Ovarian reserve markers to identify poor responders in the context of poseidon classification

It is well-known that poor ovarian reserve is a cause of infertility, poor response to gonadotrophin stimulation and poor success rate after in vitro fertilization (IVF) cycles. Some years ago a consensus was elaborated on precise criteria which can lead to a correct identification of poor responders (the Bologna criteria). More recently, the POSEIDON group has proposed a new stratified classification of patients with low prognosis, also with the aim of providing clinical indications for the management of these patients. A literature search was carried out for studies that investigated the ability of ovarian reserve markers, in particular AMH and AFC, to predict poor ovarian response in IVF cycles; secondly, studies regarding the Bologna criteria and their prognostic value were analyzed and available literature on POSEIDON classification was reported. The most recent markers of ovarian reserve (serum AMH and ultrasound AFC) have shown to provide a direct and accurate measurement of ovarian follicle pool. These markers have generally shown comparable predictive power for ovarian response and a number of retrieved oocytes in IVF cycles. \u201cAbnormal ovarian reserve test\u201d is a very important parameter both in the Bologna criteria and in the POSEIDON classification. Several studies have already been published about the reproductive outcome of patients defined as poor responders according to the ESHRE Bologna criteria: all of them agree on the poor IVF outcome and low pregnancy rate of these patients. Instead, being the POSEIDON classification of very recent publication, the efficacy of the POSEIDON approach in improving management and outcomes of POR patients has yet to be tested and validated with future prospective clinical trials. Prediction of poor response may help clinicians choose the stimulation protocol with the aim of gaining patient compliance and cost reduction, and many efforts have been made by researchers in this sense, including the formulation of the Bologna criteria and of the POSEIDON classification, in which the ovarian reserve markers (AMH and AFC) play a fundamental role

External validation of anti-Müllerian hormone based prediction of live birth in assisted conception

<p>Background - Chronological age and oocyte yield are independent determinants of live birth in assisted conception. Anti-Müllerian hormone (AMH) is strongly associated with oocyte yield after controlled ovarian stimulation. We have previously assessed the ability of AMH and age to independently predict live birth in an Italian assisted conception cohort. Herein we report the external validation of the nomogram in 822 UK first in vitro fertilization (IVF) cycles.</p> <p>Methods - Retrospective cohort consisting of 822 patients undergoing their first IVF treatment cycle at Glasgow Centre for Reproductive Medicine. Analyses were restricted to women aged between 25 and 42 years of age. All women had an AMH measured prior to commencing their first IVF cycle. The performance of the model was assessed; discrimination by the area under the receiver operator curve (ROCAUC) and model calibration by the predicted probability versus observed probability.</p> <p>Results - Live births occurred in 29.4% of the cohort. The observed and predicted outcomes showed no evidence of miscalibration (p = 0.188). The ROCAUC was 0.64 (95% CI: 0.60, 0.68), suggesting moderate and similar discrimination to the original model. The ROCAUC for a continuous model of age and AMH was 0.65 (95% CI 0.61, 0.69), suggesting that the original categories of AMH were appropriate.</p> <p>Conclusions - We confirm by external validation that AMH and age are independent predictors of live birth. Although the confidence intervals for each category are wide, our results support the assessment of AMH in larger cohorts with detailed baseline phenotyping for live birth prediction.</p&gt

Fate of Salmonella enterica Typhimurium and Listeria monocytogenes in Black Soldier Fly (Hermetia illucens) Larvae Reared on Two Artificial Diets

