A novel alternative method for long-term evaluation of male reproductive toxicity and its recovery using a pre-pubertal mouse testis organ culture system.

Successful treatment of pediatric cancers often results in long-term health complications, including potential effects on fertility. Therefore, assessing the male reproductive toxicity of anti-cancer drug treatments and the potential for recovery is of paramount importance. However, in vivo evaluations are time-intensive and require large numbers of animals. To overcome these constraints, we utilized an innovative organ culture system that supports long-term spermatogenesis by placing the testis tissue between a base agarose gel and a polydimethylsiloxane ceiling, effectively mirroring the in vivo testicular environment. The present study aimed to determine the efficacy of this organ culture system for accurately assessing testicular toxicity induced by cisplatin, using acrosin-green fluorescent protein (GFP) transgenic neonatal mouse testes. The testis fragments were treated with different concentrations of cisplatin-containing medium for 24 h and incubated in fresh medium for up to 70 days. The changes in tissue volume and GFP fluorescence over time were evaluated to monitor the progression of spermatogenesis, in addition to the corresponding histopathology. Cisplatin treatment caused tissue volume shrinkage and reduced GFP fluorescence in a concentration-dependent manner. Recovery from testicular toxicity was also dependent on the concentration of cisplatin received. The results demonstrated that this novel in vitro system can be a faithful replacement for animal experiments to assess the testicular toxicity of anti-cancer drugs and their reversibility, providing a useful method for drug development.

Improvements in in vitro spermatogenesis: oxygen concentration, antioxidants, tissue-form design, and space control.

Incorporation of bovine serum-derived albumin formulation (AlbuMAX) into a basic culture medium, MEMα, enables the completion of in vitro spermatogenesis through testicular tissue culture in mice. However, this medium was not effective in other animals. Therefore, we sought an alternative approach for in vitro spermatogenesis using a synthetic medium without AlbuMAX and aimed to identify its essential components. In addition to factors known to be important for spermatogenesis, such as retinoic acid and reproductive hormones, we found that antioxidants (vitamin E, vitamin C, and glutathione) and lysophospholipids are vital for in vitro spermatogenesis. Moreover, based on our experience with microfluidic devices (MFD), we developed an alternative approach, the PDMS-ceiling method (PC method), which involves simply covering the tissue with a flat chip made of PDMS, a silicone resin material used in MFD. The PC method, while straightforward, integrates the advantages of MFD, enabling improved and uniform oxygen and nutrient supply via tissue flattening. Furthermore, our studies underscored the significance of lowering the oxygen concentration to 10-15%. Using an integrated cultivation method based on these findings, we successfully achieved in vitro spermatogenesis in rats, which has been a long-standing challenge. Further improvements in culture conditions would pave the way for spermatogenesis completion in diverse animal species.

In vitro reconstitution of the whole male germ-cell development from mouse pluripotent stem cells.

Mammalian male germ-cell development consists of three distinct phases: primordial germ cell (PGC) development, male germ-cell specification for spermatogonium development, and ensuing spermatogenesis. Here, we show an in vitro reconstitution of whole male germ-cell development by pluripotent stem cells (PSCs). Mouse embryonic stem cells (mESCs) are induced into PGC-like cells (mPGCLCs), which are expanded for epigenetic reprogramming. In reconstituted testes under an optimized condition, such mPGCLCs differentiate into spermatogonium-like cells with proper developmental transitions, gene expression, and cell-cycle dynamics and are expanded robustly as germline stem cell-like cells (GSCLCs) with an appropriate androgenetic epigenome. Importantly, GSCLCs show vigorous spermatogenesis, not only upon transplantation into testes in vivo but also under an in vitro culture of testis transplants, and the resultant spermatids contribute to fertile offspring. By uniting faithful recapitulations of the three phases of male germ-cell development, our study creates a paradigm for the in vitro male gametogenesis by PSCs.

Gap between UAS and ureteroscope predicts renal stone-free rate after flexible ureteroscopy with the fragmentation technique.

