Non-invasive Cell Research Articles

RNA-seq in DMD urinary stem cells recognized muscle-related transcription signatures and addressed the identification of atypical mutations by whole-genome sequencing.

SummaryUrinary stem cells (USCs) are a non-invasive, simple, and affordable cell source to study human diseases. Here we show that USCs are a versatile tool for studying Duchenne muscular dystrophy (DMD), since they are able to address RNA signatures and atypical mutation identification. Gene expression profiling of DMD individuals’ USCs revealed a profound deregulation of inflammation, muscle development, and metabolic pathways that mirrors the known transcriptional landscape of DMD muscle and worsens following USCs’ myogenic transformation. This pathogenic transcription signature was reverted by an exon-skipping corrective approach, suggesting the utility of USCs in monitoring DMD antisense therapy. The full DMD transcript profile performed in USCs from three undiagnosed DMD individuals addressed three splicing abnormalities, which were decrypted and confirmed as pathogenic variations by whole-genome sequencing (WGS). This combined genomic approach allowed the identification of three atypical and complex DMD mutations due to a deep intronic variation and two large inversions, respectively. All three mutations affect DMD gene splicing and cause a lack of dystrophin protein production, and one of these also generates unique fusion genes and transcripts. Further characterization of USCs using a novel cell-sorting technology (Celector) highlighted cell-type variability and the representation of cell-specific DMD isoforms. Our comprehensive approach to USCs unraveled RNA, DNA, and cell-specific features and demonstrated that USCs are a robust tool for studying and diagnosing DMD.

Bright near-infrared aggregation-induced emission dots for long-term bioimaging in vitro/vivo

Advanced hepatocarcinoma is almost incurable and has caused a huge number of deaths every year. Noninvasive long-term cell tracing is an efficient and important tool to understand genesis, development and evolution of cancer cells. Bright organic luminescent dots (named TNZ2tPPI-Tat NPs) with unique aggregation-induced near-infrared red emission (NIR-AIE), prepared in this contribution, enjoy the advantages of desirable biocompatibility, great water dispersibility, high photoluminescent quantum efficiency (32 %), large Stokes shift (166 nm), anti-background interference and great photostability. Moreover, the thus-obtained AIE-active NPs show ultralong fluorescent bioimaging (7 days) in MHCC97-H cells, and still keep clear fluorescent signals after 5 generations in three hepatic cells (Lo2, MHCC97-H, Hep 3B). In vivo fluorescent imaging demonstrates that TNZ2tPPI-Tat NPs still maintain strong brightness over 26 days, suggest great stability of such bright NPs. Further compare with the counterparts of commercial fluorescent probes such as Cy5 and AF647, such AIE dots enjoy more superior performances in the aspect of fluorescent intensity and long-term bioimaging efficiency, which is promising candidate to serve as noninvasive long-term cell tracker.

Peroxidasin protein expression and enzymatic activity in metastatic melanoma cell lines are associated with invasive potential

Peroxidasin, a heme peroxidase, has been shown to play a role in cancer progression. mRNA expression has been reported to be upregulated in metastatic melanoma cell lines and connected to the invasive phenotype, but little is known about how peroxidasin acts in cancer cells. We have analyzed peroxidasin protein expression and activity in eight metastatic melanoma cell lines using an ELISA developed with an in-house peroxidasin binding protein. RNAseq data analysis confirmed high peroxidasin mRNA expression in the five cell lines classified as invasive and low expression in the three non-invasive cell lines. Protein levels of peroxidasin were higher in the cell lines with an invasive phenotype. Active peroxidasin was secreted to the cell culture medium, where it accumulated over time, and peroxidasin protein levels in the medium were also much higher in invasive than non-invasive cell lines. The only well-established physiological role of peroxidasin is in the formation of a sulfilimine bond, which cross-links collagen IV in basement membranes via catalyzed oxidation of bromide to hypobromous acid. We found that peroxidasin secreted from melanoma cells formed sulfilimine bonds in uncross-linked collagen IV, confirming peroxidasin activity and hypobromous acid formation. Moreover, 3-bromotyrosine, a stable product of hypobromous acid reacting with tyrosine residues, was detected in invasive melanoma cells, substantiating that their expression of peroxidasin generates hypobromous acid, and showing that it does not exclusively react with collagen IV, but also with other biomolecules.

