Systematic molecular analyses for 115 karyotypically normal men with isolated non-obstructive azoospermia.

STUDY QUESTION: Do copy-number variations (CNVs) in the azoospermia factor (AZF) regions and monogenic mutations play a major role in the development of isolated (non-syndromic) non-obstructive azoospermia (NOA) in Japanese men with a normal 46, XY karyotype? SUMMARY ANSWER: Deleterious CNVs in the AZF regions and damaging sequence variants in eight genes likely constitute at least 8% and approximately 8% of the genetic causes, respectively, while variants in other genes play only a minor role. WHAT IS KNOWN ALREADY: Sex chromosomal abnormalities, AZF-linked microdeletions, and monogenic mutations have been implicated in isolated NOA. More than 160 genes have been reported as causative/susceptibility/candidate genes for NOA. STUDY DESIGN, SIZE, DURATION: Systematic molecular analyses were conducted for 115 patients with isolated NOA and a normal 46, XY karyotype, who visited our hospital between 2017 and 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS: We studied 115 unrelated Japanese patients. AZF-linked CNVs were examined using sequence-tagged PCR and multiplex ligation-dependent probe amplification, and nucleotide variants were screened using whole exome sequencing (WES). An optimized sequence kernel association test (SKAT-O), a gene-based association study using WES data, was performed to identify novel disease-associated genes in the genome. The results were compared to those of previous studies and our in-house control data. MAIN RESULTS AND THE ROLE OF CHANCE: Thirteen types of AZF-linked CNVs, including the hitherto unreported gr/gr triplication and partial AZFb deletion, were identified in 63 (54.8%) cases. When the gr/gr deletion, a common polymorphism in Japan, was excluded from data analyses, the total frequency of CNVs was 23/75 (30.7%). This frequency is higher than that of the reference data in Japan and China (11.1% and 14.7%, respectively). Known NOA-causative AZF-linked CNVs were found in nine (7.8%) cases. Rare damaging variants in known causative genes (DMRT1, PLK4, SYCP2, TEX11, and USP26) and hemizygous/multiple-heterozygous damaging variants in known spermatogenesis-associated genes (TAF7L, DNAH2, and DNAH17) were identified in nine cases (7.8% in total). Some patients carried rare damaging variants in multiple genes. SKAT-O detected no genes whose rare damaging variants were significantly accumulated in the patient group. LIMITATIONS, REASONS FOR CAUTION: The number of participants was relatively small, and the clinical information of each patient was fragmentary. Moreover, the pathogenicity of identified variants was assessed only by in silico analyses. WIDER IMPLICATIONS OF THE FINDINGS: This study showed that various AZF-linked CNVs are present in more than half of Japanese NOA patients. These results broadened the structural variations of AZF-linked CNVs, which should be considered for the molecular diagnosis of spermatogenic failure. Furthermore, the results of this study highlight the etiological heterogeneity and possible oligogenicity of isolated NOA. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by Grants from the Japan Society for the Promotion of Science (21K19283 and 21H0246), the Japan Agency for Medical Research and Development (22ek0109464h0003), the National Center for Child Health and Development, the Canon Foundation, the Japan Endocrine Society, and the Takeda Science Foundation. The results of this study were based on samples and patient data obtained from the International Center for Reproductive Medicine, Dokkyo Medical University Saitama Medical Center, Koshigaya, Japan. The authors have no conflicts of interest to disclose. TRIAL REGISTRATION NUMBER: N/A.

Preoperative testosterone and follicle stimulating hormone levels are important predictors for sperm retrieval by microdissection testicular sperm extraction in non-mosaic Klinefelter syndrome.

