Testosterone production by Leydig cells (LCs) plays a crucial role in male reproduction. The functional degeneration of LCs can cause testosterone deficiency, ultimately resulting in primary male hypogonadism. Transplantation of exogenous LCs with the ability to produce testosterone in response to the regulation of the hypothalamus–pituitary–gonad axis could be a promising alternative option to treat male primary hypogonadism. Recent studies have shown that it is possible to generate Leydig-like cells from stem cells by various approaches. In addition, somatic cells, such as embryonic or adult fibroblasts, have also been successfully reprogrammed into Leydig-like cells. In this review, we summarized the recent advances in the generation of Leydig-like cells, with an emphasis on comparing the effectiveness and safety of different protocols used and the cells generated. By further analyzing the characteristics of Leydig-like cells generated from fibroblasts based on small signaling molecules and regulatory factors, we found that although the cells may produce testosterone, they are significantly different from real LCs. For future in vivo applications, it is important that the steroidogenic cells generated be evaluated not only for their steroidogenic functions but also for their overall cell metabolic state by proteomics or transcriptomic tools.

Phospholipase C zeta (PLCζ) is a sperm-specific protein that triggers oocyte activation. The analysis of PLCζ expression in human spermatozoa can be used as a diagnostic marker for oocyte activation deficiency. Our laboratory has previously optimized a standard “in-house” assay to determine PLCζ expression in human spermatozoa. However, one study has suggested that an antigen unmasking method (AUM) would be more efficient in visualizing PLCζ in human sperm. This study aimed to compare our established assay and AUM (involving HCl, acidic Tyrode's solution [AT], and heat). The mean relative fluorescence (RF) intensity of PLCζ in frozen-thawed spermatozoa from fourteen fertile donors stained with the in-house method was significantly higher than three other AUM groups (in-house [mean ± standard error of mean]: 18.87 ± 2.39 arbitrary units [a.u.] vs non-AUM: 11.44 ± 1.61 a.u., AT-AUM: 12.38 ± 1.89 a.u., and HCl-AUM: 12.51 ± 2.16 a.u., P < 0.05, one-way analysis of variance). The mean RF intensity of PLCζ in AT- and HCl-treated spermatozoa from 12 infertile males was not significantly different from that of the non-AUM group. However, the in-house method resulted in the highest RF intensity (12.11 ± 1.36 a.u., P < 0.01). Furthermore, specificity testing of antibody-antigen binding indicated that the in-house method showed more specific binding than spermatozoa treated by the AUM. In conclusion, our in-house method showed superior visualization and reliability than the AUM, thus supporting the continued use of our in-house assay for clinical research screening.

In this study, we determined the levels of cytokine secretory inhibitors and the microbiota biofilms of semen from healthy and infertile subjects. A total of 118 clinical bacterial isolates were isolated and tested. Cytokine secretory inhibitors were determined based on the difference in cytokine content between the control and experimental samples of cell-free supernatants of isolated microorganisms. Biofilm formation was studied by determining the adhesion of microorganisms to the surface of a 96-well sterile plate and expressed as the optical density at 630 nm (OD₆₃₀). Cell-free supernatants of Staphylococcus contained higher levels of secretory inhibitor of cytokines in conditionally healthy than in infertile patients. In contrast, in infertile men, the ability to reduce cytokine levels was more characteristic of Enterococcus and Corynebacterium. Seminal Staphylococcus, Corynebacterium, and Enterococcus isolated from infertile subjects showed a greater ability to form biofilms than the same bacteria isolated from healthy men. Further research is needed on this topic, since it is necessary to determine the relationships between decreased secretory inhibitors of cytokines, production of biofilms by bacteria in semen, and infertility. It is likely that the ability of microorganisms to change the concentration of cytokines and increase the level of biofilm formation in semen may be associated with minimal impairments of fertilizing ability, which are not detected using other methods.

