Clinical data-based modeling of IVF live birth outcome and its application.

BACKGROUND: The low live birth rate and difficult decision-making of the in vitro fertilization (IVF) treatment regimen bring great trouble to patients and clinicians. Based on the retrospective clinical data of patients undergoing the IVF cycle, this study aims to establish classification models for predicting live birth outcome (LBO) with machine learning methods. METHODS: The historical data of a total of 1405 patients undergoing IVF cycle were first collected and then analyzed by univariate and multivariate analysis. The statistically significant factors were identified and taken as input to build the artificial neural network (ANN) model and supporting vector machine (SVM) model for predicting the LBO. By comparing the model performance, the one with better results was selected as the final prediction model and applied in real clinical applications. RESULTS: Univariate and multivariate analysis shows that 7 factors were closely related to the LBO (with P < 0.05): Age, ovarian sensitivity index (OSI), controlled ovarian stimulation (COS) treatment regimen, Gn starting dose, endometrial thickness on human chorionic gonadotrophin (HCG) day, Progesterone (P) value on HCG day, and embryo transfer strategy. By taking the 7 factors as input, the ANN-based and SVM-based LBO models were established, yielding good prediction performance. Compared with the ANN model, the SVM model performs much better and was selected as the final model for the LBO prediction. In real clinical applications, the proposed ANN-based LBO model can predict the LBO with good performance and recommend the embryo transfer strategy of potential good LBO. CONCLUSIONS: The proposed model involving all essential IVF treatment factors can accurately predict LBO. It can provide objective and scientific assistance to clinicians for customizing the IVF treatment strategy like the embryo transfer strategy.

The bioinfomatics analysis of the M1 macrophage-related gene CXCL9 signature in cervical cancer.

BACKGROUND: The expression and function of coexpression genes of M1 macrophage in cervical cancer have not been identified. And the CXCL9-expressing tumour-associated macrophage has been poorly reported in cervical cancer. METHODS: To clarify the regulatory gene network of M1 macrophage in cervical cancer, we downloaded gene expression profiles of cervical cancer patients in TCGA database to identify M1 macrophage coexpression genes. Then we constructed the protein-protein interaction networks by STRING database and performed functional enrichment analysis to investigate the biological effects of the coexpression genes. Next, we used multiple bioinformatics databases and experiments to overall investigate coexpression gene CXCL9, including western blot assay and immunohistochemistry assay, GeneMANIA, Kaplan-Meier Plotter, Xenashiny, TISCH2, ACLBI, HPA, TISIDB, GSCA and cBioPortal databases. RESULTS: There were 77 positive coexpression genes and 5 negative coexpression genes in M1 macrophage. The coexpression genes in M1 macrophage participated in the production and function of chemokines and chemokine receptors. Especially, CXCL9 was positively correlated with M1 macrophage infiltration levels in cervical cancer. CXCL9 expression would significantly decrease and high CXCL9 levels were linked to good prognosis in the cervical cancer tumour patients, it manifestly expressed in blood immune cells, and was positively related to immune checkpoints. CXCL9 amplification was the most common type of mutation. The CXCL9 gene interaction network could regulate immune-related signalling pathways, and CXCL9 amplification was the most common mutation type in cervical cancer. Meanwhile, CXCL9 may had clinical significance for the drug response in cervical cancer, possibly mediating resistance to chemotherapy and targeted drug therapy. CONCLUSION: Our findings may provide new insight into the M1 macrophage coexpression gene network and molecular mechanisms in cervical cancer, and indicated that M1 macrophage association gene CXCL9 may serve as a good prognostic gene and a potential therapeutic target for cervical cancer therapies.

Cervical cancer is a common gynaecological malignancy, investigating the precise gene expression regulation of M1 macrophage is crucial for understanding the changes in the immune microenvironment of cervical cancer. In our study, a total of 82 coexpression genes with M1 macrophages were identified, and these genes were involved in the production and biological processes of chemokines and chemokine receptors. Especially, the chemokine CXCL9 was positively correlated with M1 macrophage infiltration levels in cervical cancer. CXCL9 as a protective factor, it manifestly expressed in blood immune cells, and was positively related to immune checkpoints. CXCL9 amplification was the most common type of mutation. And CXCL9 expression could have an effect on the sensitivity of some chemicals or targeted drugs against cervical cancer. These findings may provide new insight into the M1 macrophage coexpression gene network and molecular mechanisms, and shed light on the role of CXCL9 in cervical cancer.