PURPOSE: To assess the effect of our new classification on surgical outcomes after flexible ureteroscopy (fURS) for kidney stones. METHODS: We retrospectively examined 128 patients after single renal fURS procedures performed using ureteral access sheaths (UASs) with the fragmentation technique. Based on the gap (calculated by subtracting the ureteroscope diameter from the UAS diameter), enrolled patients were divided into three groups: small (< 0.6 mm), medium (0.6 to < 1.2 mm), and large space groups (≥ 1.2 mm). Stone-free (SF) status was defined as either complete absence of stones (SF) or the presence of stones < 4 mm in diameter on non-contrast computed tomography (NCCT). RESULTS: The SF rate was significantly lower in the small space group (50% in small, 97.9% in medium, 89.2% in large; p = 0.001). Perioperative complications over Clavien-Dindo Grade I were observed in 16.7%, 4.2%, and 8.1% of patients, respectively (p = 0.452). The ratio of stone volume and operative time (efficiency of stone removal) was significantly higher in the large space group compared to the small and medium space groups (0.009 ± 0.003 ml/min, 0.013 ± 0.005 ml/min, 0.027 ± 0.012 ml/min, respectively; p < 0.001). CONCLUSION: Our findings that gaps > 0.6 mm (1.8 Fr), including the combination of a 9.5-Fr UAS and a small caliber ureteroscope, improve SF rates, and larger gaps facilitate stone removal efficiency providing the basis for future development of clinical protocols aimed at improving outcomes.

Spatially Fractionated Microbeam Analysis of Tissue-sparing Effect for Spermatogenesis.

Spatially fractionated radiation therapy (SFRT) has been based on the delivery of a single high-dose fraction to a large treatment area that has been divided into several smaller fields, reducing the overall toxicity and adverse effects. Complementary microbeam studies have also shown an effective tissue-sparing effect (TSE) in various tissue types and species after spatially fractionated irradiation at the microscale level; however, the underlying biological mechanism remains elusive. In the current study, using the combination of an ex vivo mouse spermatogenesis model and high-precision X-ray microbeams, we revealed the significant TSE for maintaining spermatogenesis after spatially fractionated microbeam irradiation. We used the following ratios of the irradiated to nonirradiated areas: 50:50, 150:50 and 350:50 µm-slit, where approximately 50, 75 and 87.5% of the sample was irradiated (using center-to-center distances of 100, 200 and 400 µm, respectively). We found that the 50 and 75% micro-slit irradiated testicular tissues showed an almost unadulterated TSE for spermatogenesis, whereas the 87.5% micro-slit irradiated tissues showed an incomplete TSE. This suggests that the TSE efficiency for spermatogenesis is dependent on the size of the nonirradiated spermatogonial stem cell pool in the irradiated testicular tissues. In addition, there would be a spatiotemporal limitation of stem cell migration/competition, resulting in the insufficient TSE for 87.5% micro-slit irradiated tissues. These stem cell characteristics are essential for the accurate prediction of tissue-level responses during or after SFRT, indicating the clinical potential for achieving better outcomes while preventing adverse effects.

The Tissue-Sparing Effect of Spatially Fractionated X-rays for Maintaining Spermatogenesis: A Radiobiological Approach for the Preservation of Male Fertility after Radiotherapy.

Radiotherapy can result in temporary or permanent gonadal toxicity in male cancer patients despite the high precision and accuracy of modern radiation treatment techniques. Previous radiobiological studies have shown an effective tissue-sparing response in various tissue types and species following exposure to spatially fractionated radiation. In the present study, we used an ex vivo mouse testicular tissue culture model and a conventional X-ray irradiation device to evaluate the tissue-sparing effect (TSE) of spatially fractionated X-rays for the protection of male fertility from radiotherapy-related adverse effects. We revealed a significant TSE for maintaining spermatogenesis in the ex vivo testes model following spatially fractionated X-ray irradiation. Moreover, we experimentally propose a possible mechanism by which the migration of spermatogonial cells, from the non-irradiated areas to the irradiated ones, in irradiated testicular tissue, is essential for the TSE and maintaining spermatogenesis. Therefore, our findings demonstrate that the control of TSE following spatially fractionated X-rays in the testes has a considerable potential for clinical application. Interdisciplinary research will be essential for further expanding the applicability of this method as an approach for the preservation of male fertility during or after radiotherapy.

Minimally invasive versus standard endoscopic combined intrarenal surgery for renal stones: a retrospective pilot study analysis.