Ensuring food security is one of the main challenges facing the world over the next 30 years. There is, thus, an urgent need to significantly increase the supply of sustainable protein that can be transformed into animal feed. Proteins from insects offer a valuable alternative. This article presents the results of challenge tests conducted to investigate the dynamics of the microbial load of Salmonella enterica Typhimurium and Listeria monocytogenes in black soldier fly (Hermetia illucens) larvae grown on contaminated substrates. Four separate challenge tests were performed on two substrates: the Gainesville diet and a homemade diet. The challenge test procedure was carried out in accordance with ISO/DIS 20976-2 (under development). The results of this study show that, when grown on contaminated substrates, BSF larvae do not eliminate Salmonella Typhimurium or L. monocytogenes, but can reduce their microbial load. Sanitation processes downstream of the breeding of BSF larvae are, however, required to reduce the microbiological risks of this novel food

Acid microenvironment promotes cell survival of human bone sarcoma through the activation of cIAP proteins and NF-κB pathway

Extracellular acidification is a very common cause of stress in tumor microenvironment and of Darwinian pressure. In acid areas of the tumor, most cancer cells are-albeit slowly proliferating-more resistant to cell death than those in well-perfused regions. Tumor acidosis can directly regulate the expression of pro-survival proteins since a low extracellular pH activates the caspase-dependent cell death machinery. This mechanism has never been explored in bone sarcomas. We cultured osteosarcoma and Ewing sarcoma cells under low pH (pH 6.5), and we performed deep-sequencing and protein analysis. Both in in vitro and in vivo models, acidification activity enhanced tumor cells survival. However, we did not observe any change in ERK1 phosphorylation. On the contrary, both at the mRNA and protein level, we found a significant induction of TRAF adaptor proteins and of cIAP proteins (BIRC2 and/or BIRC3). As a consequence, the downstream nuclear transcription factor kappa B (NF-κB) survival pathway was increased. Furthermore, the treatment with the cIAP inhibitor LCL161 reverted the protection from apoptosis under low pH. In vitro results were confirmed both in Ewing sarcoma xenograft and in osteosarcoma patients, since the analysis of tumor tissues demonstrated that the levels of expression of TRAF1 or NF-κB1 significantly correlate with the level of expression of the vacuolar ATPase (V-ATPase), the most important proton pump in eukaryotes. Moreover, in the tissue sections of xenograft model, the nuclear translocation of RelB, a key subunit of the NF-κB transcriptional complex, localized in the tumor region that also corresponded to the acid microenvironment associated with the highest levels of expression of LAMP2 and V-ATPase, in the internal area of the tumor, as revealed by immunohistochemistry. Our data confirm that tumor acid microenvironment activates a stress-regulated switch to promote cell survival of bone sarcoma, and support the hypothesis that this mechanism is mediated by the recruitment of TRAF/cIAP complexes. Altogether, these results suggest that TRAF/cIAP can be considered as a target for anti-cancer therapies

The Influence of Cancer Stem Cells on the Risk of Relapse in Adenocarcinoma and Squamous Cell Carcinoma of the Lung: A Prospective Cohort Study.

Purpose Lung cancer relapse may be associated with the presence of a small population of cancer stem cells (CSCs) with unlimited proliferative potential. Our study assessed the relationship between CSCs and the relapse rate in patients harboring adenocarcinoma (ADL) and squamous cell carcinoma of the lung (SCCL). Experimental design This is an observational prospective cohort study (NCT04634630) assessing the influence of CSC frequency on relapse rate after major lung resection in 35 patients harboring early (I-II) (n = 21) and locally advanced (IIIA) (n = 14) ADL and SCCL. There was a 2-year enrollment period followed by a 1-year follow-up period. Surgical tumor specimens were processed, and CSCs were quantified by cytofluorimetric analysis. Results Cancer stem cells were expressed in all patients with a median of 3.1% of the primary cell culture. Primary analysis showed no influence of CSC frequency on the risk of relapse (hazard ratio [HR] = 1.05, 95% confidence interval [CI] = 0.85-1.30). At secondary analysis, patients with locally advanced disease with higher CSC frequency had an increased risk of relapse (HR = 1.26, 95% CI = 1.14-1.39), whereas this was not observed in early-stage patients (HR = 0.90, 95% CI = 0.65-1.25). Conclusion No association was found between CSC and relapse rates after major lung resection in patients harboring ACL and SCCL. However, in locally advanced-stage patients, a positive correlation was observed between CSC frequency and risk of relapse. These results indicate a need for further molecular investigations into the prognostic role of CSCs at different lung cancer stages