Mechanical stretching induces fibroblasts apoptosis through activating Piezo1 and then destroying actin cytoskeleton.

The anatomical positions of pelvic floor organs are maintained by ligaments and muscles. Stress urinary incontinence (SUI) occurs when the pelvic floor tissues are repeatedly stimulated by excessive mechanical tension that exceeds the bearing capacity of ligaments or muscles. Besides, cells respond mechanically to mechanical stimulation by reconstituting the Piezo1 and cytoskeletal system. The aim of this study is to determine how Piezo1 and actin cytoskeleton are involved in the mechanized stretch (MS) induced apoptosis of human anterior vaginal wall fibroblasts (hAVWFs) and the mechanism. A four-point bending device was used to provide mechanical stretching to establish a cellular mechanical damage model. The apoptosis of hAVWFs cells in non-SUI patients was significantly increased by MS, which exhibited apoptosis rates comparable to those of SUI patients. Based on these findings, Piezo1 connects the actin cytoskeleton to the apoptosis of hAVWFs cells, providing an idea for the clinical diagnosis and treatment of SUI. However, the disassembly of the actin cytoskeleton suppressed the protective effect of Piezo1 silencing on MS. Based on these findings, Piezo1 connects the actin cytoskeleton to apoptosis of hAVWFs, providing new insight for the clinical diagnosis and treatment of SUI.

Identification of CD28 and PTEN as novel prognostic markers for cervical cancer.

Cervical cancer (CC) is the most common malignant tumor with poor clinical outcome among women. Identification of novel biomarkers could be beneficial for the clinical diagnosis and treatment of CC. This study aimed to identify prognostic biomarkers for the prediction of prognostic status of CC patients, and explore the effect of the corresponding methylated genes in the occurrence and development of CC. The methylation microarray data of CC was extracted from The Cancer Genome Atlas (TCGA) dataset. The methylation genes associated with the prognostic status were identified based on the information of the relapse-free survival (RFS) of the CC patients. The prognostic gene pairs were further identified. Then, the prognostic signature was identified by the forward search algorithm based on the C-index method. The results were validated by independent dataset. Finally, the functional analysis was performed on the methylation genes. A total of 276 methylation genes and 2508 gene pairs associated with the prognostic status of the CC were identified. A signature composed of eight methylation gene pairs was obtained to predict the prognostic status of cervical patients. A series of genes that played an important role in the occurrence and development of CC were obtained by the functional enrichment analysis. To summary, a prognostic signature consisting of eight methylation gene pairs was obtained. Of note, the CD28 and PTEN gene pair were found to play important roles in the occurrence and development of CC.

Overexpression of MALAT1 contributes to cervical cancer progression by acting as a sponge of miR-429.

Cervical cancer remains a malignant type of tumor and is the fourth leading cause of cancer-related death among females. MALAT1 has been identified as a tumor oncogene in various cancers. Our present study aimed to explore the biological role of MALAT1 in cervical cancer. We observed that MALAT1 was significantly upregulated in human cervical cancer cell lines compared with the ectocervical epithelial cells. MALAT1 was repressed by transfection with LV-shMALAT1, whereas increased by LV-MALAT1 in HeLa and Caski cells. Silencing of MALAT1 obviously reduced cervical cell viability, induced cell apoptosis, and repressed cell invasion capacity. Conversely, overexpression of MALAT1 exhibited an opposite phenomenon. Furthermore, miR-429 was predicted as a direct target of MALAT1, and it was dramatically decreased in cervical cancer cells. It has been shown that miR-429 plays a crucial role in cervical cancer progression. In our current study, the targeting correlation between MALAT1 and miR-429 was confirmed by luciferase reporter assays and RIP experiments. Finally, in vivo animal models were established, and we indicated that MALAT1 inhibited cervical cancer progression via targeting miR-429. These findings revealed that MALAT1 can sponge miR-429 and regulate cervical cancer pathogenesis in vivo and in vitro. In conclusion, we indicated that the MALAT1/miR-429 axis was involved in cervical cancer development.