Spermiogenesis is a complex and tightly regulated process, consisting of acrosomal biogenesis, condensation of chromatin, flagellar assembly, and disposal of extra cytoplasm. Previous studies have reported that sperm flagellar 2 (SPEF2) deficiency causes severe asthenoteratozoospermia owing to spermiogenesis failure, but the underlying molecular mechanism in humans remains unclear. Here, we performed proteomic analysis on spermatozoa from three SPEF2 mutant patients to study the functional role of SPEF2 during sperm tail development. A total of 1262 differentially expressed proteins were detected, including 486 upregulated and 776 downregulated. The constructed heat map of the differentially expressed proteins showed similar trends. Among these, the expression of proteins related to flagellar assembly, including SPEF2, sperm associated antigen 6 (SPAG6), dynein light chain tctex-type 1 (DYNLT1), radial spoke head component 1 (RSPH1), translocase of outer mitochondrial membrane 20 (TOM20), EF-hand domain containing 1 (EFHC1), meiosis-specific nuclear structural 1 (MNS1) and intraflagellar transport 20 (IFT20), was verified by western blot. Functional clustering analysis indicated that these differentially expressed proteins were specifically enriched for terms such as spermatid development and flagellar assembly. Furthermore, we showed that SPEF2 interacts with radial spoke head component 9 (RSPH9) and IFT20 in vitro, which are well-studied components of radial spokes or intra-flagellar transport and are essential for flagellar assembly. These results provide a rich resource for further investigation into the molecular mechanism underlying the role that SPEF2 plays in sperm tail development and could provide a theoretical basis for gene therapy in SPEF2 mutant patients in the future.

The present study aimed to evaluate the clinical outcomes of magnetic-activated cell sorting (MACS) in sperm preparation for male subjects with a sperm DNA fragmentation index (DFI) ≥30%. A total of 86 patients who had undergone their first long-term long protocol were selected. The protocol involved in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) cycles, and the patients were divided into the MACS or control groups. The MACS group included sperm samples analyzed with MACS that were combined with density gradient centrifugation (DGC) and the swim-up (SU) technique (n = 39), and the control group included sperm samples prepared using standard techniques (DGC and SU; n = 41). No differences were noted with regard to basic clinical characteristics, number of oocytes retrieved, normal fertilization rate, cleavage rate, or transplantable embryo rate between the two groups in IVF/ICSI. In addition, the clinical pregnancy and implantation rates of the first embryo transfer cycles indicated no significant differences between the two groups. However, there was a tendency to improve the live birth rate (LBR) of the first embryo transfer cycle (63.2% vs 53.9%) and the cumulative LBR (79.5% vs 70.7%) in the MACS group compared with the control group. Moreover, the number of transferred embryos (mean ± standard deviation [s.d.]: 1.7 ± 0.7 vs 2.3 ± 1.6) and the transfer number of each retrieved cycle (mean ± s.d.: 1.2 ± 0.5 vs 1.6 ± 0.8) were significantly lower in the MACS group than those in the control group. Thus, the selection of nonapoptotic spermatozoa by MACS for higher sperm DFI could improve assisted reproductive clinical outcomes.

This paper presents a meta-analysis regarding the detection rate (DR) of fluorine-18 (¹⁸F)-labeled prostate-specific membrane antigen positron emission tomography/computed tomography (PSMA PET/CT) in the management of patients with prostate cancer (PCa). Relevant studies regarding ¹⁸F-PSMA PET/CT in the management of PCa published until June 1, 2021, were electronically searched in online databases including EMBASE, PubMed, and Web of Science. The primary outcome was the DR of ¹⁸F-PSMA PET/CT in managing PCa patients, while the secondary outcome was the DR of ¹⁸F-PSMA PET/CT according to Gleason scores and serum prostate-specific antigen (PSA) level. The pooled DR was calculated on a per-patient basis, with pooled odd ratios and 95% confidence intervals (CIs). In total, 17 observational studies evaluating 1019 patients with PCa met the inclusion criteria. The DR of ¹⁸F-PSMA PET/CT was 0.83 (95% CI: 0.78–0.88), in the random-effects model. Subsequently, the analysis of DR of ¹⁸F-PSMA PET/CT in PCa patients using Gleason score (≤7 vs ≥8), showed a significant difference in PCa patients. Based on the above results, the higher Gleason score of PCa patients, the higher DR of ¹⁸F-PSMA PET/CT. The DR of ¹⁸F-PSMA PET/CT in PCa was 0.57 for PSA <0.5 ng ml⁻¹; 0.75 for PSA ≥0.5 ng ml^-1 and <1.0 ng ml^-1; 0.93 for PSA ≥1.0 ng ml^-1 and <2.0 ng ml^-1; and 0.95 for PSA ≥2.0 ng ml⁻¹. Therefore, the significant diagnostic value was found in terms of the DR of ¹⁸F-PSMA PET/CT in managing PCa patients and was associated with Gleason score and serum PSA level.

