Early cumulus cell removal increases cumulative live birth rate while having no negative effect on the malformation rate in in vitro fertilization: a propensity score-matched cohort study.

PURPOSE: The aim of this study was to investigate the efficacy and safety of early cumulus cell removal (ECCR) during human in vitro fertilization (IVF). METHODS: A retrospective analysis was performed between January 2011 and December 2019. The study enrolled 1131 couples who underwent IVF treatment with ECCR. After propensity score matching at a 1:1 ratio, 1131 couples who underwent overnight coincubation of gametes were selected. The main outcome measure was the cumulative live birth rate. Secondary outcome measures included the cumulative pregnancy rate, polyspermy rate, available embryo rate, miscarriage rate, malformation rate, time to live birth, and oocyte-to-baby rate. RESULTS: There were no significant differences found between the two groups in the polyspermy rate, available embryo rate, miscarriage rate, time to live birth, oocyte-to-baby rate, and neonatal congenital anomalies rate. The results of the study showed that ECCR was associated with a significantly higher cumulative live birth rate and cumulative pregnancy rate, along with a significantly lower fertilization rate. CONCLUSIONS: ECCR tended to confer increased cumulative live birth rate and had no negative effect on the neonatal malformation rate.

Nasopharyngeal carcinoma patient-derived xenograft mouse models reveal potential drugs targeting cell cycle, mTOR, and autophagy pathways.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is associated with Epstein-Barr virus (EBV) infection. To test preclinical NPC drugs, we established two patient-derived xenograft (PDX) mouse models, EBV-positive PDX-B13 and EBV-negative PDX-Li41, for drug screening. METHODS: Based on next generation sequencing (NGS) studies, PDX-B13 had CCND1 copy number (CN) gain but CDKN2A CN loss, whereas PDX-Li41 had CDKN2A and RB1 CN loss, TSC1 (negative regulator of mTOR) frameshift deletion mutation, and increased activation of mTOR, a serine/threonine kinase that governs metabolism, autophagy, and apoptosis. Increased mTOR was also associated with poor NPC prognosis. RESULTS: Everolimus, an mTOR inhibitor, suppressed tumor growth in the two PDX NPC models and had an additive antitumor effect with palbociclib, a CDK4/6 inhibitor. PDX tumors treated with various drugs or untreated were subjected to RNA sequencing, transcriptome profile analysis, and selective Western blotting to understand the interactions between these drugs and gene expression profiles. Palbociclib also suppressed EB viral nuclear antigen (EBNA1) expression in PDX-B13. Everolimus together with autophagy inhibitor, hydroxychloroquine, had additive anti-tumor effect on PDX-B13 tumor. Immunohistochemistry revealed that high mTOR levels were correlated with poor overall survival in patients with metastatic NPC (N = 90). CONCLUSIONS: High mTOR levels are a poor prognostic factor in NPC, and cell cycle, mTOR and autophagy pathways may serve as therapeutic targets in NPC. In addition, PDX models can be used for efficiently testing potential NPC drugs.

C-type Lectin Domain Family 4 Member G (CLEC4G) Is a Negative Marker for CD34 in the Evolution of Liver Pathogenesis.

OBJECTIVE: This study aimed to explore the expression of the C-type lectin domain family 4 member G (CLEC4G) gene in the liver sinusoidal endothelial cells (LSECs) during liver pathogenesis, and to evaluate its correlation with CD34 and clinical significance in hepatocellular carcinoma patients. METHODS: We conducted bioinformatics analysis of the differential expression of CLEC4G in various human organs, carcinomatous and adjacent tissues. Then, mRNA and protein expression levels of CD34 in hepatocellular carcinoma (HCC) samples were detected via real-time quantitative reverse transcription PCR (qRT-PCR) and immunohistochemical (IHC), respectively. ELISA was applied to detect serum levels of CLEC4G in healthy controls, liver fibrosis and HCC patients. RESULTS: The expressions of mRNA and protein levels of CLEC4G were higher in normal liver tissues, moderately expressed in cirrhotic and para-cancerous tissues (P<0.001), and lowest in HCC tissues (P<0.001). We also found high CD34 expression in tumors, which was negatively correlated with CLEC4G at both mRNA and protein levels. Compared to the healthy controls, the CLEC4G levels in liver fibrosis patients and HCC patients gradually became lower (P<0.001). CONCLUSIONS: The low expression of CLEC4G is potentially correlated with LSEC capillarization and the appearance of micro-vessels. Such a phenomenon may serve as a reliable diagnostic marker for hepatocellular carcinoma.