LINC00657 expedites neuropathic pain development by modulating miR-136/ZEB1 axis in a rat model.

Long non-coding RNAs (lncRNAs) are involved in the progression of several diseases. The interactions among lncRNAs, microRNA (miRNAs) or their targeting genes are reported to play crucial roles in the development of diseases. LINC00657 is observed to be upregulated in several cancers. However, the biological role of LINC00657 in neuropathic pain progress is unclear. Hence, in our study, we aimed to investigate the function of LINC00657 in neuropathic pain development. A chronic constriction injury (CCI) rat model was established, and we found that LINC00657 was greatly increased in CCI rats associated with a decrease of miR-136. Inhibition of LINC00657 suppressed neuropathic pain via alleviating mechanical and thermal hyperalgesia. In addition, miR-136 overexpression can also inhibit the neuropathic pain development. MiR-136 was predicted to serve as a miRNA target of LINC00657, and dual-luciferase reporter assay confirmed the correlation between LINC00657 and miR-136. Moreover, we observed that the decrease of LINC00657 was able to inhibit the neuroinflammation of CCI rats by targeting expression of cyclooxygenase-2, tumor necrosis factor-α and interleukin-1ß while miR-136 inhibitors reversed this phenomenon. Next, by using bioinformatics analysis, ZEB1 was predicted as a direct target of miR-136, and miR-136 could negatively modulate ZEB1 expression. Besides these, ZEB1 was remarkably increased in the CCI rats. Knockdown of ZEB1 can inhibit neuropathic pain development, while miR-136 inhibitors can reverse it. In conclusion, it was implied that LINC00657 can induce the neuropathic pain development via regulating miR-136/ZEB1 axis.

The YY1-HOTAIR-MMP2 Signaling Axis Controls Trophoblast Invasion at the Maternal-Fetal Interface.

We aimed to determine the effect of YY1 expression on the expression profile of long noncoding RNAs (lncRNAs) in trophoblasts, and we studied the involvement of certain lncRNAs and YY1 in the pathogenesis of recurrent miscarriage (RM). RT2 lncRNA PCR arrays revealed that YY1 overexpression in trophoblasts significantly promoted the expression of the HOX transcript antisense RNA HOTAIR and demonstrated that HOTAIR expression was significantly lower in the RM trophoblasts than in control trophoblasts. Ectopic HOTAIR overexpression and knockdown experiments revealed that it was a novel target of YY1. Bioinformatics analysis identified two YY1-binding sites in the HOTAIR promoter region, and chromatin immunoprecipitation (ChIP) analysis verified that YY1 binds directly to its promoter region. Interestingly, HOTAIR overexpression enhanced trophoblast invasion in an ex vivo explant culture model, while its knockdown repressed these effects. Furthermore, liquid chromatography-tandem mass spectrometry (LC-MS/MS) label-free quantitative proteomics screening revealed that HOTAIR overexpression activated phosphatidylinositol 3-kinase-protein kinase B (PI3K-AKT) signaling in trophoblasts. In an ex vivo explant culture model, HOTAIR overexpression effectively elevated matrix metalloproteinase 2 (MMP2) expression via the PI3K-AKT signaling pathway, enhancing trophoblast migration and invasion. These findings reveal a new regulatory pathway in which YY1 activates PI3K-AKT signaling via HOTAIR, promoting MMP2 expression, suggesting that HOTAIR is a potential therapeutic target for RM.

An increased risk of ovarian cancer associated with polymorphism in BRCC5 gene in Caucasian populations.

