might be widely used in the future.

Modified stepwise mini-incision microdissection testicular sperm extraction: a useful technique for patients with a history of orchidopexy affected by non-obstructive azoospermia.

Non-obstructive azoospermia (NOA), which is defined as the absence of spermatozoa in the ejaculate secondary to impaired spermatogenesis within the testis, may be caused by a variety of etiologies, including varicocele-induced testicular damage, cryptorchidism, prior testicular torsion, post-pubertal mumps orchitis, gonadotoxic effects from medications, genetic abnormalities, chemotherapy/radiation, and other unknown causes currently classified as idiopathic (Cocuzza et al., 2013). The microdissection testicular sperm extraction (micro-TESE) technique involves a meticulous microsurgical exploration of the testicular parenchyma to identify and selectively extract larger seminiferous tubules that carry a higher probability of complete spermatogenesis (Schlegel, 1999). The Cornell group evaluated the efficacy of micro-TESE in 152 NOA patients with an associated history of cryptorchidism. In their series, spermatozoa were successfully retrieved in 116/181 attempts (64%), and the resulting pregnancy rate was 50% with a delivery rate of 38% (Dabaja and Schlegel, 2013). Franco et al. (2016) described a stepwise micro-TESE approach in NOA patients, which was considered to reduce the cost, time, and effort associated with the surgery. Alrabeeah et al. (2016) further reported that a mini-incision micro-TESE, carried through a 1-cm equatorial testicular incision, can be useful for micro-TESE candidates, particularly in patients with cryptozoospermia. We conducted a retrospective study of 20 consecutive NOA patients with a history of orchidopexy from May 2015 to March 2017.

Microsurgical subinguinal varicocelectomy with spermatic cord double traction and vein stripping.

We retrospectively reviewed data for 286 patients with varicocele who underwent microsurgical subinguinal varicocelectomy from March 2015 to May 2017 in Shanghai General Hospital (Shanghai, China). In this surgical approach, the testis was delivered, and the gubernacular and external cremasteric veins were stripped. In addition, the spermatic cord was delivered downward with continuous double traction away from the external ring. The remaining procedure was similar to the conventional approach. We followed patients for at least 3 months and evaluated postoperative semen parameters, pain symptoms, and complications. We excluded data for 32 men due to inadequate follow-up (<3 months). Of the remaining 254 patients, 73 had oligoasthenospermia, 121 had nonobstructive azoospermia, and 60 had symptomatic varicoceles. Total progressive sperm counts increased in the oligoasthenospermic patients from a median preoperative value of 9.15 × 106 ml-1 to 25.33 × 106 ml-1 (n= 34), and 35.6% (26/73) initially oligoasthenospermic men contributed to unassisted pregnancies. Sperm returned to the ejaculate in 12.4% (15/121) azoospermia patients. In patients with scrotal pain (n = 60), 43 (71.7%) reported complete resolution of pain, 16 (26.7%) reported partial resolution, and 1 (1.7%) reported no change. No patients experienced varicocele recurrence. This double-traction strategy avoids opening the external oblique aponeurosis, and results in less damage and faster recovery. In addition, the stripping strategy eliminates potential damage to the testis caused by the varicose veins. Our results showed that microsurgical subinguinal varicocelectomy using spermatic cord double traction in conjunction with testicular delivery for vein stripping is a safe and effective approach for varicocele repair.

[Spatio-temporal expression and function of KIFC1 in acrosomogenesis].

OBJECTIVE: To investigate the spatio-temporal expression and function of the end-directed KIFC1 (kinesin family member C1) protein during acrosomogenesis. METHODS: The expression and location of KIFC1 were analyzed by flow separation and immunofluorescence, and the small interference RNA (RNAi1) of KIFC1 with high-interference efficiency was screened using in vitro GC2-spd cell lines. The KIFC1 RNAi1 mixed with 0.5% trypan blue solution was microinjected into the testicular seminiferous tubules and negative Con-RNAi1 into the contralateral testis of a 3-week-old Balb/c mouse, followed by morphological analysis of the sperm collected from the testis tail at 3 weeks after injection. RESULTS: The expression of KIFC1 was observed mainly in the cytoplasm of the sperm cells in the early stage of sperm deformation, in the acrosomal vesicle and acrosome in the middle stage, and in the residual body in the late stage, but vanished during sperm maturation. The sperm head deformity rate was significantly higher in the RNAi1 than in the negative control group (ï¼»32.12 ± 0.25ï¼½% vs ï¼»7.06 ± 1.25ï¼½%, P < 0.01). CONCLUSIONS: The KIFC1 protein may play an important role in the formation of spermatozoa, mainly affecting acrosomogenesis.

[Qilin Pills promote testicular spermatogenesis in azoospermia mice].

OBJECTIVE: To investigate the effect of Qilin Pills (QP) in facilitating the recovery of spermatogenic function in azoospermia (AS) mice and to explore its mechanism of regulating testicular spermatogenesis. METHODS: Fifteen 4-week-old male mice were equally randomized into an AS model control, a low-dose QP and a high-dose QP group. The AS model was established in the mice by intraperitoneal injection of busulfan at 35 mg/kg. After modeling, the animals in the low- and high-dose QP groups were treated with Qilin Pills intragastrically at 2 000 and 8 000 mg/kg/d respectively while those in the model control group fed on a normal diet, all for 28 days. Then, all the mice were sacrificed for examination of the ultrastructures of the epididymis and testis by HE staining, detection of the specific markers of spermatogenic, Sertoli and Leydig cells by Western blot, and determination of the expressions of these markers in the testis tissue by immunofluorescence assay. RESULTS: The number of spermatogenic cells in the testis tissue was significantly decreased in the AS model controls, with no spermatozoa in most of the seminiferous tubules in the epididymis (Johnsen's score: 5.2 ± 0.5). In the high-dose QP group, spermatogenic cells were tightly arranged with distinct layers in the seminiferous tubules, with a large number of spermatozoa but no non-sperm cells in the lumens of the epididymis (Johnsen's score: 9.4 ± 0.6). The number of spermatogenic cells in the testis was increased in the low-dose QP group with some spermatozoa in the seminiferous tubules as compared with that in the model control, but lower than in the high-dose group (Johnsen's score: 7.6 ± 0.6). The Johnsen's score was significantly lower in the model control than in the high- and low-dose QP groups (P < 0.01), and higher in the high-dose than in the low-dose QP group (P < 0.05). The expressions of the specific markers of Sertoli cells SCF, BMP4, SYCP3, DMC1 and Ki67 were also remarkably lower in the model control than in the high- and low-dose QP groups (P < 0.01), and higher in the high-dose than in the low-dose QP group (P < 0.05 or P < 0.01). No statistically significant differences were observed among the three groups of mice in the markers of spermatogonial stem cells (SSC) and undifferentiated SSCs UCHL1, STRA8, NGN3 and PLZF3 (P > 0.05). The expressions of the spermatocyte markers DMC1 and SYCP3 were markedly lower in the model control than in the high- and low-dose QP groups (P < 0.05 or P < 0.01), and higher in the high-dose than in the low-dose QP group (P < 0.05 or P < 0.01). The Ki67 fluorescence signals were distributed in the spermatogonia, with a higher intensity in the model control than in the high- and low-dose QP groups. The acrosome marker PNA was found mainly in the seminiferous tubules, with abundant fluorescence signals in the high- and low-dose QP groups but no obvious dot signals in the model controls. CONCLUSIONS: Qilin Pills may contribute to the meiosis of spermatogonia and promote spermatogenesis by improving the function of Sertoli cells in the testis.