Prostate cancer (PCa) is one of the most frequent cancers in men, and its biomolecular targets have been extensively studied. This study aimed to analyze the expression of toll-like receptor 9 (TLR9) and vascular endothelial growth factor C (VEGF-C) and the clinical value of the coexpression of TLR9 and VEGF-C in PCa. We retrospectively evaluated 55 patients with clinically localized, intermediate-risk, or high-risk PCa who underwent laparoscopic radical prostatectomy (LRP) and extended pelvic lymph node dissection (ePLND) without neoadjuvant hormonal therapy at a single institution from June 2013 to December 2016. In all 55 patients, the median number of lymph nodes (LNs) resected was 23 (range: 18–31), and a total of 1269 LNs were removed, of which 78 LNs were positive. Seventeen patients had positive LNs, with a positive rate of 30.9%. In addition, the immunohistochemical results in the above patients revealed that high TLR9 expression was correlated with higher Gleason score (GS) (P = 0.049), increased LN metastasis (P = 0.004), and more perineural invasion (PNI) (P = 0.033). Moreover, VEGF-C expression was associated with GS (P = 0.040), pathological stage (pT stage) (P = 0.022), LN metastasis (P = 0.003), and PNI (P = 0.001). Furthermore, a significant positive correlation between TLR9 and VEGF-C was found (P < 0.001), and the TLR9/VEGF-C phenotype was associated with LN metastasis (P = 0.047). Collectively, we propose that TLR9 stimulation may promote LN metastasis in PCa cells through the upregulation of VEGF-C expression, thereby affecting the prognosis of PCa patients. Therefore, these markers may serve as valuable targets for the treatment of PCa.

We describe and summarize the diagnosis, treatment, and reasons for delayed treatment of children with cryptorchidism torsion in Children's Hospital of Chongqing Medical University. The study included 19 cases of cryptorchidism torsion. The age of the children ranged from 16 days to 12 years (median: 6 years). The interval from diagnosis to surgery varied from 4 h to 16 days (median: 3 days). Ultrasound was performed in all cases. Fifteen cases had cryptorchidism torsion, 2 cases had a soft tissue mass in the inguinal region, and 2 cases had an inguinal/abdominal teratoma. Five cases were treated with an orchidopexy, 12 cases were treated with orchiectomy, and 2 cases received resection of a testicular tumor. The 5 children with an orchidopexy were followed up from 1 month to 7 years (median: 3 years), with 1 child having a testis retraction and no blood supply. Of the 12 children who had an orchiectomy, three had delayed diagnosis due to family unawareness of the condition, while other delays were due to delayed referral from primary care facilities. The relative rarity and insufficient awareness of cryptorchidism torsion resulted in a low rate of testicular salvage. Therefore, hospitals of all levels should be fully aware of cryptorchidism with torsion and ensure a male child's genital system and inguinal region are examined to improve the success rate of testicular salvage.

Postfinasteride syndrome (PFS) is a term coined to characterize a constellation of reported undesirable sexual, physical, and neuropsychiatric side effects. In the present study, we conducted the meta-analysis to demonstrate whether the use of 5α-reductase inhibitors (5ARIs) increases the risk of PFS-like adverse effects. A search of studies published until May 10, 2020, was performed using PubMed, EMBASE, and the Cochrane Library. We included randomized controlled trials with at least one comparison between male patients receiving 5ARIs versus placebo for the treatment of benign prostatic hyperplasia (BPH) or androgenetic alopecia (AGA), and identified 34 studies from 28 articles that met our eligibility criteria. In the random-effects model, the overall use of 5ARIs exhibited a 1.87-fold risk of PFS-like adverse effects during the trial (95% confidence interval [CI]: 1.64–2.14). Regarding specific types of adverse effects, the use of 5ARIs had a 1.89-fold risk of sexual adverse effects (95% CI: 1.74–2.05) and was associated with an increased risk of physical adverse effects (relative risk [RR]: 1.31, 95% CI: 0.80–2.15), albeit without statistical significance. This meta-analysis helped to better define the adverse effects caused by 5ARIs. We concluded that the overall use of 5ARIs significantly increased the risk of PFS-like adverse effects in men with AGA or BPH during treatment. Enhanced awareness of and education on the PFS-like adverse effects are necessary for clinicians.