PURPOSE: The effect of combining miniaturization with endoscopic combined intrarenal surgery (ECIRS) is unclear. Thus, we compared the treatment outcomes between minimally invasive ECIRS (mini-ECIRS) using 16.5 Fr percutaneous access sheath and standard ECIRS using 24 Fr access sheath for renal stones MATERIALS AND METHODS: We retrospectively analyzed consecutive patients who underwent single session mini or standard-ECIRS in the modified Valdivia position for renal stones between April 2009 and May 2016. To adjust for patient characteristics, 77 pairs were matched using preoperative parameters including age, sex, history of febrile urinary tract infection (UTI), stone surface area, number of involved calyces, and staghorn calculi. RESULTS: The stone free rate (SFR) was similar between mini and standard ECIRS according to non-contrast computed tomography (61.1% vs. 52.0%, p = 0.388). The rate of perioperative complications exceeding grade 2 based on the Clavien-Dindo classification was similar in both groups (19.5% vs. 26.0%, p = 0.442). Severe complications exceeding grade 3 were also similar in both groups (2.6% vs. 3.9%, p > 0.99). Two cases of septic shock were noted in each group. Although there was no difference regarding bleeding-related complications (2.6% vs. 6.5%, p = 0.442), pseudoaneurysm or blood transfusion was not observed in the mini-ECIRS group. Pain visual analog scale values in the perioperative period were lower in the mini-ECIRS group (1.34 ± 1.08 vs. 1.69 ± 1.23, p = 0.062). CONCLUSIONS: This study demonstrated that, compared to standard ECIRS, mini-ECIRS maintained SFR without increasing perioperative complications, tended to reduce postoperative pain and had a potential to reduce bleeding-related complications. This report suggests the advantages of ECIRS miniaturization for renal stones.

Development and internal validation of a nomogram to predict perioperative complications after flexible ureteroscopy for renal stones in overnight ureteral catheterization cases.

PURPOSE: To identify risk factors by developing and internally validating a nomogram for preventing perioperative complications in overnight ureteral catheterization cases after fURS for kidney stones. METHODS: We retrospectively examined 309 patients with overnight ureteral catheterization after single fURS procedures for renal stones. fURS procedures were performed based on the fragmentation technique. The ureteral catheter was removed on postoperative day 1. Within this group, patients who experienced perioperative complications (complication group) were compared with those who did not experience complications (non-complication group). The complication group included 77 patients whose Clavien-Dindo classification score was I, II, III, or IV and/or those whose body temperature during hospitalization was over 37.5 °C. RESULTS: The overall stone volume, stone-free rate, incidence of perioperative complications, and procedure duration were 1.39 mL, 94.8%, 24.9%, and 62 min, respectively. Severe complications of a Clavien-Dindo level III or IV were observed in only four cases (1.3%). Multivariate assessment revealed five independent predictors of perioperative complications after fURS with overnight catheterization: age (p = 0.11), sex (p = 0.067), stone volume (p = 0.33), Hounsfield units (p = 0.16), and narrow ureter (p = 0.018). We developed a nomogram to predict perioperative complications after fURS using these parameters. CONCLUSIONS: We developed a predictive model for perioperative complications of patients with overnight catheterization after fURS for renal stones. This model could select patients who were at a low risk of complications.

High-precision microbeam radiotherapy reveals testicular tissue-sparing effects for male fertility preservation.

Microbeam radiotherapy (MRT) is based on a spatial fractionation of synchrotron X-ray microbeams at the microscale level. Although the tissue-sparing effect (TSE) in response to non-uniform radiation fields was recognized more than one century ago, the TSE of MRT in the testes and its clinical importance for preventing male fertility remain to be determined. In this study, using the combination of MRT techniques and a unique ex vivo testes organ culture, we show, for the first time, the MRT-mediated TSE for the preservation of spermatogenesis. Furthermore, our high-precision microbeam analysis revealed that the survival and potential migration steps of the non-irradiated germ stem cells in the irradiated testes tissue would be needed for the effective TSE for spermatogenesis. Our findings indicated the distribution of dose irradiated in the testes at the microscale level is of clinical importance for delivering high doses of radiation to the tumor, while still preserving male fertility.

Generating Genetically Engineered Mice Using a Spermatogonial Stem Cell-Mediated Method.