Capsaicin suppresses hepatocarcinogenesis by inhibiting the stemness of hepatic progenitor cells via SIRT1/SOX2 signaling pathway.

BACKGROUND & AIMS: Capsaicin, a functional component of chili pepper, possesses anti-inflammatory, analgesic, and anti-cancer properties. This study aimed to determine the property of capsaicin against hepatocarcinogenesis in vivo and investigate the role of the SIRT1/SOX2 pathway in the mode of action of capsaicin in hepatic progenitor cells (HPCs), which is related to hepatocarcinogenesis. MATERIALS & METHODS: We prepared a diethylnitrosamine-induced liver cancer model in rats to examine hepatocarcinogenesis, and delivered liposomal capsaicin through the subcutaneous transposition of the spleen to the liver. Liver sections from rats and hepatocarcinoma patients were stained for the markers of HPCs or SIRT1/SOX2 signaling. SIRT1/SOX2 signalling expression was measured using immunoprecipitation and western blot. RESULTS: We found that capsaicin significantly inhibited hepatocarcinogenesis. Notably, capsaicin inhibited HPCs activation in vivo but did not induce apoptosis in the normal hepatic progenitor cell line in rats in vitro. This suggests that capsaicin suppresses hepatocarcinogenesis by inhibiting the stemness of HPCs. Moreover, capsaicin can induce this inhibition by reducing the stability of SOX2. SIRT1 is overexpressed in liver cancer and acts as a tumor promoter via SOX2 deacetylation. Using immunoprecipitation, we identified direct binding between SIRT1 and SOX2. The capsaicin treatment resulted in SIRT1 downregulation which reduced deacetylation, and increased nuclear export as well as subsequent ubiquitous degradation of SOX2. CONCLUSIONS: Altogether, we report that capsaicin suppresses hepatocarcinogenesis by inhibiting the stemness of HPCs via SIRT1/SOX2 signaling. It may serve as a promising therapeutic candidate for liver cancer.

Obstetric and neonatal outcomes after frozen-thawed embryos transfer with laser-assisted hatching: a retrospective cohort study.

PURPOSE: To ascertain if assisted hatching (AH) increases the risk of placenta-associated diseases and perinatal outcomes after frozen-thawed cleavage-stage embryo transfer. METHODS: We retrospectively evaluated 924 women who conceived with frozen-thawed cleavage-stage embryos transfer with (n = 390) or without (n = 534) laser-AH between 2013 and 2015. Data were obtained from the database on in vitro fertilization (IVF) patients in Shanghai First Maternity and Infant Hospital. We assessed neonatal (preterm birth, low birthweight, fetal macrosomia, stillbirth) and obstetric (miscarriage, ectopic pregnancy, post-term pregnancy, gestational diabetes (GDM), preeclampsia, intrahepatic cholestasis (ICP), placenta previa, placental abruption, premature rupture of membranes) outcomes. RESULTS: In twins, the median birthweight was lower in the AH group than that in the control group, and the prevalence of low birthweight (< 2500 g) was significantly higher in the AH group; after adjusting for maternal age, body mass index, mode of fertilization, and parity, no significant difference was found. In twins, no significant difference was detected in the prevalence of stillbirth or preterm pregnancy. In singleton births, there was no significant difference in the prevalence of low birthweight, macrosomia, preterm pregnancy or post-term pregnancy between the two groups. In singletons and twins, there were no significant differences in the prevalence of miscarriage, ectopic pregnancy, preeclampsia, GDM, ICP, or placenta abruption between the two groups. CONCLUSIONS: AH is a relatively safe method and our study provides important information for using this method in carefully selected patients.