Creating stem cell-derived neuromuscular junctions in vitro.

Recent development of novel therapies has improved mobility and quality of life for people suffering from inheritable neuromuscular disorders. Despite this progress, the majority of neuromuscular disorders are still incurable, in part due to a lack of predictive models of neuromuscular junction (NMJ) breakdown. Improvement of predictive models of a human NMJ would be transformative in terms of expanding our understanding of the mechanisms that underpin development, maintenance, and disease, and as a testbed with which to evaluate novel therapeutics. Induced pluripotent stem cells (iPSCs) are emerging as a clinically relevant and non‐invasive cell source to create human NMJs to study synaptic development and maturation, as well as disease modeling and drug discovery. This review will highlight the recent advances and remaining challenges to generating an NMJ capable of eliciting contraction of stem cell‐derived skeletal muscle in vitro. We explore the advantages and shortcomings of traditional NMJ culturing platforms, as well as the pioneering technologies and novel, biomimetic culturing systems currently in use to guide development and maturation of the neuromuscular synapse and extracellular microenvironment. Then, we will explore how this NMJ‐in‐a‐dish can be used to study normal assembly and function of the efferent portion of the neuromuscular arc, and how neuromuscular disease‐causing mutations disrupt structure, signaling, and function.

Label-free imaging for quality control of cardiomyocyte differentiation

Human pluripotent stem cell (hPSC)-derived cardiomyocytes provide a promising regenerative cell therapy for cardiovascular patients and an important model system to accelerate drug discovery. However, cost-effective and time-efficient platforms must be developed to evaluate the quality of hPSC-derived cardiomyocytes during biomanufacturing. Here, we develop a non-invasive label-free live cell imaging platform to predict the efficiency of hPSC differentiation into cardiomyocytes. Autofluorescence imaging of metabolic co-enzymes is performed under varying differentiation conditions (cell density, concentration of Wnt signaling activator) across five hPSC lines. Live cell autofluorescence imaging and multivariate classification models provide high accuracy to separate low (< 50%) and high (≥ 50%) differentiation efficiency groups (quantified by cTnT expression on day 12) within 1 day after initiating differentiation (area under the receiver operating characteristic curve, 0.91). This non-invasive and label-free method could be used to avoid batch-to-batch and line-to-line variability in cell manufacturing from hPSCs.

The association between Annexin A2 and epithelial cell adhesion molecule in breast cancer cells.

BackgroundThe epithelial cell adhesion molecule (EpCAM) is a type I transmembrane and glycosylated protein, which is overexpressed in many neoplasms. However, EpCAM has no known ligand partners and the mechanisms by which it functions are not fully understood.AimThis study was performed to discover novel partners of EpCAM, which may provide a better understanding of its functions.MethodsThe membrane fraction of the ERα+ noninvasive breast cancer cell line ZR‐75‐1 and MCF‐7 was extracted and followed by co‐immunoprecipitation of EpCAM using C‐10, a mouse monoclonal antibody raised against amino acids 24–93 of the EpCAM molecule. As a negative control, MDA‐MB‐231 and Hs578T were used since they express a negligible amount of EpCAM and are known as EpCAM−/low ERα−/low invasive and tumorigenic breast cancer cell lines.ResultsAnnexin A2 (ANXA2) was found to be selectively and differentially co‐immunoprecipitated with EpCAM in the ERα+ breast cancer cells MCF‐7 and ZR‐75‐1. ANXA2 is a multifunctional protein and known to act as a co‐receptor for tissue plasminogen activator (tPA) on the surface of endothelial and cancer cells, thereby affecting fibrinolytic activity and neoangiogenesis as well as invasive and metastatic properties. In this study, the association between EpCAM and ANXA2 was found to affect the activity of tPA.ConclusionThis study concludes that ANXA2 co‐localizes with EpCAM at the plasma membrane, and the co‐localization may have functional implications. Data suggest that EpCAM supports ANXA2 to function as a co‐receptor for the tPA, and that EpCAM has a regulatory function on the expression and subcellular localization of ANXA2.