Challenges in Clinical Development of Mesenchymal Stromal/Stem Cells: Concise Review

Identified 50 years ago, mesenchymal stromal/stem cells (MSCs) immediately generated a substantial interest among the scientific community because of their differentiation plasticity and hematopoietic supportive function. Early investigations provided evidence of a relatively low engraftment rate and a transient benefit for challenging congenital and acquired diseases. The reasons for these poor therapeutic benefits forced the entire field to reconsider MSC mechanisms of action together with their ex vivo manipulation procedures. This phase resulted in advances in MSCs processing and the hypothesis that MSC-tissue supportive functions may be prevailing their differentiation plasticity, broadening the spectrum of MSCs therapeutic potential far beyond their lineage-restricted commitments. Consequently, an increasing number of studies have been conducted for a variety of clinical indications, revealing additional challenges and suggesting that MSCs are still lagging behind for a solid clinical translation. For this reason, our aim was to dissect the current challenges in the development of still promising cell types that, after more than half a century, still need to reach their maturity. Stem Cells Translational Medicine 2019;8:1135–1148

Targeting GD2-positive glioblastoma by chimeric antigen receptor empowered mesenchymal progenitors

Tumor targeting by genetically modified mesenchymal stromal/stem cells (MSCs) carrying anti-cancer molecules represents a promising cell-based strategy. We previously showed that the pro-apoptotic agent tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can be successfully delivered by MSCs to cancer sites. While the interaction between TRAIL and its receptors is clear, more obscure is the way in which MSCs can selectively target tumors and their antigens. Several neuroectoderm-derived neoplasms, including glioblastoma (GBM), sarcomas, and neuroblastoma, express high levels of the tumor-associated antigen GD2. We have already challenged this cell surface disialoganglioside by a chimeric antigen receptor (CAR)-T cell approach against neuroblastoma. With the intent to maximize the therapeutic profile of MSCs delivering TRAIL, we here originally developed a bi-functional strategy where TRAIL is delivered by MSCs that are also gene modified with the truncated form of the anti-GD2 CAR (GD2 tCAR) to mediate an immunoselective recognition of GD2-positive tumors. These bi-functional MSCs expressed high levels of TRAIL and GD2 tCAR associated with a robust anti-tumor activity against GD2-positive GBM cells. Most importantly, the anti-cancer action was reinforced by the enhanced targeting potential of such bi-functional cells. Collectively, our results suggest that a truncated anti-GD2 CAR might be a powerful new tool to redirect MSCs carrying TRAIL against GD2-expressing tumors. This affinity-based dual targeting holds the promise to combine site-specific and prolonged retention of MSCs in GD2-expressing tumors, thereby providing a more effective delivery of TRAIL for still incurable cancers

Inducible Caspase9-mediated suicide gene for MSC-based cancer gene therapy

Cellular therapies based on mesenchymal stromal/stem cells (MSC) are promising strategies in regenerative medicine and oncology. Despite encouraging results, there is still some level of concerns on inoculating MSC in cancer patients. To face this issue, one possibility resides in engineering MSC by incorporating a suicide gene in order to control their fate once infused. Strategies based on Herpes Simplex Virus Thymidine Kinase (HSV-TK) and the Cytosine Deaminase genes have been developed and more recently a novel suicide gene, namely, iCasp9, has been proposed. This approach is based on a variant of human Caspase9 that binds with high affinity to a synthetic, bioinert small molecule (AP20187) leading to cell death. Based on this technology so far marginally applied to MSC, we tested the suitability of iCasp9 suicide strategy in MSC to further increase their safety. MSC have been transfected by a lentiviral vector carrying iCasp9 gene and then tested for viability after AP20187 treatment in comparison with mock-transfected cells. Moreover, accounting our anti-tumor approaches based on MSC expressing potent anti-cancer ligand TNF-Related Apoptosis-Inducing Ligand (TRAIL), we generated adipose MSC co-expressing iCasp9 and TRAIL successfully targeting an aggressive sarcoma type. These data show that anti-cancer and suicide mechanisms can coexist without affecting cells performance and hampering the tumoricidal activity mediated by TRAIL. In conclusion, this study originally indicates the suitability of combining a MSC-based anti-cancer gene approach with iCasp9 demonstrating efficiency and specificity