Combination of Epithelial Growth Factor Receptor Blockers and CDK4/6 Inhibitor for Nasopharyngeal Carcinoma Treatment.

BACKGROUND: Nasopharyngeal carcinoma (NPC) involves host genetics, environmental and viral factors. In clinical observations, patients of young and old ages were found to have higher recurrence and metastatic rates. METHODS: Cytokine array was employed to screen druggable target(s). The candidate target(s) were confirmed through patient-derived xenografts (PDXs) and a new EBV-positive cell line, NPC-B13. RESULTS: Overexpression of epithelial growth factor (EGF) and EGF receptor (EGFR) was detected in young patients than in older patients. The growth of NPC PDX tumors and cell lines was inhibited by EGFR inhibitors (EGFRi) cetuximab and afatinib when used separately or in combination with the cell cycle blocker palbociclib. Western blot analysis of these drug-treated PDXs demonstrated that the blockade of the EGF signaling pathway was associated with a decrease in the p-EGFR level and reduction in PDX tumor size. RNA sequencing results of PDX tumors elucidated that cell cycle-related pathways were suppressed in response to drug treatments. High EGFR expression (IHC score ≥ grade 3) was correlated with poor survival in metastatic patients (p = 0.008). CONCLUSIONS: Our results provide encouraging preliminary data related to the combination treatment of EGFRi and palbociclib in patients with NPC.

Integrated genomic analyses in PDX model reveal a cyclin-dependent kinase inhibitor Palbociclib as a novel candidate drug for nasopharyngeal carcinoma.

BACKGROUND: Patient-derived xenograft (PDX) tumor model has become a new approach in identifying druggable tumor mutations, screening and evaluating personalized cancer drugs based on the mutated targets. METHODS: We established five nasopharyngeal carcinoma (NPC) PDXs in mouse model. Subsequently, whole-exome sequencing (WES) and genomic mutation analyses were performed to search for genetic alterations for new drug targets. Potential drugs were applied in two NPC PDX mice model to assess their anti-cancer activities. RNA sequencing and transcriptomic analysis were performed in one NPC PDX mice to correlate with the efficacy of the anti-cancer drugs. RESULTS: A relative high incident rate of copy number variations (CNVs) of cell cycle-associated genes. Among the five NPC-PDXs, three had cyclin D1 (CCND1) amplification while four had cyclin-dependent kinase inhibitor CDKN2A deletion. Furthermore, CCND1 overexpression was observed in > 90% FFPE clinical metastatic NPC tumors (87/91) and was associated with poor outcomes. CNV analysis disclosed that plasma CCND1/CDKN2A ratio is correlated with EBV DNA load in NPC patients' plasma and could serve as a screening test to select potential CDK4/6 inhibitor treatment candidates. Based on our NPC PDX model and RNA sequencing, Palbociclib, a cyclin-dependent kinase inhibitor, proved to have anti-tumor effects by inducing G1 arrest. One NPC patient with liver metastatic was treated with Palbociclib, had stable disease response and a drop in Epstein Barr virus (EBV) EBV titer. CONCLUSIONS: Our integrated information of sequencing-based genomic studies and tumor transcriptomes with drug treatment in NPC-PDX models provided guidelines for personalized precision treatments and revealed a cyclin-dependent kinase inhibitor Palbociclib as a novel candidate drug for NPC.

Inactivation of the tight junction gene CLDN11 by aberrant hypermethylation modulates tubulins polymerization and promotes cell migration in nasopharyngeal carcinoma.