Chemical inhibitor anticancer drugs regulate mechanical properties and cytoskeletal structure of non-invasive and invasive breast cancer cell lines: Study of effects of Letrozole, Exemestane, and Everolimus

Regardless of their target and mechanism, anticancer drugs directly influence biological behavior of cancer cells by activating chemical signaling pathways. Due to the complex interaction between diverse signaling pathways, these drugs may profoundly impact the physical characteristics of cancer cells and regulate their mechanical properties. In this study, the effects of two Aromatase Inhibitor (Letrozole and Exemestane), and one mTOR Inhibitor (Everolimus) on cell mechanical properties, actin content/distribution, and nuclear areas of two invasive and non-invasive breast cancer cell line after 24 h treatment with concentrations previously reported were investigated. While metabolic activity of cell lines was highly affected by drug treatment, significant alterations in Young's modulus of cell bodies, nuclear areas, and actin content and distribution were reported with higher impact on invasive cells. It was concluded that regulation of mechanical behavior of cells by all three drugs emphasizes the cross talk between chemical and physical signaling cascades, and describes a correlation between biological and physical behaviors of cancer cells which might give an insight to a better understanding of mechanisms by which anti-cancer drugs function to enhance their performances.

The Role of Interleukin-1-Receptor-Antagonist in Bladder Cancer Cell Migration and Invasion.

Background: The interleukin-1-receptor antagonist IL1RA (encoded by the IL1RN gene) is a potent competitive antagonist to interleukin-1 (IL1) and thereby is mainly involved in the regulation of inflammation. Previous data indicated a role of IL1RA in muscle-invasive urothelial carcinoma of the bladder (UCB) as well as an IL1-dependent decrease in tissue barrier function, potentially contributing to cancer cell invasion. Objective: Based on these observations, here we investigated the potential roles of IL1RA, IL1A, and IL1B in bladder cancer cell invasion in vitro. Methods: Cell culture, real-time impedance sensing, invasion assays (Boyden chamber, pig bladder model), qPCR, Western blot, ELISA, gene overexpression. Results: We observed a loss of IL1RA expression in invasive, high-grade bladder cancer cell lines T24, UMUC-3, and HT1197 while IL1RA expression was readily detectable in the immortalized UROtsa cells, the non-invasive bladder cancer cell line RT4, and in benign patient urothelium. Thus, we modified the invasive human bladder cancer cell line T24 to ectopically express IL1RA, and measured changes in cell migration/invasion using the xCELLigence Real-Time-Cell-Analysis (RTCA) system and the Boyden chamber assay. The real-time observation data showed a significant decrease of cell migration and invasion in T24 cells overexpressing IL1RA (T24-IL1RA), compared to cells harboring an empty vector (T24-EV). Concurrently, tumor cytokines, e.g., IL1B, attenuated the vascular endothelial barrier, which resulted in a reduction of the Cell Index (CI), an impedance-based dimensionless unit. This reduction could be reverted by the simultaneous incubation with IL1RA. Moreover, we used an ex vivo porcine organ culture system to evaluate cell invasion capacity and showed that T24-IL1RA cells showed significantly less invasive capacity compared to parental T24 cells or T24-EV. Conclusions: Taken together, our results indicate an inverse correlation between IL1RA expression and tumor cell invasive capacity and migration, suggesting that IL1RA plays a role in bladder carcinogenesis, while the exact mechanisms by which IL1RA influences tumor cells migration/invasion remain to be clarified in future studies. Furthermore, we confirmed that real-time impedance sensing and the porcine ex vivo organ culture methods are powerful tools to discover differences in cancer cell migration and invasion.

Imaging Beta-Cell Function in the Pancreas of Non-Human Primates Using a Zinc-Sensitive MRI Contrast Agent.

Non-invasive beta cell function measurements may provide valuable information for improving diabetes diagnostics and disease management as the integrity and function of pancreatic beta cells have been found to be compromised in Type-1 and Type-2 diabetes. Currently, available diabetes assays either lack functional information or spatial identification of beta cells. In this work, we introduce a method to assess the function of beta cells in the non-human primate pancreas non-invasively with MRI using a Gd-based zinc(II) sensor as a contrast agent, Gd-CP027. Additionally, we highlight the role of zinc(II) ions in the paracrine signaling of the endocrine pancreas via serological measurements of insulin and c-peptide. Non-human primates underwent MRI exams with simultaneous blood sampling during a Graded Glucose Infusion (GGI) with Gd-CP027 or with a non-zinc(II) sensitive contrast agent, gadofosveset. Contrast enhancement of the pancreas resulting from co-release of zinc(II) ion with insulin was observed focally when using the zinc(II)-specific agent, Gd-CP027, whereas little enhancement was detected when using gadofosveset. The contrast enhancement detected by Gd-CP027 increased in parallel with an increased dose of infused glucose. Serological measurements of C-peptide and insulin indicate that Gd-CP027, a high affinity zinc(II) contrast agent, potentiates their secretion only as a function of glucose stimulation. Taken in concert, this assay offers the possibility of detecting beta cell function in vivo non-invasively with MRI and underscores the role of zinc(II) in endocrine glucose metabolism.