Several reports on the association between the BRCC5 gene polymorphism and ovarian cancer risk have been published recently, but the estimates of the risk vary widely. We thus performed a meta-analysis in an effort to determine the association. To identify the eligible studies, we searched the PubMed, Embase, and CNKI databases, and reviewed all original studies retrieved as well as their citations. The risk of ovarian cancer was estimated using odds ratio (OR) and its 95 % confidence interval (CI). Meta-analysis of seven comparisons revealed an obvious rise in the risk of ovarian cancer under the CC vs. GG contrast model (OR = 1.52, 95 % CI = 1.07-2.16, P OR = 0.020). A similar increase was also indicated in the CC vs. GC + GG model (OR = 2.10, 95 % CI = 1.51-2.93, P OR < 0.001). Our meta-analysis indicates that the BRCC5 polymorphism may be a candidate modifier of ovarian cancer risk in Caucasians.

A novel universal multiplex PCR improves detection of AZFc Y-chromosome microdeletions.

PURPOSE: To determine the frequencies and the characteristics of Y chromosome microdeletions (pl) in infertile men from central China to perform appropriate therapeutic choices by updated multiplex-PCR. METHODS: In this study, we established a novel universal primer-multiplex-PCR (U-M-PCR) method to overcome the disadvantages of traditional multiplex PCR (M-PCR). We chose 15 sequence-tagged sites (STS) for detection of Y chromosome microdeletions. 540 infertile male patients and 100 healthy male controls were selected in the study. RESULTS: Of the 540 male infertility patients, 48 Y-chromosome microdeletions were detected, with a total deletion rate of 8.9 %. Of these deletions, the rate of AZFa deletions (sY84) was 0.5 % (3/540), the rate of AZFb deletions (sY143) was 0.7 % (4/540) and the rate of AZFc deletions (sY242, sY254 and sY255) was 7.6 % (41/540). Compared with AZF deletion rates by M-PCR, we found U-M-PCR could detect AZFc deletion more specifically (1.0 % & 7.6 %). No Y-chromosome microdeletions were detected in the 100 males with normal semen (the control group). CONCLUSIONS: U-M-PCR method was more specific to detect AZFc microdeletions. It is necessary to use the U-M-PCR method to offer genetic screening and counseling to infertile men prior to intracytoplasmic sperm injection (ICSI) or in-vitro fertilization (IVF).

MicroRNA-215 is a potential prognostic marker for cervical cancer.

Recently, microRNAs (miRNAs) have been shown to be involved in multiple biological pathways that can influence tumor progression and metastasis and they can serve as prognostic biomarkers in many cancers. The present study examined the prognostic significance of miR-215 in cervical cancer. The paraffin-embedded paired cervical scrape samples and tumor tissue samples from 302 patients with stage II cervical cancer were detected for the expression of miR-215 by using qRT-PCR. A miR-215-based classifier was established by using the Cox regression model. The prognostic and predictive accuracy of this classifier was determined in both the internal testing group of 138 patients, and the external independent group of 280 patients. Moreover, cervical cancer HeLa cells overexpressing miR-215 (HeLa-miR-215) were constructed and subcutaneously injected into the nude mice to examine the effect of miR-215 on tumor growth and metastasis in vivo. The results showed that the expression level of miR-215 was significantly higher in cervical cancer tissues than in paired normal tissues (P<0.0001). When patients were classified into high- and low-risk cancer progression groups according to miR-215 level, the 5-year disease-free survival in high- and low-risk groups were 43% (95% CI: 32.1-51.6) and 67% (95% CI: 48.6-77.3) (hazard ratio [HR] 2.02, 95% CI: 1.16-3.52; P=0.013) respectively. Moreover, the expression level of miR-215 was negatively associated with survival rate in patients at TNM stage T3 (HR: 3.317; 95% CI: 1.18-5.14, P=0.017) and TNM stage T4 (HR: 3.48; 95% CI: 1.49-4.45, P=0.008). Tumor volume in nude mice injected with HeLa-miR-215 cells was significantly larger than that in mice injected with control HeLa cells. It was concluded that the expression level of miR-215 is associated with cervical tumor progression and worse survival rate, suggesting that it may serve as a potential prognostic marker to identify patients at higher risk of recurrence.

The activation of Piezo1 channel promotes invasion and migration via the release of extracellular ATP in cervical cancer.

