Despite extensive prior research on prostate cancer (PCa) transcriptomics, the molecular mechanisms underlying the disease’s progression, particularly in the castration-resistant or metastatic stages, remain incompletely understood. The majority of recent research has concentrated on bulk RNA sequencing, which could mask the variation found in tumor microenvironments. This study aims to address this gap by integrating single-cell RNA sequencing (scRNA-seq) and bulk RNA sequencing with weighted gene co-expression network analysis (WGCNA) to investigate the molecular mechanisms of PCa at a higher resolution. In order to further individualized treatment plans for PCa, we aim to discover important genes and signaling pathways that could be used as therapeutic targets. We first preprocessed expression profile data from prostate cancer tissue samples, selecting 9,809 high-quality cells from a dataset. Following batch correction with Harmony and dimensionality reduction with principal component analysis (PCA), we used the Louvain clustering algorithm to divide the cells into discrete subtypes. The clusters were then visualized using t-SNE. This resulted in 16 cellular subtypes categorized into five major cell types: epithelial cells, monocytes, endothelial cells, CD8 + T-cells, and fibroblasts. Analysis of receptor-ligand pairs uncovered significant interactions between monocytes and both tumor cells and endothelial cells. Applying the high-dimensional WGCNA (hdWGCNA) method to construct a gene co-expression network, we detected seven gene modules, four of which were highly expressed in tumor cell subtypes and contained 380 key genes. Combining pathway analysis, we ultimately screened six key genes: CNPY2, CPE, DPP4, IDH1, NIPSNAP3A, and WNK4. We used Cox univariate regression and least absolute shrinkage and selection operator (lasso) regression techniques to build a prognostic prediction model that included these six important genes based on clinical data gathered from PCa patients. The prognostic prediction model constructed in this study demonstrated excellent predictive performance in both the training set and an external validation set, with the high-risk group showing significantly lower overall survival (OS) than the low-risk group. Furthermore, there was a substantial correlation found between risk scores and several immune-related gene sets, chemotherapeutic drug sensitivity, and tumor immune infiltration. High- and low-risk groups exhibited significant differences in immune cell content, immune factor levels, and immune dysfunction. Further analysis revealed significant correlations between the expression levels of model genes and multiple disease-related genes. Through Gene Set Variation Analysis (GSVA) and Gene Set Enrichment Analysis (GSEA), we uncovered perturbations in multiple signaling pathways in high- and low-risk groups, potentially impacting the prognosis of PCa patients. This study uncovers key genes and signaling pathways in the prostate cancer tumor microenvironment, particularly genes such as CNPY2, CPE, DPP4, IDH1, NIPSNAP3A and WNK4, which have potential as therapeutic targets. Our findings provide new insights into personalized treatment strategies for PCa and warrant further clinical validation in the future.

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.

Background: Cryopreservation of extremely few spermatozoa is still a major challenge for male fertility preservation. This study aims to evaluate the cooling rate, recovery rate, and retrieval rate, along with other parameters of spermatozoa that cryopreserved using Cryopiece, a novel carrier, for individual sperm cryopreservation. Methods: Semen samples from 60 fertile donors were collected, and each semen sample was screened for motile sperm and mixed with cryoprotective agent (CPA), and then frozen using Cryopiece, micro-straw, and mini-straws. The cooling rate, retrieval rate, and recovery rate, morphology, DNA fragmentation index (DFI) and mitochondrial membrane potential (MMP), were compared among the un-frozen sperm and the sperm cryopreserved using these carriers. Results: Cryopiece possessed the fastest cooling rate. After freeze-thaw, the average retrieval rate of sperm cryopreserved using Cryopiece was 96.25%, and the average recovery rate was 64.40%, which were higher than that of sperm cryopreserved using the other two carriers (71.42% and 54.30% for micro-straw, and 63.54% and 58.04% for mini-straw, respectively). There was no significant impact on DFI after sperm cryopreservation, and no significant difference in morphology between sperm cryopreserved using these carriers was observed. Though MMP of sperm changed significantly after cryopreservation, micro-straw maintained sperm MMP better than Cryopiece and mini-straw did, while no significant difference was observed in MMP between sperm cryopreserved using Cryopiece and mini-straw. Conclusions: Cryopiece produced satisfying retrieval and recovery rates in sperm cryopreservation and should be an ideal carrier for cryopreservation of small number of sperm.