18F]FDG-labelled stem cell PET imaging in different route of administrations and multiple animal species

Stem cell therapy holds great promise for tissue regeneration and cancer treatment, although its efficacy is still inconclusive and requires further understanding and optimization of the procedures. Non-invasive cell tracking can provide an important opportunity to monitor in vivo cell distribution in living subjects. Here, using a combination of positron emission tomography (PET) and in vitro 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) direct cell labelling, the feasibility of engrafted stem cell monitoring was tested in multiple animal species. Human mesenchymal stem cells (MSCs) were incubated with phosphate-buffered saline containing [18F]FDG for in vitro cell radiolabelling. The pre-labelled MSCs were administrated via peripheral vein in a mouse (n = 1), rats (n = 4), rabbits (n = 4) and non-human primates (n = 3), via carotid artery in rats (n = 4) and non-human primates (n = 3), and via intra-myocardial injection in rats (n = 5). PET imaging was started 10 min after cell administration using a dedicated small animal PET system for a mouse and rats. A clinical PET system was used for the imaging of rabbits and non-human primates. After MSC administration via peripheral vein, PET imaging revealed intense radiotracer signal from the lung in all tested animal species including mouse, rat, rabbit, and non-human primate, suggesting administrated MSCs were trapped in the lung tissue. Furthermore, the distribution of the PET signal significantly differed based on the route of cell administration. Administration via carotid artery showed the highest activity in the head, and intra-myocardial injection increased signal from the heart. In vitro [18F]FDG MSC pre-labelling for PET imaging is feasible and allows non-invasive visualization of initial cell distribution after different routes of cell administration in multiple animal models. Those results highlight the potential use of that imaging approach for the understanding and optimization of stem cell therapy in translational research.

Ultrasound based noninvasive real-time cell proliferation process monitoring.

The proliferation process's efficiency is related to the number of cells grown in culture and the maximum efficiency obtained at the stationary phase. Since the culture's growth speed is different for various cells and even for subgroups of the same cells, it is essential to monitor the process properly to obtain maximum efficiency. In this work, ultrasonic velocity measurement was performed noninvasively for wireless real-time monitoring of the suspension cell culture using a single integrated device to get maximum efficiency from the process by determining the phases. Using the advantage of the developed device's portability and wireless connectivity, the cells are monitored in the incubator without interfering with the actual process. Therefore, a real-time highly sampled growth curve is obtained, which was not possible to obtain with the currently used methods or the offline methods that are based on taking samples from the culture invasively. Filtering and curve fitting methods are also applied to the data to obtain a clean growth curve. The method developed as a result of this study ensures that the suspension cell culture was monitored most conveniently in the actual growth medium in real-time and noninvasively.

Evaluation of Rare Types of Breast Cancer among Indian Patients - a Retrospective Study from a Regional Cancer Center.

Breast cancer (BC) is the most common cancer among Indian women and invasive duct cell carcinoma (IDCC) the most common histology (80-90%) followed by noninvasive duct cell carcinoma (non-IDCC) subtypes (about 10%). Most of the non-IDCC are rare (<1-2%), and literature on this topic is sparse. Age is an important prognostic factor and varies with ethnicity. The aim of our study is to evaluate the incidence and age at presentation of different types of BC. This is a single institute retrospective observational study evaluating BC over the last 7 years at our institute. We evaluated the demographic and pathological features. There were a total of 2725 patients, of these 89.7% had IDCC and 10.2% had non-IDCC. There were 13 subtypes of non-IDCC, the most common being sarcoma/phyllodes (2.4%) and lobular (2.3%). The other subtypes were very rare (<1.5% each). The non-IDCC male BC was very rare (0.07%). The median age at presentation was similar for IDCC and non-IDCC types (50years). The p value for age at presentation was significant for mucinous (0.0001), phyllodes (<0.0001), and sarcoma breast (0.009) when compared with IDCC. The incidence of IDCC, non-IDCC, and rare subtypes of non-IDCC are comparable to literature except lobular (lower) and phyllodes/sarcoma (higher). Indian patients presented a decade earlier for both IDCC and non-IDCC types. Tubular and papillary presented at an age comparable to IDCC, in contrast to the West. Given these differences, the prognosis of non-IDCC needs to be evaluated in future studies on Indian BC patients.