Resistance to neoplastic transformation of ex-vivo expanded human mesenchymal stromal cells after exposure to supramaximal physical and chemical stress

The risk of malignant transformation of ex-vivo expanded human mesenchymal stromal cells (huMSCs) has been debated in the last years; however, the biosafety of these cells after exposure to supramaximal physical and chemical stress has never been systematically investigated. We established an experimental in vitro model to induce supramaximal physical (ionizing radiation, IR) and chemical (starvation) stress on ex-vivo expanded bone marrow (BM)-derived huMSCs and investigated their propensity to undergo malignant transformation. To this aim, we examined MSC morphology, proliferative capacity, immune-phenotype, differentiation potential, immunomodulatory properties and genetic profile before and after stressor exposure. Furthermore, we investigated the cellular mechanisms underlying MSC response to stress. MSCs were isolated from 20 healthy BM donors and expanded in culture medium supplemented with 5% platelet lysate (PL) up to passage 2 (P2). At this stage, MSCs were exposed first to escalating doses of IR (30, 100, 200 Gy) and then to starvation culture conditions (1% PL). With escalating doses of radiation, MSCs lost their typical spindle-shaped morphology, their growth rate markedly decreased and eventually stopped (at P4-P6) by reaching early senescence. Irradiated and starved MSCs maintained their typical immune-phenotype, ability to differentiate into adipocytes/osteoblasts and to inhibit mitogen-induced T-cell proliferation. The study of the genetic profile of irradiated/ starved MSCs did not show any alteration. While the induction of supramaximal stress triggered production of ROS and activation of DNA damage response pathway via multiple mechanisms, our data indicate that irradiated/starved MSCs, although presenting altered morphology/growth rate, do not display increased propensity for malignant transformation

Ibrutinib modifies the function of monocyte/macrophage population in chronic lymphocytic leukemia

In lymphoid organs, nurse-like cells (NLCs) show properties of tumor-associated macrophages, playing a crucial role in chronic lymphocytic leukemia (CLL) cell survival. Ibrutinib, a potent inhibitor of Bruton's tyrosine kinase (BTK), is able to counteract pro-survival signals in CLL cells. Since the effects on CLL cells have been studied in the last years, less is known about the influence of ibrutinib on NLCs properties. We sought to determine how ibrutinib modifies NLCs functions focusing on the balance between immunosuppressive and inflammatory features. Our data show that ibrutinib targets BTK expressed by NLCs modifying their phenotype and function. Treatment with ibrutinib reduces the phagocytic ability and increases the immunosuppressive profile of NLCs exacerbating the expression of M2 markers. Accordingly, ibrutinib hampers LPS-mediated signaling, decreasing STAT1 phosphorylation, while allows IL-4-mediated STAT6 phosphorylation. In addition, NLCs treated with ibrutinib are able to protect CLL cells from drug-induced apoptosis partially through the secretion of IL-10. Results from patient samples obtained prior and after 1 month of treatment with ibrutinib show an accentuation of CD206, CD11b and Tie2 in the monocytic population in the peripheral blood. Our study provides new insights into the immunomodulatory action of ibrutinib on monocyte/macrophage population in CLL