BACKGROUND: The mechanosensitive ion channel Piezo1 has emerged as a potential prognostic and therapeutic target in different types of cancers. The aim of this study was to determine the expression levels and underlying mechanisms of Piezo1 in the invasion and migration processes in cervical cancer. METHODS: Initially, we employed qRT-PCR, western blot, and immunohistochemical staining techniques to assess the disparity in Piezo1 expression in cervical cancer tissues and cells. Subsequently, we conducted wound healing, transwell assays and phalloidin staining to observe the effects of stable Piezo1 silencing and Piezo1 selective agonist Yoda1 on the invasion and migration capabilities. The release of extracellular ATP was assessed using the enhanced ATP assay kit. Furthermore, we conducted rescue experiments to investigate whether the activation of Piezo1 facilitates cervical cancer invasion and migration through extracellular ATP. Finally, we constructed xenograft tumor models to determine weather the Piezo1 selective agonist Yoda1 influenced the tumor growth in vivo. RESULTS: In our study, we found that Piezo1 expression was elevated in both cervical cancer tissues and cells, with the highest levels observed in patients with lymph node metastasis. Knocking down Piezo1 resulted in a significant reduction in the invasion and migration capabilities of cervical cancer cells, whereas the use of the Piezo1 selective agonist Yoda1 enhanced these capabilities. Moreover, the activation of Piezo1 channels was found to regulate the release of extracellular ATP. Mechanistically, the activation of Piezo1 might facilitate cervical cancer invasion, migration, and pseudopodium formation through the release of extracellular ATP. And Piezo1 was an important molecule for the tumor growth of cervical cancer in vivo. CONCLUSION: Our findings revealed that Piezo1 facilitated the invasion and migration of cervical cancer by releasing extracellular ATP, which might hold potential as a valuable target for prognostic and therapeutic interventions in cervical cancer.

IL-4-induced M2 macrophages inhibit fibrosis of endometrial stromal cells.

BACKGROUND: Intrauterine adhesions (IUA) refers to endometrial fibrosis caused by irreversible damage of the endometrial basal layer. As the key regulators in tissue repair, regeneration, and fibrosis, macrophages play an essential role in endometrial regeneration and repair during the normal menstrual cycle. However, the mechanism of macrophages involved in IUA remains unclear. METHODS: In the late stages of proliferation, the endometrium was collected to make paraffin sections. HE and Masson staining were used to observing endometrial morphology and endometrial fibrosis. Immunohistochemistry and Western blotting were used to detect the expression level of fibrosis indexes COL1A1 and α-SMA. The macrophage infiltration was evaluated by immunohistochemistry for the expression levels of CD 206 and CD163. Next, we cultured the primary human endometrial stromal cells (HESCs), and then an IUA cell model was established with 10 ng/ml TGF-ß1 for 72 h. THP 1 cells were differentiated by 100 ng/ml PMA into macrophages for 48 h, then macrophages were polarized to M2 macrophages by 20 ng/ml IL-4 for 24 h. The culture supernatants (M(IL-4) -S) of M2 macrophages were applied to the IUA cell model. The expression of fibrosis markers was then assessed using immunofluorescence and Western blotting. RESULTS: The results show that Patients with IUA have fewer endometrial glands and significantly increased fibrosis levels. Moreover, the infiltration of CD206-positive (M2) macrophages was significantly reduced in IUA patients, and negatively correlated with the expression of endometrial fibrosis indexes α-SMA and COL1A1. In addition, the primary HESCs treated with 10 ng/ml TGF-ß1 for 72 h were found to have significantly increased levels of fibrosis indexes. Furthermore, supernatants from IL4-induced M2 macrophages inhibit the TGF-ß1-induced fibrosis of HESCs. CONCLUSIONS: M2 macrophages may negatively regulate the expression of COL1A1 and α-SMA, inhibiting the TGF-ß1-induced fibrosis of HESCs. Our study suggests that targeting macrophage phenotypes and promoting the polarization of macrophages to M2 may become a novel strategy for the clinical treatment of IUA.

MLK4 as an immune marker and its correlation with immune infiltration in Cervical squamous cell carcinoma and endocervical adenocarcinoma(CESC).