ISOLATION AND REPROGRAMMING OF URINARY PROGENITOR CELLS FROM A PATIENT WITH MUTATION IN GENE CYFIP2 THAT CAUSES INTELLECTUAL DISABILITY

<h3>Background</h3> West syndrome (WS) is an infantile neural disorder that causes a delay in development and infantile spasms. Mutations in the cytoplasmic FMRP-interacting protein 2 (CYFIP2) gene were recently described as possible causes of WS. CYFIP2 has a role in actin polymerization through WAVE regulatory complex (WRC), especially on dendritic spines and synaptosomes. To study the molecular mechanism of mutation in CYFIP2 and to propose a possible therapy, this study aimed to establish a cell model based on induced pluripotent stem cells (iPSC), through a non-invasive cell sample collected from the patient and its subsequent reprogramming. <h3>Methods</h3> The urine sample was collected for a 2 years old Brazilian female patient characterized with the R87C variant of CYFIP2, with the parent's consent. For isolation, the sample was washed, centrifuged, and the cellular pellet was cultivated until confluence. For reprogramming, the episomal vectors expressing hSK, hOCT4, shp53, hUL, and EBNA1 were electroporated in cells by Nucleofector. Cells were cultivated until reprogrammed cell colonies appeared. Colonies were labeled with anti-TRA-1-60 and anti-TRA-1-81 antibodies on days 15 and 24 to follow up. <h3>Results</h3> Isolation and expansion of urine-derived cells lasted 21 days and based on its morphology and growth rate, cells were classified as type II urinary progenitor cells (UPC), according to the literature. After 15 days of reprogramming, pluripotent stem cells-like colonies can be visualized in culture. The TRA-1-60 and TRA-1-81 markers were even more evident in colonies on day 24, compared to day 15. On day 26 after reprogramming, colonies were manually harvested, and the cells were expanded until confluence. <h3>Conclusions</h3> The isolation and reprogramming of UPC collected from a patient with the CYFIP2 R87C variant were possible, even with the possibility of CYFIP2 mutation impaired cell growth or reprogramming. Also, the reprogrammed cells expressed two of the pluripotent markers, TRA-1-60 and TRA-1-81. The next step is the characterization of the reprogrammed cells, through expression analysis for different pluripotent markers, differentiation in the three germ layers, and karyotype. After the establishment of the iPSC as a study model for CYFIP2, cells will be differentiated into neural cells, making possible the study of the molecular mechanisms and drug screening.

Genome-wide DNA methylation and RNA expression differences correlate with invasiveness in melanoma cell lines.

Aims& objectives: The aim of this study was to investigate the role of DNA methylation in invasiveness in melanoma cells. Materials& methods: The authors carried out genome-wide transcriptome (RNA sequencing) and reduced representation bisulfite sequencing methylome profiling between noninvasive (n=4) and invasive melanoma cell lines (n=5). Results:The integration of differentially expressed genes and differentially methylated fragments (DMFs) identified 12 DMFs (two in AVPI1, one in HMG20B, two in BCL3, one in NTSR1, one in SYNJ2, one in ROBO2 and four in HORMAD2) that overlapped with either differentially expressed genes (eight DMFs and six genes) or cis-targets of lncRNAs (five DMFs associated with cis-targets and four differentially expressed lncRNAs). Conclusions:DNA methylation changes are associated with a number of transcriptional differences observed in noninvasive and invasive phenotypes in melanoma.