Objective:To evaluate the safety and effectiveness of The Stork OTC conception aid kit for the treatment of male infertility compared with natural sexual intercourse.Methods:A multi-center, prospective, open, self-controlled, randomly crossed trial was performed in 57 infertility couples who were unable to naturally conceive, and were selected in Shanghai General Hospital, Shanghai Jiao Tong University, Reproductive Medicine Center,Tongji Medical College, Huazhong University of Science and Technology and Anhui Provincial Hospital from July 2017 to September 2018. Patients were required to participate both the conception aid kit and natural sexual intercourse in two consecutive periods of ovulation. A random envelope method was used to determine the application sequences of two methods. Twenty-nine couples were treated with the conception aid kit in the first month of ovulation, and applied the natural sexual intercourse in the second month of ovulation as group A. Twenty-eight couples were applied the natural sexual intercourse in the first month of ovulation, and were treated with the conception aid kit in the second month of ovulation as group B. Test results collected from all couples who used the conception aid kit were categorized as the test group data. Meanwhile, test results out of those couples who completed natural sexual intercourse were collected as control group data. Compared with its self-controlled natural sexual intercourse two months before and after, the effectiveness and safety of the conception aid kit was verified by general vaginal examination, routine leucorrhea examination, motile sperm score from post coital test (PCT), pregnancy rate comparison and other methods.Results:The trial was conducted at three clinical trial centers. A total of 57 infertility couples were enrolled. A total of 54 couples were validated eventually including 26 couples in group A and 28 infertility couples in group B. Effectiveness was evaluated. The PCT results showed that the sperm score of test group was 6.11±3.00, and the score of the sperm score of control group was 1.22±0.79. The motile sperm score of test group was 5 times ( P<0.001) than that in control group. In the follow-up, 7 (13.0%) of the couples successfully achieved pregnancy with the conception aid kit, 6 (85.7%) of them had healthy offspring, 1 (14.3%) had spontaneous abortion. No couples were pregnant after natural sexual intercourse. Safety was evaluated. The general examination of the vagina and the routine tests of the vaginal leukorrhea showed no difference between test group and control group. All 54 effective couples who completed the test can successfully use the conception aid kid to place the seminal vesicles containing semen and remove them with the attached pulling thread on time without obvious discomfort. Conclusion:The Stork OTC conception aid kits can effectively treat infertility, significantly increase the number of forward-moving sperm entering the cervix, and significantly increase the pregnancy rate. It is safe, non-invasive, simple, can be easily operate by patients themselves, and meets the needs of clinical applications.

Busulfan and other chemotherapeutic drugs used in the treatment of cancer may result in temporary or even permanent damage to spermatogenesis. During spermatogenesis, the rapidly dividing spermatogonia are highly susceptible to chemotherapy. Consequently, there is significant interest in developing an approach that could provide stimulation and regenerate spermatogenesis after chemotherapy. In a previous study, we suggested the potential application for vascular endothelial growth factor C (VEGFC) because of its key role in stimulating the proliferation of spermatogonia. However, methods to facilitate the recovery of spermatogenesis in such patients using VEGFC, or other regulatory factors, are sorely lacking because of the rapid degradation of these proteins and restrictions created by the blood-testis- barrier. To this end, we loaded VEGFC into polyanion dextran sulfate incorporated in a polycation chitosan shell to produce VEGFC sustained-release ultrafine particles (UFPs, CS-DS-VEGFC). We tested such particles in an azoospermic mouse model, created using busulfan. For each mouse, CS-DS-VEGFC was injected into the seminiferous tubules of one testis, while unloaded UFPs (CS-DS), or the VEGFC protein alone, was injected into the opposite testis as a control. All mice were sacrificed and evaluated 5 weeks later. Spermatogenesis in the tubules that were injected with CS-DS-VEGFC was clearly better than those injected with controls, and contained more spermatogonia and spermatocytes, along with Ki67 and PCNA positive- cells per tubule. In addition, the phosphorylation levels of AKT and MAPK in these tubules were also higher than in controls, indicating that CS-DS-VEGFC could induce the sustained activation of these pathways. In conclusion, CS-DS-VEGFC, combined with the efferent tubule injection technique, is a feasible approach with which to improve the regeneration of spermatogenesis in busulfan-induced azoospermic 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.