Large numbers of microbes can be present in seminal fluid, and there are differences in the semen microbiota between normal and abnormal semen samples. To evaluate the semen microbiota in patients with leukocytospermia, 87 seminal fluid samples, including 33 samples with a normal seminal leukocyte count and 54 samples with leukocytospermia, were obtained for a cross-sectional analysis. Twenty samples with a normal seminal leukocyte count had normal sperm parameters (Control group), and 13 samples with a normal seminal leukocyte count were from asthenozoospermia patients (Ast group). However, 32 samples with leukocytospermia were from asthenozoospermia patients (LA group), and only 22 samples with leukocytospermia had normal sperm parameters (Leu group). The 16S ribosomal RNA (rRNA) gene sequencing method was used to sequence the microbiota in the seminal fluid, and multiple bioinformatics methods were utilized to analyze the data. Finally, the results showed that the worse sperm parameters were observed in the leukocytospermia-related groups. Semen microbiota analysis found that there was increased alpha diversity in the leukocytospermia-related groups. Firmicutes, Proteobacteria, Actinobacteria, and Bacteroidetes were the primary phyla in the seminal fluid. Two microbiota profiles, namely, Lactobacillus-enriched and Streptococcus-enriched groups, were identified in this study. The majority of the samples in the groups with a normal seminal leukocyte count could be categorized as Lactobacillus-enriched, whereas the majority of the leukocytospermia samples could be categorized as Streptococcus-enriched. Our study indicated that males with leukocytospermia have worse sperm parameters and a different semen microbiota composition compared to males with a normal seminal leukocyte count.

To analyze the performance of the Prostate Health Index (phi) and its derivatives for predicting Gleason score (GS) upgrading between prostate biopsy and radical prostatectomy (RP) in the Chinese population, an observational, prospective RP cohort consisting of 351 patients from two medical centers was established from January 2017 to September 2020. Pathological reclassification was determined by the Gleason Grade Group (GG). The area under the receiver operating characteristic curve (AUC) and logistic regression (LR) models were used to evaluate the predictive performance of predictors. In clinically low-risk patients with biopsy GG ≤2, phi (odds ratio [OR] = 1.80, 95% confidence interval [95% CI]: 1.14–2.82, P = 0.01) and its derivative phi density (PHID; OR = 2.34, 95% CI: 1.30–4.20, P = 0.005) were significantly associated with upgrading to GG ≥3 after RP, and the results were confirmed by multivariable analysis. Similar results were observed in patients with biopsy GG of 1 for the prediction of upgrading to RP GG ≥2. Compared to the base model (AUC = 0.59), addition of the phi or PHID could provide additional predictive value for GS upgrading in low-risk patients (AUC = 0.69 and 0.71, respectively, both P < 0.05). In conclusion, phi and PHID could predict GS upgrading after RP in clinically low-risk patients.

Glycosylphosphatidylinositol-anchored sperm hyaluronidases have long been believed to assist in sperm penetration through the cumulus–oocyte complex (COC); however, their role in mammalian fertilization remains unclear. Previously, we have shown that hyaluronidase 5 (Hyal5)/Hyal7 double-knockout (dKO) mice produce significantly fewer offspring than their wild-type (WT) counterparts because of defective COC dispersal. Male infertility is mainly caused by a low sperm count. It can be further exacerbated by the deficiency of sperm hyaluronidase, which disperses the cumulus cells of the outer layer of the COC. In the current study, we evaluated the effects of a low count of Hyal-deficient sperm and conditions of ovulated oocytes on the fertilization rate using a mouse model. Our results demonstrated that a low sperm count further decreases the in vitro fertilization (IVF) rate of Hyal-deficient dKO spermatozoa. In addition, the dKO spermatozoa resulted in a fertilization rate of 12.5% upon fertilizing COCs with a thick cumulus layer, whereas the IVF rate was comparable to that of WT spermatozoa when oocytes with a thin or no cumulus layer were fertilized. Finally, we proved that the IVF rate of dKO spermatozoa could be recovered by adding rat spermatozoa as a source of sperm hyal. Our results suggest that a deficiency of proteins involved in fertilization, such as sperm hyal, has a vital role in fertilization.