Advances in surface‐enhanced Raman scattering bioprobes for cancer imaging

AbstractSurface‐enhanced Raman scattering (SERS) technology with feature of high sensitivity, selective enhancement, in situ detection, nondestructive, label free, and fingerprint spectrum has been widely utilized in various practical applications, especially for bioanalysis. Cancer diagnosis and therapy based on SERS bioimaging has been reported as a promising approach in recent decades, and various types of material‐based SERS bioprobes are rapidly designed continuously. SERS bioimaging has been successfully employed in cancer screening, diagnosis, and componential analysis in both in vitro and in vivo cancer tissue samples, which simultaneously provides visual morphology and biochemical information. Precise tumor tissue excision, real‐time monitoring of cellular uptake process, and noninvasive cell tracking and labeling are realized due to the effective Raman image bioprobes, satisfying the requirements of precision medicine. Here, an overview of SERS bioprobes based on noble metals, metal oxides, and composite materials is presented by highlighting their respective, unique advantages and recent achievements in cancer imaging realm. This review also offers a distinctive perspective on designing multifunctional micro–nanoparticles with remarkable SERS activity, and proposing novel strategies for establishing high‐performance sensors and imaging bioprobes derived from the deep insight of enhancement mechanism for different types of SERS substrates.

Grainyhead-Like 3 Influences Migration and Invasion of Urothelial Carcinoma Cells.

Invasive urothelial carcinomas of the bladder (UCB) characteristically show a loss of differentiation markers. The transcription factor Grainyhead-like 3 (GRHL3) plays an important role in the development and differentiation of normal urothelium. The contribution to UCB progression is still elusive. Differential expression of GRHL3 was assessed in normal human urothelium and in non-invasive and invasive bladder cancer cell lines. The contribution of GRHL3 to cell proliferation, viability and invasion in UCB cell lines was determined by gain- and loss-of-function assays in vitro and in an organ culture model using de-epithelialized porcine bladders. GRHL3 expression was detectable in normal human urothelial cells and showed significantly higher mRNA and protein levels in well-differentiated, non-invasive RT4 urothelial carcinoma cells compared to moderately differentiated RT112 cells. GRHL3 expression was absent in anaplastic and invasive T24 cells. Ectopic de novo expression of GRHL3 in T24 cells significantly impaired their migration and invasion properties in vitro and in organ culture. Its downregulation improved the invasive capacity of RT4 cells. The results indicate that GRHL3 may play a role in progression and metastasis in UCB. In addition, this work demonstrates that de-epithelialized porcine bladder organ culture can be a useful, standardized tool to assess the invasive capacity of cancer cells.

Quantitative phase microscopy for non-invasive live cell population monitoring

We present here a label-free development based on preexisting Quantitative Phase Imaging (QPI) that allows non-invasive live monitoring of both individual cells and cell populations. Growth, death, effect of toxic compounds are quantified under visible light with a standard inverted microscope. We show that considering the global biomass of a cell population is a more robust and accurate method to assess its growth parameters in comparison to compiling individually segmented cells. This is especially true for confluent conditions. This method expands the use of light microscopy in answering biological questions concerning live cell populations even at high density. In contrast to labeling or lysis of cells this method does not alter the cells and could be useful in high-throughput screening and toxicity studies.

Improved clinical outcomes after non-invasive oocyte selection and Day 3 eSET in ICSI patients