Mixed pedigree kinase 4 (MLK4) is a member of the serine/threonine kinases mixed pedigree kinase (MLKs) family. Few reports on immune-related targets in Cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), and the role of MLK4 in cervical cancer remains to be studied. The expression of MLK4 in CESC was analyzed by TCGA database containing 306 CESC tissues and 3 peritumoral tissue samples, and the effect of MLK4 on immune invasion was evaluated using the Deseq2 package(Benjamini-Hochberg corrected p-value < 0.05 and log2 fold change ≥|2|). Tissue microarray was used to verify the expression of MLK4 in CESC patients, and it was found that MLK4 was significantly overexpressed in CESC, and significantly correlated with WHO grade. Multiple analysis algorithms revealed that the high expression of MLK4 was negatively correlated with immune cell infiltration in CESC. Analysis showed that MLK4 expression was negatively correlated with the infiltration of various immune cells including CD8+T cells, and MLK4 mRNA expression was positively correlated with immune checkpoints PD-L1,CTLA4, LAG3, and negatively correlated with immune promotion genes CD86 and CD80. Furthermore, vitro assays were performed to investigate the biological characteristics of MLK4 in C33A cells. The EDU and transwell assays demonstrated that the decrease in MLK4 expression in C33A cells resulted in a decrease in cell proliferation and invasion. The silencing of MLK4 resulted in a significant increase in the expression of inflammatory cytokines IL-1ß(p<0.05), TNF-α(p<0.01), and IL-6 (p<0.05). The results of cell assays indicate that knocking down MLK4 would inhibit the expression of established biochemical markers CEA, AFP and HCG. Hence, it is plausible that MLK4 could potentially exert a significant influence on the development and progression of Cervical cancer.

Diagnostic, prognostic, and immunological roles of CD177 in cervical cancer.

BACKGROUND: CD177, an indicator of prognosis in diverse cancers, is involved in the physiological processes of various tumor cells, and acts as an immune molecule with novel functions in cancer pathogenesis. However, the diagnostic, prognostic, and immunological role of CD177 in cervical cancer remains unclear. METHODS: Utilizing publicly available databases and integrating several bioinformatics analysis methods, we evaluated the expression level of CD177 in cervical cancer by GENT2, HPA, and GEO databases. And the experiments of western blot and immunohistochemical staining were used to test the hypothesis. The Kaplan-Meier Plotter database, Xena Shiny, and the constructed nomogram were clearly demonstrated its prognostic value for patients. Gene set enrichment analysis explored the relationship between CD177 and cervical cancer immune responses and immune cells infiltration level. In addition, we investigated the association between CD177 expression and stromal score, immune score, immune checkpoint, and drug sensitivity by TCGA RNA-seq data. RESULTS: CD177 was apparently expressed at low levels in cervical cancer and predicted a poor survival rate for patients. CD177 significantly activated immune-related signaling pathways and had a positive relationship with immune cell infiltration level. The high CD177 expression group possessed the high stromal score and immune score. CD177 had potential interactions with CTLA4, CD27, BLTA, CD200R1, CD80, NRP1, TNFRSF25, TIGIT, ICOS, and TNFSF9 checkpoint markers. And CD177 expression was positively relevant with drug sensitivity for Lapatinib, Belinostat, ATRA, Gefitinib, Navitoclax, and Tamoxifen. SIGNIFICANCE: These findings may shed light on the vital role of CD177 in cervical cancer diagnosis, prognosis, and immunological functions, and it may be a promising predictor and potential factor for cervical cancer patients.

Machine Learning-Based Modeling of Ovarian Response and the Quantitative Evaluation of Comprehensive Impact Features.