This study aimed to assess outcomes of microdissection testicular sperm extraction (MD-TESE) and identify predictors for sperm retrieval (SR) in patients with non-mosaic Klinefelter syndrome (NM-KFS). We retrospectively evaluated 37 patients with NM-KFS who underwent MD-TESE. Data of age at operation, body mass index (BMI), testicular volume, serum luteinizing hormone (LH), follicle stimulating hormone (FSH), preoperative and postoperative testosterone levels with reduction ratio between the two values, and FSH/preoperative testosterone ratio were analysed. These patients were divided into two groups according to success or failure of SR: the successful and failure groups. Factors related to SR were evaluated by statistical analyses using the Mann-Whitney U test and logistic regression modelling. Regarding these factors, the cut-off level was specified using the receiver operating characteristics (ROC) curve. Moreover, the percentage of SR at that level was assessed. A simple scoring model was developed based on the multivariate analysis. Fourteen patients underwent successful SR, whereas 23 experienced failure SR. Statistical analysis found preoperative testosterone and FSH levels to be significant factors associated with SR. On the ROC curve, the cut-off levels for preoperative testosterone and FSH were 2.34 ng/ml and 33.2 mIU/ml respectively. A new scoring model was developed, consisting of preoperative testosterone (≥2.34 ng/ml) and FSH (≤33.2 mIU/ml). The sperm retrieval rates (SRRs) were clearly discriminated by stratification according to the scoring model. The SRR of the cases of scores of 2, 1 and 0 were 87.5%, 31.6% and 10% respectively. At our hospital, the SRR of MD-TESE in patients with NM-KFS was 37.8%. The patients with high testosterone and low FSH levels tended to demonstrate successful SR.

Testicular volume in non-obstructive azoospermia with a history of bilateral cryptorchidism may predict successful sperm retrieval by testicular sperm extraction.

PURPOSE: Cryptorchidism is one of the most common causes of non-obstructive azoospermia (NOA) in adulthood. Even if early orchidopexy is performed to preserve fertility potential, some patients still suffer from azoospermia. Fertility potential is significantly lower in bilateral than unilateral cryptorchidism. The aims of this study were to identify clinical parameters that predict the likely success of sperm recovery by microscopic testicular sperm extraction (micro-TESE) and also the likely outcome of intracytoplasmic sperm injection using sperm from NOA patients who submitted to bilateral orchidopexy. METHODS: Fifty-two NOA patients with a history of bilateral cryptorchidism underwent micro-TESE. The following clinical parameters were evaluated as predictive factors for successful sperm recovery: age at micro-TESE; age at orchidopexy; period from orchidopexy to micro-TESE; luteinizing hormone (LH); follicle-stimulating hormone (FSH); testosterone; average testicular volume; and body mass index. RESULTS: In the successful sperm retrieval group, average testicular volume was significantly greater, while serum LH and FSH, and body mass index were significantly lower. In a multivariate analysis, average testicular volume was positively correlated with successful sperm recovery. CONCLUSION: Our results indicate that testicular volume in NOA patients with bilateral cryptorchidism is a predictor for successful sperm recovery.

[Sperm Cryopreservation for a Gender Identity Disorder Patient Prior to Sex Reassignment Surgery : A Case Report].

We describe the first case of a 25-year-old patient with gender identity disorder (GID) who underwent cryopreservation of sperm prior to male-to-female (MTF) sex reassignment surgery in Japan. The patient wanted to freeze sperm to keep open the option of conceiving a child in the future. The ethics committee of our institution discussed the case and officially approved cryopreservation of sperm before sex reassignment surgery. Compared with foreign countries, sperm cryopreservation of GID is not recognized and guidelines have yet been published in Japan. Here, we report sperm cryopreservation for MTF before sex reassignment surgery.

Hydroxyapatites: Key Structural Questions and Answers from Dynamic Nuclear Polarization.

We demonstrate that NMR/DNP (Dynamic Nuclear Polarization) allows an unprecedented description of carbonate substituted hydroxyapatite (CHAp). Key structural questions related to order/disorder and clustering of carbonates are tackled using distance sensitive DNP experiments using 13C-13C recoupling. Such experiments are easily implemented due to unprecedented DNP gain (orders of magnitude). DNP is efficiently mediated by quasi one-dimensional spin diffusion through the hydroxyl columns present in the CHAp structure (thought of as "highways" for spin diffusion). For spherical nanoparticles and ϕ < 100 nm, it is numerically shown that spin diffusion allows their study as a whole. Most importantly, we demonstrate also that the DNP study at 100 K leads to data which are comparable to data obtained at room temperature (in terms of spin dynamics and line shape resolution). Finally, all 2D DNP experiments can be interpreted in terms of domains exhibiting well identified types of substitution: local order and carbonate clustering are clearly favored.