Mouse spermatogonial stem cells (SSCs) can be grown in culture for long periods. Cultured SSCs, also called germline stem (GS) cells, maintain themselves by self-renewing proliferation while retaining the ability to differentiate into sperm. Thus, when transplanted into the seminiferous tubules of a host mouse testis, they settle in the basal compartment of the tubules and establish spermatogenenic colonies. The sperm produced in the host are competent to produce offspring. This can be exploited for the generation of genetically modified mice, through the transplantation of genetically modified GS cells. In this section, we describe a method of genome editing-mediated GS cell modification and transplantation.

Outcome of flexible ureteroscopy for renal stone with overnight ureteral catheterization: a propensity score-matching analysis.

PURPOSE: To evaluate the influence of overnight ureteral catheterization and determine if routine long-term post-stenting can be avoided in flexible ureterorenoscopy (fURS) procedure for kidney stone. METHODS: Three hundred ninety-three patients who underwent single fURS for kidney stone between January 2013 and June 2016 at a single institute were retrospectively analyzed. The stone-free (SF) and perioperative complication rates in patients with routine long-term post-stenting after fURS (long-term stent group) were compared with those of patients with overnight ureteral catheterization (short-term stent group). Propensity score-matching analysis was used to adjust the difference in baseline preoperative parameters between the two groups. All preoperative parameters were chosen to develop the propensity score, and 74 patients in the short-term stent group were retrospectively matched with the patients in the long-term stent group at a 1:1 ratio. RESULTS: Patient characteristics included age, sex, side of involvement, height, body weight, body mass index, number of stone(s), stone volume, Hounsfield units of stone, preoperative white blood cell count, preoperative C-reactive protein, preoperative creatinine, pretreatment, pre-stenting, stenosis of the ureter, and procedure duration. The SF rates were 91.9 and 93.2% in the short-term and long-term stent groups, respectively. Perioperative complications were 14.9 and 12.2%. No difference was noted between the two groups in terms of SF and perioperative complication rates. CONCLUSIONS: Short-term post-stenting using overnight ureteral catheterization in uncomplicated cases after fURS for kidney stone was as effective as conventional long-term post-stenting in reducing postoperative complications. These preliminary data suggest the possibility that routine long-term post-stenting was unnecessary.

Low-dose radiation-induced risk in spermatogenesis.

PURPOSE: To discuss low-dose radiation-induced risks to male fertility focusing on potential mechanisms of low-dose radiation-induced damage on spermatogenesis, epidemiological studies of environmental radiation effects on sperm parameters and transgenerational effects following exposure of spermatogonial stem cells (SSCs). BACKGROUND: Spermatogenesis produces mature male gametes, spermatozoa, which fertilize their counterpart female gametes, oocytes. The robust maintenance system of spermatogenesis is essential for genomic conservation; however, male fertility can be readily impacted by exposure to environmental, chemical and physical factors including ionizing radiation. The mammalian testes are known to be radiosensitive yet the underlying molecular mechanisms of low-dose radiation-induced risks for spermatogenesis remain unclear. Furthermore, evidence characterizing transgenerational effects following exposure of SSCs remain controversial. CONCLUSIONS: Current concerns over the possible effects of low-dose radiation exposure on spermatogenesis requires further elucidation that may be resolved comparing and integrating observed experimental and epidemiological data.

SHISA6 Confers Resistance to Differentiation-Promoting Wnt/ß-Catenin Signaling in Mouse Spermatogenic Stem Cells.

In the seminiferous tubules of mouse testes, a population of glial cell line-derived neurotrophic factor family receptor alpha 1 (GFRα1)-positive spermatogonia harbors the stem cell functionality and supports continual spermatogenesis, likely independent of asymmetric division or definitive niche control. Here, we show that activation of Wnt/ß-catenin signaling promotes spermatogonial differentiation and reduces the GFRα1+ cell pool. We further discovered that SHISA6 is a cell-autonomous Wnt inhibitor that is expressed in a restricted subset of GFRα1+ cells and confers resistance to the Wnt/ß-catenin signaling. Shisa6+ cells appear to show stem cell-related characteristics, conjectured from the morphology and long-term fates of T (Brachyury)+ cells that are found largely overlapped with Shisa6+ cells. This study proposes a generic mechanism of stem cell regulation in a facultative (or open) niche environment, with which different levels of a cell-autonomous inhibitor (SHISA6, in this case) generates heterogeneous resistance to widely distributed differentiation-promoting extracellular signaling, such as WNTs.