BACKGROUND: Aberrant hypermethylation of cellular genes is a common phenomenon to inactivate genes and promote tumorigenesis in nasopharyngeal carcinoma (NPC). METHODS: Methyl binding domain (MBD)-ChIP sequencing of NPC cells, microarray data of NPC biopsies and gene ontology analysis were conducted to identify a potential tumor suppressor gene CLDN11 that was both hypermethylated and downregulated in NPC. Bisulfite sequencing, qRT-PCR, immunohistochemistry staining of the NPC clinical samples and addition of methylation inhibitor, 5'azacytidine, in NPC cells were performed to verify the correlation between DNA hypermethylation and expression of CLDN11. Promoter reporter and EMSA assays were used to dissect the DNA region responsible for transcription activator binding and to confirm whether DNA methylation could affect activator's binding, respectively. CLDN11 was transiently overexpressed in NPC cells followed by cell proliferation, migration, invasion assays to characterize its biological roles. Co-immunoprecipitation experiments and proteomic approach were carried out to identify novel interacting protein(s) and the binding domain of CLDN11. Anti-tumor activity of the CLDN11 was elucidated by in vitro functional assay. RESULTS: A tight junction gene, CLDN11, was identified as differentially hypermethylated gene in NPC. High methylation percentage of CLDN11 promoter in paired NPC clinical samples was correlated with low mRNA expression level. Immunohistochemistry staining of NPC paired samples tissue array demonstrated that CLDN11 protein expression was relatively low in NPC tumors. Transcription activator GATA1 bound to CLDN11 promoter region - 62 to - 53 and its DNA binding activity was inhibited by DNA methylation. Re-expression of CLDN11 reduced cell migration and invasion abilities in NPC cells. By co-immunoprecipitation and liquid chromatography-tandem mass spectrometry LC-MS/MS, tubulin alpha-1b (TUBA1B) and beta-3 (TUBB3), were identified as the novel CLDN11-interacting proteins. CLDN11 interacted with these two tubulins through its intracellular loop and C-terminus. Furthermore, these domains were required for CLDN11-mediated cell migration inhibition. Treatment with a tubulin polymerization inhibitor, nocodazole, blocked NPC cell migration. CONCLUSIONS: CLDN11 is a hypermethylated and downregulated gene in NPC. Through interacting with microtubules TUBA1B and TUBB3, CLDN11 blocks the polymerization of tubulins and cell migration activity. Thus, CLDN11 functions as a potential tumor suppressor gene and silencing of CLDN11 by DNA hypermethylation promotes NPC progression.

Aberrantly hypermethylated Homeobox A2 derepresses metalloproteinase-9 through TBP and promotes invasion in Nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is notorious for its high invasiveness and metastatic ability. In this study, we identified a differential hypermethylated transcription repressor, Homeobox A2 (HOXA2), which may render NPC cells invasive and metastatic. Aberrant hypermethylation of HOXA2 led to low RNA expression in NPC tumors and cells. Addition of methylation inhibitor 5'Aza restored HOXA2 RNA expression in NPC cells. Methylated HOXA2 promoter reduces the binding affinity of the transcriptional co-activator p300, causing transcriptional repression of HOXA2. In NPC cells, re-expression of ectopic HOXA2 was correlated with decreased invasive ability and reduced metalloproteinase MMP-9 RNA and protein expression. Promoter, ChIP and DNA-pull down assays indicated that HOXA2 competes with the transcription activator, TATA-box binding protein (TBP) for a recognition sequence near the MMP-9 transcription start site, and suppresses MMP-9 transcription. Thus, HOXA2 acts as a suppressor or TBP-antagonist to inhibit MMP-9 expression; while methylation-mediated inactivation of HOXA2 in NPC derepresses MMP-9 production and increases invasion of NPC cells. In NPC plasma samples, increased plasma EBV copy number was correlated with increased in cell-free HOXA2 hypermethylation and elevated MMP-9 levels. Plasma EBV DNA and methylated cell-free HOXA2 can be used as biomarkers for monitoring NPC treatment.