In vitro apatite formation on nano-crystalline titania layer aligned parallel to Ti6Al4V alloy substrates with sub-millimeter gap.

Pure titanium substrates were chemically oxidized with H2O2 and subsequent thermally oxidized at 400 °C in air to form anatase-type titania layer on their surface. The chemically and thermally oxidized titanium substrate (CHT) was aligned parallel to the counter specimen such as commercially pure titanium (cpTi), titanium alloy (Ti6Al4V) popularly used as implant materials or Al substrate with 0.3-mm gap. Then, they were soaked in Kokubo's simulated body fluid (SBF, pH 7.4, 36.5 °C) for 7 days. XRD and SEM analysis showed that the in vitro apatite-forming ability of the contact surface of the CHT specimen decreased in the order: cpTi > Ti6Al4V > Al. EDX and XPS surface analysis showed that aluminum species were present on the contact surface of the CHT specimen aligned parallel to the counter specimen such as Ti6Al4V and Al. This result indicated that Ti6Al4V or Al specimens released the aluminum species into the SBF under the spatial gap. The released aluminum species might be positively or negatively charged in the SBF and thus can interact with calcium or phosphate species as well as titania layer, causing the suppression of the primary heterogeneous nucleation and growth of apatite on the contact surface of the CHT specimen under the spatial gap. The diffusion and adsorption of aluminum species derived from the half-sized counter specimen under the spatial gap resulted in two dimensionally area-selective deposition of apatite particles on the contact surfaces of the CHT specimen.

Calcium phosphate crystallization on titania in a flowing Kokubo solution.

Dry titania layers on air-oxidized titanium substrates have been found to be active enough to cause apatite to be deposited in Kokubo's simulated body fluid (SBF) in narrow confined spaces, such as those in narrow grooves and thin gaps. Such in vitro apatite deposition is the basis of the GRAPE(®) technique. The aim of the present study is to determine why GRAPE conditions favor apatite deposition when laminar SBF flow (at 0.01-0.3 ml/min) passes through a shallow channel (0.5 mm) between a pair of titanium substrates each with a dry layer of titania. Assessing the factors that control the heterogeneous nucleation process led to the proposal of the working hypothesis that there are nucleation pre-embryos, ion assemblies that can be stabilized to form embryos, on the titania layer but that they are removed by the SBF flow. Specimens were subjected to different combinations of processes. One combination was that titania layers were exposed to still or flowing SBF, and the other was that half of a specimen, the inlet or outlet side, was exposed to still or flowing SBF with the other half being covered. The surface morphologies of the specimens were then compared in detail. The conclusion was that exposure to still SBF for 2 days before exposure to flowing SBF was required for apatite to be deposited. Some complicated apatite deposition modes were observed, e.g., apatite was deposited even on areas unexposed to still SBF. All of the results were successfully interpreted using the working hypothesis. The conclusion was that the GRAPE(®) technique depends on the confined space holding pre-embryo and embryo assemblies.

A unified in vitro evaluation for apatite-forming ability of bioactive glasses and their variants.

The aim of this study was to propose and validate a new unified method for testing dissolution rates of bioactive glasses and their variants, and the formation of calcium phosphate layer formation on their surface, which is an indicator of bioactivity. At present, comparison in the literature is difficult as many groups use different testing protocols. An ISO standard covers the use of simulated body fluid on standard shape materials but it does not take into account that bioactive glasses can have very different specific surface areas, as for glass powders. Validation of the proposed modified test was through round robin testing and comparison to the ISO standard where appropriate. The proposed test uses fixed mass per solution volume ratio and agitated solution. The round robin study showed differences in hydroxyapatite nucleation on glasses of different composition and between glasses of the same composition but different particle size. The results were reproducible between research facilities. Researchers should use this method when testing new glasses, or their variants, to enable comparison between the literature in the future.

Liquid phase deposited titania coating to enable in vitro apatite formation on Ti6Al4V alloy.

