Investigation on the mechanisms of human sperm DNA damage based on the proteomics analysis by SWATH-MS.

BACKGROUND: Spermatozoa have the task of delivering an intact paternal genome to the oocyte and supporting successful embryo development. The detection of sperm DNA fragmentation (SDF) has been emerging as a complementary test to conventional semen analysis for male infertility evaluation, but the mechanism leading to SDF and its impact on assisted reproduction remain unclear. Therefore, the study identified and analyzed the differentially expressed proteins of sperm with high and low SDF. METHODS: Semen samples from men attended the infertility clinic during June 2020 and August 2020 were analyzed, and sperm DNA fragmentation index (DFI) was detected by the sperm chromatin structure assay. Semen samples with low DFI (< 30%, control group) and high DFI (≥ 30%, experimental group) were optimized by density gradient centrifugation (DGC), and the differentially expressed proteins of obtained sperm were identified by the Sequential Window Acquisition of All Theoretical Mass Spectra Mass Spectrometry (SWATH-MS) and performed GO and KEGG analysis. RESULTS: A total of 2186 proteins were identified and 1591 proteins were quantified, of which 252 proteins were identified as differentially expressed proteins, including 124 upregulated and 128 downregulated. These differentially expressed proteins were involved in metabolic pathways, replication/recombination/repair, acrosomal vesicles, kinase regulators, fertilization, tyrosine metabolism, etc. Western blotting results showed that the expression levels of RAD23B and DFFA proteins and the levels of posttranslational ubiquitination and acetylation modifications in the experimental group were significantly higher than those in the control group, which was consistent with the results of proteomics analysis. CONCLUSIONS: Proteomic markers of sperm with high DNA fragmentation can be identified by the SWATH-MS and bioinformatic analysis, and new protein markers and posttranslational modifications related to sperm DNA damage are expected to be intensively explored. Our findings may improve our understanding of the basic molecular mechanism of sperm DNA damage.

Investigation of the mechanisms leading to human sperm DNA damage based on transcriptome analysis by RNA-seq techniques.

RESEARCH QUESTION: What are the molecular mechanisms leading to human sperm DNA damage? DESIGN: Semen samples were collected and the sperm DNA fragmentation index (DFI) was assessed. Differentially expressed RNA in spermatozoa with a high (DFI ≥30%, experimental group) or normal (DFI <30%, control group) DFI were identified by RNA-sequencing (RNA-seq) technology, and Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was performed. Three differentially expressed RNA related to sperm DNA damage and repair, namely PMS1, TP53BP1 and TLK2, were validated using real-time quantitative (RT-qPCR). RESULTS: A total of 19,970 expressed RNA were detected in the two groups. Compared with the control group, the expression levels of 189 RNA in the experimental group were significantly increased and those of 163 genes decreased. Gene Ontology enrichment analysis showed that these RNA were mainly concentrated in the ATPase-dependent transmembrane transport complex, extracellular exosome, somatic cell DNA recombination, protein binding, cytoplasm and regulation of localization. KEGG pathway analysis showed that these RNA were mainly related to the PI3K-Akt signalling pathway, endocytosis, p53 signalling pathway and cGMP-PKG signalling pathway. The RT-qPCR results showed that the expression levels of PMS1, TP53BP1 and TLK2 in the experimental group were significantly lower than in the control group (P = 0.01, 0.015 and 0.004, respectively), which was identical to the results of RNA sequencing. CONCLUSIONS: Differentially expressed RNA related to sperm DNA damage and repair may be identified by RNA-seq technology, which provides new insights into the understanding of sperm DNA damage and repair, and will help to discover new biomarkers related to sperm DNA damage.

Correlation between different endometrial preparation protocols and pregnancy outcome of frozen embryo transfer in patients with polycystic ovary syndrome: a retrospective study.

OBJECTIVE: We retrospectively analyzed the correlation between different endometrial preparation protocols and pregnancy outcomes in patients with polycystic ovary syndrome (PCOS) who underwent frozen embryo transfer (FET). METHODS: A total of 200 PCOS patients who underwent FET were divided into HRT group (n = 65), LE group (n = 65), GnRHa + HRT group (n = 70) according to different endometrial preparation protocols. The endometrial thickness on the day of endometrial transformation, the number of embryos transferred, and the number of high-quality embryos transferred were compared among the three groups. The pregnancy outcomes of FET in the three groups were compared and analyzed, and a further multivariate logistic regression model was used to analyze the factors influencing FET pregnancy outcomes in PCOS patients. RESULTS: Endometrial thickness on the day of endometrial transformation, clinical pregnancy rate and live birth rate in GnRHa + HRT group were higher than those in the HRT group and LE group. The results of multivariate regression analysis showed that the pregnancy outcome of PCOS patients undergoing FET was significantly associated with the patient's age, endometrial preparation protocols, number of embryos transferred, endometrial thickness, and duration of infertility. CONCLUSION: Compared with HRT or LE alone, GnRHa + HRT protocol results in higher levels of endometrial thickness on the day of endometrial transformation, clinical pregnancy rate, and live birth rate. Female age, endometrial preparation protocols, number of embryos transferred, endometrial thickness, and duration of infertility are determined as factors influencing pregnancy outcomes in PCOS patients undergoing FET.