Cryopreservation of testis tissues and in vitro spermatogenesis.

Cancer treatments, either chemo- or radiotherapy, may cause severe damage to gonads which could lead to the infertility of patients. In post-pubertal male patients, semen cryopreservation is recommended to preserve the potential to have their own biological children in the future; however, it is not applicable to prepubertals. The preservation of testis tissue which contains spermatogonial stem cells (SSCs) but not sperm would be an alternative measure. The tissues or SSCs have to be transplanted back into patients to obtain sperm; however, this procedure remains experimental, invasive, and is accompanied with the potential risk of re-implantation of cancer cells. Recently, we developed an organ culture system which supports the spermatogenesis of mice up to sperm formation from SSCs. It was also shown that the tissues could be frozen for later sperm production, which resulted in the generation of offspring. Thus, it could be useful as a clinical application for preserving the reproductive potential of male pediatric cancer patients. The establishment of an optimized cryopreservation method and the development of a culture system for human testis tissue are expected in the future.

Genome Editing in Mouse Spermatogonial Stem Cell Lines Using TALEN and Double-Nicking CRISPR/Cas9.

Mouse spermatogonial stem cells (SSCs) can be cultured for multiplication and maintained for long periods while preserving their spermatogenic ability. Although the cultured SSCs, named germline stem (GS) cells, are targets of genome modification, this process remains technically difficult. In the present study, we tested TALEN and double-nicking CRISPR/Cas9 on GS cells, targeting Rosa26 and Stra8 loci as representative genes dispensable and indispensable in spermatogenesis, respectively. Harvested GS cell colonies showed a high targeting efficiency with both TALEN and CRISPR/Cas9. The Rosa26-targeted GS cells differentiated into fertility-competent sperm following transplantation. On the other hand, Stra8-targeted GS cells showed defective spermatogenesis following transplantation, confirming its prime role in the initiation of meiosis. TALEN and CRISPR/Cas9, when applied in GS cells, will be valuable tools in the study of spermatogenesis and for revealing the genetic mechanism of spermatogenic failure.

Spermatogonial stem cells: Progress and prospects.

Twenty years ago, the transplantation of spermatogonial stem cells (SSCs) from a mouse to other recipient mice was shown to be feasible, which clearly demonstrated the functional identity of SSCs. Since then, several important new findings and other technical developments have followed, which included a new hypothesis on their cell kinetics and spermatogonial hierarchy in the testis, a culture method allowing their self-renewal and proliferation, a testis tissue organ culture method, which induced their complete differentiation up to sperm, and the in vitro induction of germ cells from embryonic stem cells and induced pluripotent stem cells. These advancements reinforced or advanced our understanding of this unique cell. Nonetheless, there are many unresolved questions in the study of spermatogonial stem cells and a long road remains until these cells can be used clinically in reproductive medicine.

Offspring production with sperm grown in vitro from cryopreserved testis tissues.

With the increasing cure rate of paediatric cancers, infertility, as one of the adverse effects of treatments, has become an important concern for patients and their families. Since semen cryopreservation is applicable only for post-pubertal patients, alternative pre-pubertal measures are necessary. Here we demonstrate that testis tissue cryopreservation is a realistic measure for preserving the fertility of an individual. Testis tissues of neonatal mice were cryopreserved either by slow freezing or by vitrification. After thawing, they were cultured on agarose gel and showed spermatogenesis up to sperm formation. Microinsemination was performed with round spermatids and sperm, leading to eight offspring in total. They grew healthily and produced progeny upon natural mating between them. This strategy, the cryopreservation of testis tissues followed by in vitro spermatogenesis, is promising to preserve the fertility of male paediatric cancer patients in the future.

Evaluation and validation of the core lower urinary tract symptom score as an outcome assessment tool for the treatment of benign prostatic hyperplasia: effects of the α1-adrenoreceptor antagonist silodosin.