A recently developed "GRAPE(®) technology" provides titanium or titanium alloy implants with spontaneous apatite-forming ability in vitro, which requires properly designed gaps and optimum heat treatment in air. In this study, titanium alloy and commercially pure (cp) titanium substrates were thermally oxidized in air before aligning pairs of specimens in the GRAPE(®) set-up, i.e., titanium alloy and cp titanium substrates were aligned parallel to each other with optimum gap width (spatial design). A liquid phase deposition (LPD) technique was employed for titania coatings on titanium alloy substrate. Then, they were soaked in Kokubo's simulated body fluid (SBF, pH 7.4, 36.5 °C) for 7 days to confirm the in vitro apatite formation on the substrates under the specific spatial design. Anatase-type titania coatings fabricated by using LPD technique led to the deposition of apatite particles within 7 days and showed apatite X-ray diffraction. On the other hand, thermally oxidized titanium alloy substrate in air and non-treated specimens did not show any apatite X-ray diffraction. These results indicated that the heterogeneous nucleation of apatite induced on anatase-type titania coating prepared by LPD technique when it was aligned parallel to thermally oxidized cp titanium substrate with optimum gap width.

Two tumor types in a unilateral testis in a patient with severe oligozoospermia and a history of cryptorchidism surgery: A case report.

Testicular cancer, the most common cancer among young male adults, is associated with infertility. A 38-year-old male patient was admitted to Dokkyo Medical University Saitama Medical Center, Japan, with infertility associated with severe oligozoospermia. Scrotal ultrasonography revealed two distinct tumors in the left testis: A mass with abundant blood flow on the cranial side and a mass with poor blood flow on the caudal side. Additional analysis revealed mild elevation of intact human chorionic gonadotropin (hCG) levels (tumor marker level assessment), high testosterone and low luteinizing hormone and follicle-stimulating hormone levels (hormonal level assessment) and severe oligoasthenozoospermia (semen assessment). The preoperative diagnosis was left-sided testicular cancer and severe oligoasthenozoospermia and the patient underwent left high orchiectomy and oncological testicular sperm extraction. Based on the pathological assessment, the cranial tumor was diagnosed as a seminoma with syncytiotrophoblastic cells, whereas the caudal tumor had only scar tissue with germ cell neoplasia in situ in the adjacent parenchyma. Following surgery, intact hCG and hormone levels of the patient were normalized, and the semen parameters (semen volume, sperm density, and motility) improved dramatically. To the best of our knowledge, the present case is the first report of two types of testicular tumor in a unilateral testis in a patient with a history of cryptorchidism surgery. The present case demonstrated that scrotal ultrasonography should be performed in patients with abnormal semen results to rule out testicular tumors.

An Injectable Hydrogel Composing Anti-Inflammatory and Osteogenic Therapy toward Bone Erosions Microenvironment Remodeling in Rheumatoid Arthritis.

Healing bone erosions in rheumatoid arthritis (RA) remains greatly challenging via biomaterial strategies. Given the unsuccessful innate bone erosion healing due to an inflammatory disorder, over-activated osteoclasts, and impaired osteoblasts differentiation, RA pathogenesis-guided engineering of an innovative hydrogel platform is needed for remodeling osteoimmune and osteogenic microenvironment of bone erosion healing. Herein, in situ adaptable and injectable interpenetrating polymer network (IPN) hydrogel is developed through an ingenious combination of a bio-orthogonal reaction between hyaluronic acid (HA) and collagen, along with effective electrostatic interactions leveraging bisphosphonate (BP)-functionalized HA macromers (HABP) and nanorod shaped zinc (Zn)-doped biphasic calcium phosphate (ZnBCP). IPN hydrogel exhibits exceptional adaptability to the local shape complexity at bone erosions, and by integrating ZnBCP and HABP, a multi-stage releasing platform is engineered, facilitating controlled cargo delivery for remodeling more anti-inflammatory M2 cells and reducing over-activated osteoclastic activities, thereby reconstructing the bone regeneration microenvironment. Sustainedly co-delivering multiple ions (calcium and phosphate) can display excellent osteogenic properties and be conducive to the bone formation process, by effects of osteogenesis-associated cell differentiation. Overall, the introduced bioactive IPN hydrogel therapy remodels the osteoimmune environment by synergistic pro-inflammation-resolving, osteogenesis, and anti-osteoclastic activities, displaying excellent bone reconstruction in the collagen-induced arthritis rabbit model.

Robot-assisted radical prostatectomy using the novel hinotoriTM surgical robot system: initial experience and operation learning curve at a single institution.