Mesh erosion causes small bowel obstruction: a rare complication of laparoscopic inguinal hernia repair: case description and review of literature.

Transabdominal preperitoneal (TAPP) and total extraperitoneal (TEP) are the two types of laparoscopic repair of the inguinal hernia. The main advantages of laparoscopic repair, as compared to open repair, are a shorter hospital stay and a quicker recovery to normal activities. However, we cannot be overlooked or neglected the complication of laparoscopic inguinal hernia repair although it brings us lots of benefits. We report a case of small bowel obstruction caused by a displaced mesh used for the laparoscopic inguinal hernia repair and review of literature.

[Gossypol acetic acid induces DNA double-strand breaks in human mucoepidermoid carcinoma cell MEC-1].

The present study was conducted to investigate the effects of gossypol acetic acid (GAA) on the proliferation of human mucoepidermoid carcinoma cell line MEC-1 in vitro and its possible molecular mechanisms of DNA double-strand breaks (DSB). MTT assay was performed to test the inhibition of proliferation of MEC-1 cells by GAA. DSB and γH2AX foci formation induced by GAA were detected by neutral comet assay and immunostaining. GAA (5-40 µmol/L) inhibited the growth of MEC-1 cells in a dose- and time-dependent manner. One of the indexes of comet assay, percentage of head DNA was decreased, however other indexes, including tail length, percentage of tail DNA, tail moment (TM) and Olive tail moment (OTM) were increased when treated with 2.5- 40 µmol/L GAA for 24 h or 20 µmol/L GAA for 3-48 h, compared with those in control. The percentage of γH2AX-positive cells was also increased when MEC-1 was treated with 2.5-20 µmol/L GAA for 24 h or 20 µmol/L GAA for 3-48 h, compared with that in control. All these results show that GAA inhibits the proliferation of MEC-1, and DSB maybe one of the mechanisms of inhibitory effect of GAA on the growth of tumor cells.

Intestinal ischemic preconditioning reduces liver ischemia reperfusion injury in rats.

The aim of the current study was to investigate whether intestinal ischemic preconditioning (IP) reduces damage to the liver during hepatic ischemia reperfusion (IR). Sprague Dawley rats were used to model liver IR injury, and were divided into the sham operation group (SO), IR group and IP group. The results indicated that IR significantly increased Bax, caspase 3 and NF­κBp65 expression levels, with reduced expression of Bcl­2 compared with the IP group. Compared with the IR group, the levels of AST, ALT, MPO, MDA, TNF­α and IL­1 were significantly reduced in the IP group. Immunohistochemistry for Bcl­2 and Bax indicated that Bcl­2 expression in the IP group was significantly increased compared with the IR group. In addition, IP reduced Bax expression compared with the IR group. The average liver injury was worsened in the IR group and improved in the IP group, as indicated by the morphological evaluation of liver tissues. The present study suggested that IP may alleviates apoptosis, reduce the release of pro­inflammatory cytokines, ameloriate reductions in liver function and reduce liver tissue injury. To conclude, IP provided protection against hepatic IR injury.

miR-20b overexpression is predictive of poor prognosis in gastric cancer.

BACKGROUND: miR-20b has been shown to be aberrantly expressed in several tumor types. However, the clinical significance of miR-20b in the prognosis of patients with gastric cancer (GC) is poorly understood, and the exact role of miR-20b in GC remains unclear. MATERIALS AND METHODS: The expression of miR-20b was detected in 102 patients with GC by a SYBR Green assay and was compared with the expression in matched adjacent normal tissue specimens. The aim of the present study was to investigate the association of the expression of miR-20b with the clinicopathological characteristics and the overall survival of patients with GC as analyzed by Kaplan-Meier analysis and Cox proportional hazards regression models. RESULTS: Our results showed that miR-20b expression was upregulated in GC tissue compared with normal mucosa (P=0.00). Furthermore, miR-20b expression was positively correlated with advanced lymph node metastasis (P=0.041), tumor node metastasis stage (P=0.000), and deeper and distant metastasis (P=0.031). The overall survival rate of patients with GC was significantly lower in those whose tumors expressed high levels of miR-20b mRNA compared with those whose tumors expressed low levels of miR-20b mRNA (P=0.019). CONCLUSION: miR-20b may serve as a potential molecular marker for the prognosis of GC.

Clinicopathological Significance of MicroRNA-20b Expression in Hepatocellular Carcinoma and Regulation of HIF-1α and VEGF Effect on Cell Biological Behaviour.

miRNA-20b has been shown to be aberrantly expressed in several tumor types. However, the clinical significance of miRNA-20b in the prognosis of patients with hepatocellular carcinoma (HCC) is poorly understood, and the exact role of miRNA-20b in HCC remains unclear. The aim of the present study was to investigate the association of the expression of miR-20b with clinicopathological characteristics and overall survival of HCC patients analyzed by Kaplan-Meier analysis and Cox proportional hazards regression models. Meanwhile, the HIF-1α and VEGF targets of miR-20b have been confirmed. We found not only miR-20b regulation of HIF-1α and VEGF in normal but also regulation of miR-20b in hypoxia. This mechanism would help the tumor cells adapt to the different environments thus promoting the tumor invasion and development. The whole study suggests that miR-20b, HIF-1α, and VEGF serve as a potential therapeutic agent for hepatocellular carcinoma.