Appropriate ovarian responses to the controlled ovarian stimulation strategy is the premise for a good outcome of the in vitro fertilization cycle. With the booming of artificial intelligence, machine learning is becoming a popular and promising approach for tailoring a controlled ovarian stimulation strategy. Nowadays, most machine learning-based tailoring strategies aim to generally classify the controlled ovarian stimulation outcome, lacking the capacity to precisely predict the outcome and evaluate the impact features. Based on a clinical cohort composed of 1365 women and two machine learning methods of artificial neural network and supporting vector regression, a regression prediction model of the number of oocytes retrieved is trained, validated, and selected. Given the proposed model, an index called the normalized mean impact value is defined and calculated to reflect the importance of each impact feature. The proposed models can estimate the number of oocytes retrieved with high precision, with the regression coefficient being 0.882% and 89.84% of the instances having the prediction number ≤ 5. Among the impact features, the antral follicle count has the highest importance, followed by the E2 level on the human chorionic gonadotropin day, the age, and the Anti-Müllerian hormone, with their normalized mean impact value > 0.3. Based on the proposed model, the prognostic results for ovarian response can be predicted, which enables scientific clinical decision support for the customized controlled ovarian stimulation strategies for women, and eventually helps yield better in vitro fertilization outcomes.

Long-term culture and transplantation of spermatogonial stem cells from BALB/c mice.

Development of a culture system that supports self-renewal and proliferation of spermatogonial stem cells (SSCs) is enormously valuable for experimental research and potential treatment for male infertility. Although several research groups had reported their successes in SSC isolation and culture, the two current accepted culture systems are different in cell enrichment methods, serum and growth factors. Previous researches also indicated SSCs from different mouse strains required different culture conditions. Here we report for the first time that SSCs from BALB/c mice could be cultured in an improved culture system for 3 months. The modified culture system consisted of an improved enzymatic procedure, the enrichment of undifferentiated spermatogonia by differential adherence selection of isolated SSCs, mouse embryonic fibroblast feeder cells, StemPro-34 SFM medium supplemented with glial cell line-derived neurotrophic factor (GDNF), basic fibroblast growth factor and GDNF-family receptor alpha1 (GFRalpha1). The improved digestion method increased the viability and enrichment efficiency of isolated testis cells. Furthermore, basal culture medium with 10% fetal bovine serum as selected medium could increase the number of germ cell colonies in the initiation stage of culture. Cultured SSCs were characterized morphologically and formed typical colonies. Immunocytochemical staining and RT-PCR showed that cultured SSCs expressed Oct-4, GFRalpha1, Sox2 and several other special genes resembling undifferentiated spermatogonia. Spermatogonia transplantation further confirmed that cultured SSCs were functionally normal and could restore complete spermatogenesis. The culture methods described here could serve as a paradigm to establish conditions for the culture of SSCs from other species, allowing identification of universal factors necessary for proliferation of SSCs.

EGFR Gene Mutation and Methodological Evaluation in 399 Patients with Non-small Cell Lung Cancer.

The purpose of the present study was to study the characteristics of epidemic growth factor receptor (EGFR) gene distribution in patients with non-small cell lung cancer (NSCLC), and to detect the mutation rate of EGFR gene by Sanger sequencing and amplification refractory mutation system (ARMS)-PCR. Paraffin-embedded sections of NSCLC tissues from 399 NSCLC patients diagnosed in Renmin Hospital of Wuhan University were collected, 103 of them were detected for exons 18-21 mutation of EGFR by Sanger sequencing method, 296 cases were detected for exons 18-21 mutation by ARMS-PCR method. DNA extraction of both groups was performed with Qiagen QLAamp DNA FFPE Tissue KIT. Comparisons of detection rates between the two methods were conducted by row X list chi-square test. The total mutation rate of EGFR gene detected by Sanger sequencing was 21.4%, exons 18-21 and combined mutation rates were 1.0%, 9.7%, 1.0%, 7.8% and 2.0%, respectively. And the proportions were 4.7%, 45.2%, 4.7%, 36.3% and 9.4% respectively. The total mutation rate detected by ARMS-PCR was 51.4%, exons 18-21 and combined mutation rates were 2.7%, 27%, 1.7%, 18.2% and 1.7%, respectively. The proportions were 5.3%, 52.6%, 3.3%, 35.5% and 3.3% respectively. Further analysis of mutation rate showed that there was significant difference between the two methods in detecting total mutation of EGFR gene (P<0.001). There were significant differences in mutation detection rates of exons 19 and 21 (P<0.001, P<0.05), but there were no significant differences in other exons. And there was no significant difference in mutation detection rates between the two methods. The mutation rate of EGFR gene in NSCLC patients was 50%. And exon 19 deletion was the most common mutation type, followed by exon 21 mutation. Compared with Sanger sequencing method, ARMS method is more sensitive with significant advantages in detecting exon 19 deletions and exon 21 mutations, which can be widely used in clinical detection of EGFR gene mutations. The results of this study will further guide patients with advanced NSCLC to select TKI targeted drugs, and provide clinical diagnostic basis for targeted therapy of NSCLC patients.