Background: The hinotoriTM surgical robot system (HSRS) is the first made-in-Japan robotic system used for radical prostatectomy. Here, we report initial results and describe our learning curve (skill development) implementing robot-assisted radical prostatectomy using HSRS (h-RARP). Methods: Between November 2021 and December 2022, 97 patients who underwent h-RARP at our institution were enrolled in this study. We retrospectively evaluated the surgical outcomes of the initial cases using h-RARP, comparing those of RARP using da Vinci surgical robot system (d-RARP) in our institution. Furthermore, the learning curves of two surgeons with the highest number of h-RARP were analyzed. Patients treated by each surgeon were categorized into two groups: 1-15 cases (earlier group) and >15 cases (later group). Preoperative patient characteristics, operation parameters, and complication rates were compared between the two groups. Results: In terms of surgical outcome, h-RARP was comparable to d-RARP. The procedures performed by the HSRS were successfully completed in all cases. There was no complication of grade 3 or higher. Comparing the two surgeons, surgeon 1, who had performed 40 d-RARP procedures, had time using robot system of the later group that was significantly shorter than that of the earlier group. However, for surgeon 2 with more than 100 d-RARP procedures, there was no statistically significant difference in time using robot system between groups. Other parameters showed no difference between earlier and later groups for the two surgeons. Conclusions: Our results show that surgical outcomes of h-RARP are comparable to those of d-RARP during the initial experience of clinical application. In addition, the surgeons' learning curves for the total RARP experience suggest that the experience of d-RARP can carry over to performance using the novel HSRS.

Sol-gel-derived silicate nano-hybrids for biomedical applications.

Organic-inorganic hybrids of poly(dimethyl siloxane), gelatin, and chitosan with such silanes as tetraethoxysilane and 3-glycidoxytriethoxysilane are derived via the sol-gel routes. Their biomedical applications are discussed from biomimetic deposition of bone-like apatite, cell culture, and in vivo behavior.

A Case Report of Successful Kidney Transplantation in a Patient With Chronic Myelogenous Leukemia (CML) Who Has Been in Remission for 15 Years on Imatinib.

For chronic myeloid leukemia (CML), a Philadelphia chromosome-positive myeloproliferative neoplasm, the introduction of tyrosine kinase inhibitors has transformed CML from a lethal disease into a manageable chronic disease with a close-to-normal life expectancy. Active malignancy is an absolute contraindication to kidney transplantation. However, it is controversial whether kidney transplantation can be safely performed in patients with a history of CML who are in remission. We describe the clinical course of a 64-year-old male patient with chronic kidney disease from diabetic nephropathy (DMN) who underwent living donor kidney transplantation. The patient was diagnosed with CML 15 years ago and promptly achieved cytogenetic and molecular biological remission after starting imatinib. After that, he continued imatinib treatment for 15 years and was in remission, but his chronic kidney disease from DMN gradually worsened. A preemptive living donor kidney transplant was performed in July 2020. Imatinib for CML was discontinued because the patient maintained deep molecular remission (DMR) of major molecular response for more than 15 years before kidney transplantation. After kidney transplantation, the transplanted kidney function remained good at approximate serum creatinine levels of 1.1 mg/dL without histopathologic rejection, and the 3 monthly BCR-ABL1 measurement results were negative and are in progress. Thus, he continues to maintain treatment-free remission status without imatinib for 26 months after renal transplantation. In conclusion, this result suggests that CML with long-lasting DMR on imatinib therapy can be considered an inactive malignancy and therefore a relative indication for kidney transplantation.

A regressed germ cell tumor discovered secondary to azoospermia.

Introduction: Regressed germ cell tumors are a rare disease commonly diagnosed with metastatic symptoms without local symptoms in the testis. Case presentation: A 33-year-old man with azoospermia was referred to our hospital. His right testis was slightly swollen, and ultrasonography revealed hypoechogenicity of the right testis with decreased blood flow. Right high orchiectomy was performed. Pathologically, the seminiferous tubules were absent or highly atrophied with vitrification degeneration; however, no neoplastic lesion was confirmed. One-month post-surgery, the patient noticed a mass in the left supraclavicular fossa, of which a biopsy revealed seminoma. The patient was diagnosed with a regressed germ cell tumor and underwent systemic chemotherapy. Conclusion: We reported the first case of a regressed germ cell tumor discovered due to complaints of azoospermia.