BackgroundNon-invasive oocyte quality scoring, based on cumulus gene expression analysis, in combination with morphology scoring, can increase the clinical pregnancy (CPR) and live birth rates (LBR) in Day 3 eSET (elective single embryo transfer) ICSI patients. This was first investigated in a pilot study and is now confirmed in a large patient cohort of 633 patients. It was investigated whether CPR, LBR and time-to-pregnancy could be improved by analyzing the gene expression profile of three predictive genes in the cumulus cells, compared to patients with morphology-based embryo selection only.MethodsA large interventional, non-randomized, assessor-blinded cohort study with 633 ICSI patients was conducted in a tertiary fertility center. Non-PCOS patients, 22–39 years old, with good ovarian reserve, were stimulated with HP-hMG using a GnRH antagonist protocol and planned for fresh Day 3 eSET. The cumulus cells from individually denuded oocytes were ranked by a lab-developed cumulus cell test: qRT-PCR for three predictive genes (CAMK1D, EFNB2 and SASH1) and two control genes (UBC, B2M). The embryo selected for transfer was highest ranked from the pool of morphologically transferable Day 3 embryos. Patients in the control (n = 520) and experimental arm (n = 113) were compared for clinical pregnancy and live birth, using a weighted generalized linear model, and time-to-pregnancy using Kaplan-Meier curves.ResultsThe CPR was 61% in the experimental arm (n = 113) vs 29% in the control arm (n = 520, p < 0.0001). The LBR in the experimental arm (50%) was significantly higher than in the control arm (27%,p < 0.0001). Time-to-pregnancy was significantly shortened by 3 transfer cycles independent of the number of embryos available on Day 3 (Kaplan-Meier, p < 0.0001).Cumulus cell tested patients < 35 years (n = 65) or ≥ 35 years (n = 48) had a CPR of 62 and 60% respectively (ns). For cumulus cell tested patients with 2, 3–4, or > 4 transferable embryos, the CPR was 66, 52, and 67% (ns) respectively, and thus independent of the number of transferable embryos on Day 3.ConclusionsThis study provides further evidence of the clinical usefulness of the non-invasive cumulus cell test over time in a larger patient cohort.Trial registrationClinicaltrials.gov, NCT03659786/NCT02962466 (Registered 6Sep2018/11Nov2016, retrospectively registered.

Chemopreventive Potential of Myrtenal against Nitrosamine-Initiated, Radiation-Promoted Rat Bladder Carcinogenesis

The present study was undertaken to evaluate the chemopreventive activity of myrtenal, a natural monoterpene, against bladder carcinoma in rats induced with N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) and promoted with γ-ionizing radiation (γ-IRR) as well as to assess the involvement of inflammation, apoptosis and oxidative damage in tumor development. Histopathological examination of rat bladder revealed the presence of noninvasive papillary transitional cell carcinoma (Grade 2) in sections from BBN group indicating the credibility of the applied carcinogenesis model. Myrtenal treatment caused improvement in urinary bladder mucosa with cells more likely in Grade 1. Administration of myrtenal to BBN-treated rats exhibited downregulation in the expressions of COX-2, NF-kB and STAT-3 associated with suppression of inflammatory cytokines levels of TNF-α and IL-6 as well as biomarkers of oxidative damage (MDA & NO). In addition, myrtenal treatment caused a significant increase in caspase-3 activity and Bax/Bcl-2 ratio. Data obtained suggested that the anti-inflammatory effect and the induction of apoptosis contributed largely to the beneficial antitumor effects of myrtenal in rats with BBN/γ-IRR-induced bladder carcinoma. Present findings, in addition to benefits described in other pathologies, indicated myrtenal as a potential adjuvant natural compound for the prevention of tumor progression of bladder cancer.

Over expressions of neutrophil gelatinase-associated lipocalin and kidney injury molecule-1 in human uterine cervical neoplasms enhance tumor invasion

Objective: Accumulating evidence has demonstrated upregulation of neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1) in multiple malignancies which play a critical role in tumor progression. However, up to now little is known about the role played by NGAL in human uterine cervical carcinomas. In this retrospective study we aimed to evaluate the role of tissue expressions of NGAL and KIM-1 in a spectrum of uterine invasive and noninvasive cervical neoplasms. Methods: Immunohistochemical NGAL and KIM-1 expressions were investigated in a total of 107 formalin-fixed, paraffin-embedded uterine cervical tumor specimens and their association with different clinicopathologic parameters was evaluated. Results: In this series, cases with 30 low- and 29 high- grade cervical squamous intraepithelial lesion (SIL), 27 squamous cell carcinoma (SCC), 15 adenosquamous carcinoma (ASC) and 6 adenocarcinoma (ACs) were detected. Positive NGAL expression was detected in only inflammatory cells of cases, whereas KIM-1 expression was detected in tumor cells. Statistically it was determined that the positivity rate of strong NGAL and KIM-1 expression was prominently higher in invasive carcinomas when compared with non-invasive squamous cell neoplasms (P &lt; 0.01). KIM-1 expressions were not detected in any of the few cases with adenocarcinoma. Conclusion: Our findings have showed the presence of a correlation between membranous and cytoplasmic expressions of NGAL and KIM-1 and the invasive characteristics of uterine cervical neoplasms. As a result, expressions of NGAL and KIM-1 may be important in foreseeing the invasion and tumor progression.