We investigated the Core Lower Urinary Tract Symptom Score as an outcome assessment tool for the treatment of lower urinary tract symptoms using silodosin. In addition, the ability of the Core Lower Urinary Tract Symptom Score to detect overactive bladder in male patients with lower urinary tract symptoms was examined. The present study included 241 males with benign prostatic hyperplasia treated at 31 medical facilities between June 2009 and December 2010. All patients were given silodosin, and the effects of silodosin intake were measured using four questionnaires: the Core Lower Urinary Tract Symptom Score, International Prostate Symptom Score, Overactive Bladder Symptom Score and Quality-of-Life index. The efficacy of silodosin for treating lower urinary tract symptoms was validated according to the total scores of all four questionnaires weighted equally (P < 0.05). Spearman's ρ among the Core Lower Urinary Tract Symptom Score, International Prostate Symptom Score and Overactive Bladder Symptom Score showed a mild-high correlation. However, the correlation between the baseline values of the Core Lower Urinary Tract Symptom Score and Quality-of-Life index was low in the groups with benign prostatic hyperplasia (ρ = 0.314) and benign prostatic hyperplasia/overactive bladder (ρ = 0.244). Our findings showed the Core Lower Urinary Tract Symptom Score, both its total score and each subscore, is able to show the efficacy of silodosin, similar to other questionnaires. The Core Lower Urinary Tract Symptom Score is also useful for identifying overactive bladder symptoms in patients with benign prostatic hyperplasia. As the Core Lower Urinary Tract Symptom Score does not correlate well with the Quality-of-Life index, these two questionnaires might be better used in combination to assess treatment outcomes.

Regeneration of spermatogenesis after testicular cancer chemotherapy.

PURPOSE: Azoospermia is a common side effect of chemotherapy. Although most patients restore spermatogenesis over time, the exact time course has not been well described. We analyzed the recovery of spermatogenesis in testicular cancer patients following chemotherapy. PATIENTS AND METHODS: 49 patients, consisting of 45 treated with a bleomycin, etoposide and cisplatin (BEP) regimen and 4 with high-dose chemotherapy, were followed up with occasional semen analyses. The primary endpoint of this study was the confirmation of motile spermatozoa in the patients' semen. RESULTS: Among 45 patients treated with BEP, 44 recovered spermatogenesis. The recovery of spermatogenesis was delayed depending on the increase in BEP cycles. In groups of patients who received 1-2, 3 and 4 cycles, the recovery rates of spermatogenesis within 2 year were 83.3, 80.0 and 66.7%, respectively. In the group with 5-6 cycles of BEP, re-spermatogenesis was significantly delayed and no patients re-established spermatogenesis within 2 years. The patients' age and semen parameters before chemotherapy were not useful as predictive factors for the recovery of spermatogenesis. CONCLUSION: The recovery of spermatogenesis was rather fast and was often observed as early as several months after BEP treatment when the number of cycles was <4.

Utility and limitation of cumulative stone diameter in predicting urinary stone burden at flexible ureteroscopy with holmium laser lithotripsy: a single-center experience.

PURPOSE: To retrospectively assess the clinical utility in ureteroscopy (URS) planning of cumulative stone diameter (CSD), which does not account for stone width or depth, as a predictor of URS outcome and compare it with stone volume. MATERIALS AND METHODS: Patients with renal stones treated at a single institute by flexible URS were retrospectively evaluated. To assess the clinical utility of CSD, relationships between stone-free (SF) status and stone burden (CSD and volume) were analyzed using the area under the receiver operating characteristics (AUROC) curve. To identify stone number impact on CSD, the AUROC of CSD divided by stone number was evaluated. Correlation coefficients of CSD and stone volume were also calculated for groups by stone number. RESULTS: In cases with CSD <20.0 mm, CSD and stone volume revealed equal ability to predict SF status. In cases with CSD ≥20.0 mm, stone volume showed higher predictive ability. The ROC curves for cases with ≥4 stones showed that CSD was less predictive of SF status than stone volume. The correlation coefficients of CSD and stone volume by stone number were 0.922 for 1 stone, 0.900 for 2-3 stones, and 0.661 for ≥4 stones. CONCLUSIONS: In cases with CSD ≥20.0 mm or ≥4 stones, we should evaluate stone volume for a more predictive stone burden, and pretreatment non-contrast CT seems sufficient. In cases with CSD <20.0 mm or 1-3 stones, CSD was as valid a predictor of preoperative stone burden as stone volume, so preoperative kidney-ureter-bladder (KUB) films may be sufficient.