[Long-term culture and identification of spermatogonial stem cells from BALB/c mice in vitro].

OBJECTIVE: To establish a long-term culture system for mouse spermatogonial stem cells (SSCs) and to discuss the key factor that supports mouse SSC self-renewal and proliferation. METHODS: Testis cells from 4-6 days postpartum male transgenic BALB/c mce were collected by a modified two-step enzymatic digestion method and plated on 0. 2% elatin-coated tissue culture plates. The germ cells were enriched by differential adherence selections after respectively incubated for 1, 5 and 24 h and then plated on the mitomycin C-inactivated mouse embryonic fibroblast (MEF) feeder layer. The basal culture medium was StemPro-34 SFM supplemented with other 15 nutrient factors. The 20 ng/ml Glial cell line-derived neurotrophic factor (GDNF), 10 ng/ml basic fibroblast growth factor (bFGF) and 200 ng/ml GDNF-family receptor alpha 1 (GFRalpha1) were added to the serum-free medium to promote SSC proliferation. Several important surface markers and special genes were examined by immunocytochemical staining and RT-PCR analysis. RESULTS: After 3-4 days culture on the MEF feeder, SSCs proliferated continuously and formed typical colonies. SSCs from the BALB/c mice could be cultured in a steady state for 3 months. Immunocytochemical staining showed that Oct4 was specifically expressed in the cultured SSC nucleus and GFRalpha1 strongly expressed on the surface of the membrane. RT-PCR confirmed that the cultured SSCs expressed Oct-4, GFRalpha1, Sox2 and several other special genes resembling undifferentiated spermatogonia. CONCLUSION: SSCs from BALB/c mice could be cultured in the improved culture system for 3 months. This culture system could help further understand the regulating mechanism of SSCs and might provide an opportunity for the treatment of male infertility by SSC transplantation.

PP2A Inhibits Cervical Cancer Cell Migration by Dephosphorylation of p-JNK, p-p38 and the p-ERK/MAPK Signaling Pathway.

Protein phosphatase 2A (PP2A) was reported to play an important role in cancer development; however, the relationship between PP2A and cervical cancer development has yet to be fully understood. The present study aimed to explore the role of PP2A in the development of cervical cancer. Serum levels of PP2A were detected by ELISA in 23 patients with cervical cancer and 30 patients with benign cervical lesions. Furthermore, the PP2A activities and the mRNA and protein levels of PP2A were measured in cervical cancer (n=8) and chronic cervicitis (n=10) tissues. The results showed that the serum levels of PP2A were significantly reduced in patients with cervical cancer. Further studies showed that not only the activities of PP2A but also the mRNA and protein levels of PP2A were significantly decreased in cervical cancer tissues. Wound healing and Transwell assays demonstrated that pharmacological and genetic upregulation of PP2A could inhibit the migration of HeLa cells, but the downregulation of PP2A promoted cellular migration. The activation of PP2A also inhibited the remodeling of actin and the activity of mitogen-activated protein kinases (MAPKs) including p-JNK, p-p38 and p-ERK. Meanwhile, the activation of PP2A was found to downregulate MMP-9 levels, which further inhibited the migration and invasion of HeLa cells. In conclusion, our data suggest that the activity and expression of PP2A are significantly reduced in cervical cancer tissues, and the activation of PP2A may inhibit the migration of cervical cancer cells by inhibiting the phosphorylation of p-JNK, p-p38 and the p-ERK/MAPK signaling pathway as well as by downregulating MMP-9, implying that PP2A plays an important role in cervical cancer development.