Cystic fibrosis (CF) is one of the most common recessive genetic diseases, with a wide spectrum of phenotypes, ranging from infertility to severe pulmonary disease. Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene are considered the main genetic cause for CF. In this study, we recruited a consanguineous Iranian pedigree with four male patients diagnosed with congenital unilateral absence of the vas deferens (CUAVD), and one female patient diagnosed with congenital absence of the uterus (CAU). Testicular biopsy of one patient was performed, and hematoxylin and eosin (H and E) staining of testis sections displayed the presence of germ cell types ranging from spermatogonia to mature spermatids, indicating obstructive azoospermia. To explore the underlying genetic factor in this familial disorder, we therefore performed whole-exome sequencing (WES) on all available family members. WES data filtration and CFTR haplotype analysis identified compound heterozygous mutations in CFTR among four patients (two CUAVD patients carried p.H949Y and p.L997F, and one CUAVD and the female CAU patient carried p.H949Y and p.I148T). All these mutations were predicted to be deleterious by at least half of the prediction software programs and were confirmed by Sanger sequencing. Our study reported that CFTR compound heterozygous mutations in a consanguineous Iranian family cause infertility in both sexes.

During recent decades, the association between mutations in ubiquitin-specific protease 26 (USP26) and male infertility remains doubtful. We conducted this meta-analysis to evaluate the association between mutations in USP26 and male infertility according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) 2020 guidelines. It was registered in the International Prospective Register of Systematic Reviews (PROSPERO; CRD42021225251). PubMed, Web of Science, and Scopus were systematically searched for comparative clinical studies, which were written in English and provided eligible data. Studies were included when they compared USP26 mutations in azoospermic, oligozoospermic, and asthenozoospermic patients with controls with normal sperm parameter values or whose partners had experienced spontaneous pregnancy. Pooled odds ratio (OR) with 95% confidence interval (CI) was calculated with random effect models. Overall, twelve studies with 3927 infertility patients and 4648 healthy controls were included. The association between overall USP26 mutations and infertility was not significant (OR = 1.60, 95% CI: 0.51–5.01). For specific mutations, the pooled ORs were 1.65 (95% CI: 1.02–2.69) for cluster mutation (including 370–371insACA, 494T>C, and 1423C>T), 1.80 (95% CI: 0.35–9.15) for c.576G>A, 1.43 (95% CI: 0.79–2.56) for c.1090C>T, and 3.59 (95% CI: 2.30–5.59) for c.1737G>A. Our results suggest that several mutations (cluster mutation, c.1737G>A) may play roles in male infertility, while others (c.576G>A and c.1090C>T) do not show notable associations with male infertility. More high-quality clinical researches are needed for validation.

To explore the relationship between genetic polymorphisms of metabolic enzymes such as CYP1A1, CYP2D6, GSTM1, GSTT1, and GSTP1 and idiopathic male infertility. By observing the efficacy of antioxidants in the treatment of idiopathic male infertility, the effect of metabolic enzyme gene polymorphisms on antioxidant therapy in patients with idiopathic male infertility was prospectively studied. This case–control study included 310 men with idiopathic infertility and 170 healthy controls. The cytochrome P450 1A1 (CYP1A1), cytochrome P450 2D6 (CYP2D6), glutathione S-transferase M1 (GSTM1), glutathione S-transferase T1 (GSTT1), and glutathione S-transferase P1 (GSTP1) genotypes in peripheral blood samples were analyzed by polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (PCR-RFLP). The idiopathic male infertility group was treated with vitamin C, vitamin E, and coenzyme Q10 for 3 months and followed up for 6 months. GSTM1(−), GSTT1(−), and GSTM1/T1(−/−) in the idiopathic male infertility groups were more common than those in the control group. The sperm concentration, motility, viability, mitochondrial membrane potential (MMP), and seminal plasma total antioxidant capacity (T-AOC) level in patients with GSTM1(−), GSTT1(−), and GSTM1/T1(−/−) were lower than those in wild-type carriers, and the sperm DNA fragmentation index (DFI), 8-hydroxy-2'-deoxyguanosine (8-OH-dG), and malondialdehyde (MDA) and nitric oxide (NO) levels were higher. Therefore, oxidative damage may play an important role in the occurrence and development of idiopathic male infertility, but antioxidant therapy is not effective in male infertility patients with GSTM1 and GSTT1 gene deletions.