A Case Report of Successful Renal Transplantation After Surgically Treated Type A Aortic Dissection.

BACKGROUND: Aortoiliac lesions can influence the results of kidney transplantation and increase technical difficulties during surgery. Aortic dissection (AD) is a rare and infrequently reported event before transplantation, whereas immediate optimal perfusion is paramount for kidney transplantation. Thus, adequate blood flow imposed by the flow from the true lumen must be considered when choosing a target inflow vessel. CASE PRESENTATION: A 67-year-old man on dialysis with end-stage renal disease caused by immunoglobulin A nephropathy was referred for kidney transplantation. He had successfully undergone conventional Stanford type A AD surgery 3 years ago. Pretransplant contrast-enhanced computed tomography angiography revealed termination of the distal intimal flaps within the common iliac arteries. Dilation of the descending aorta was also observed. Based on the meticulous vascular assessment, including consultation with the cardiovascular surgery department, the right internal iliac artery (IIA) was considered usable for anastomosis. He underwent living unrelated kidney transplantation from his 66-year-old wife. The patency and blood flow in the right IIA were also verified using intraoperative findings. Without any special procedure, we used a side-to-end arterial anastomosis between the donor renal artery and recipient IIA. After vascular clamp removal, the allograft was perfused homogeneously and immediately functioned. CONCLUSION: Patients receiving previous surgery for type A AD can successfully undergo kidney transplantation if the patency of the iliac arteries from the true lumen is confirmed by perioperative evaluation, and the artery can be carefully clamped to avoid possible further dissection.

A Case Report of Successful Kidney Transplantation in a Patient With Autosomal Dominant Polycystic Kidney Disease Who Underwent Thoracic Endovascular Aortic Repair for Type B Aortic Dissection.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is associated with several cardiovascular disorders, including aortic dissection, which preferentially occurs at the thoracic or abdominal level. Because there are few case reports describing surgical repair for aortic dissection followed by renal transplantation in patients with ADPKD, kidney transplantation performed after repair for aortic dissection remains challenging. CASE PRESENTATION: A 34-year-old Japanese man with end-stage renal disease secondary to ADPKD underwent thoracic endovascular aortic repair for complicated acute type B aortic dissection 12 months earlier. A contrast computed tomography scan before transplantation revealed an aortic dissection involving the descending aorta proximal to the common iliac arteries and confirmed multiple large bilateral renal cysts. After simultaneous right native nephrectomy, the patient underwent preemptive living-donor kidney transplantation obtained from his mother. Intraoperatively, we noted that dissection of the external iliac vessels was difficult because of dense adhesions. Arterial clamping was performed immediately below the bifurcation of the internal iliac artery to prevent further aortic dissection of the external iliac artery. After end-to-end anastomosis to the internal iliac artery was completed and the vascular clamp was released, the kidney began to produce urine immediately. CONCLUSION: This case suggests that kidney transplantation in patients undergoing endovascular aortic repair for aortic dissection can be performed by adequately applying a vascular clamp proximal to the internal iliac artery during vascular anastomosis.

Preparation and Drug Release Profile of Chitosan-Siloxane Hybrid Capsules Coated with Hydroxyapatite.

Chitosan is a cationic polymer that forms polymerized membranes upon reaction with anionic polymers. Chitosan-carboxymethyl cellulose (CMC) capsules are drug delivery carrier candidates whose mechanical strength and permeability must be controlled to achieve sustained release. In this study, the capsules were prepared from chitosan-γ-glycidoxypropyltrimethoxysilane (GPTMS)-CMC. The mechanical stability of the capsules was improved by crosslinking the chitosan with GPTMS. The capsules were then coated with hydroxyapatite (HAp) by alternately soaking them in calcium chloride solution and disodium hydrogen phosphate solution to prevent rapid initial drug release. Cytochrome C (CC), as a model drug, was introduced into the capsules via two routes, impregnation and injection, and then the CC released from the capsules was examined. HAp was found to be deposited on the internal and external surfaces of the capsules. The amount of CC introduced, and the release rate were reduced by the HAp coating. The injection method was found to result in the greatest CC loading.