Heavy Metals in Follicular Fluid Affect the Ultrastructure of the Human Mature Cumulus-Oocyte Complex.

It is known that exposure to heavy metal such as lead (Pb) and cadmium (Cd) has several adverse effects, particularly on the human reproductive system. Pb and Cd have been associated with infertility in both men and women. In pregnant women, they have been associated with spontaneous abortion, preterm birth, and impairment of the development of the fetus. Since these heavy metals come from both natural and anthropogenic activities and their harmful effects have been observed even at low levels of exposure, exposure to them remains a public health issue, especially for the reproductive system. Given this, the present study aimed to investigate the potential reproductive effects of Pb and Cd levels in the follicular fluid (FF) of infertile women and non-smokers exposed to heavy metals for professional reasons or as a result of living in rural areas near landfills and waste disposal areas in order to correlate the intrafollicular presence of these metals with possible alterations in the ultrastructure of human cumulus-oocyte complexes (COCs), which are probably responsible for infertility. Blood and FF metals were measured using atomic absorption spectrometry. COCs corresponding to each FF analyzed were subjected to ultrastructural analyses using transmission electron microscopy. We demonstrated for the first time that intrafollicular levels of Pb (0.66 µg/dL-0.85 µg/dL) and Cd (0.26 µg/L-0.41 µg/L) could be associated with morphological alterations of both the oocyte and cumulus cells' (CCs) ultrastructure. Since blood Cd levels (0.54 µg/L-1.87 µg/L) were above the current reference values established by the guidelines of the Agency for Toxic Substances and Disease Registry (ATSDR) and the Environmental Protection Agency (EPA) (0.4 µg/L), whereas blood Pb levels (1.28 µg/dL-3.98 µg/dL) were below the ATSDR reference values (≤5 µg/dL), we believe that these alterations could be due especially to Cd, even if we cannot exclude a possible additional effect of Pb. Our results highlighted that oocytes were affected in maturation and quality, whereas CCs showed scarcely active steroidogenic elements. Regressing CCs, with cytoplasmic alterations, were also numerous. According to Cd's endocrine-disrupting activity, the poor steroidogenic activity of CCs might correlate with delayed oocyte cytoplasmic maturation. So, we conclude that levels of heavy metals in the blood and the FF might negatively affect fertilization, embryo development, and pregnancy, compromising oocyte competence in fertilization both directly and indirectly, impairing CC steroidogenic activity, and inducing CC apoptosis.

Petroclival Clinoidal Folds and Arachnoidal Membranes of the Anteromedial Incisural Space: Clinical Anatomy for Neuro Critical Care.

A systematic and narrative literature review was performed, focusing attention on the anatomy of the area located at the junction of the sphenoid and the basal portion of the temporal bone (petrous bone, petrous apex, upper petro-clival region) encircled by the free edge of the tentorium, the insertion of the tentorium itself to the petrous apex and the anterior and posterior clinoid processes that give rise to three distinct dural folds or ligaments: the anterior petroclinoid ligament, the posterior petroclinoid ligament and the interclinoid ligament. These dural folds constitute the posterior portion of the roof of the cavernous sinus denominated "the oculomotor triangle". The main purpose of this review study was to describe this anatomical region, particularly in the light of the relationships between the anterior margin of the free edge of the tentorium and the above-mentioned components of the sphenoid and petrous bone.

MST1 mediates doxorubicin-induced cardiomyopathy by SIRT3 downregulation.

Heart failure is a major side effect of doxorubicin (DOX) treatment in patients with cancer. However, the mechanisms underlying the development of DOX-induced heart failure need to be addressed. This study aims to test whether the serine/threonine kinase MST1, a major Hippo pathway component, contributes to the development of DOX-induced myocardial injury. C57BL/6J WT mice and mice with cardiomyocyte-specific dominant-negative MST1 (kinase-dead) overexpression received three weekly injections of DOX, reaching a final cumulative dose of 18 mg/kg. Echocardiographic, histological and biochemical analyses were performed six weeks after the first DOX administration. The effects of MST1 inhibition on DOX-induced cardiomyocyte injury were also tested in vitro. MST1 signaling was significantly activated in cardiomyocytes in response to DOX treatment in vitro and in vivo. Wild-type (WT) mice treated with DOX developed cardiac dysfunction and mitochondrial abnormalities. However, these detrimental effects were abolished in mice with cardiomyocyte-specific overexpression of dominant-negative MST1 (DN-MST1) or treated with XMU-MP-1, a specific MST1 inhibitor, indicating that MST1 inhibition attenuates DOX-induced cardiac dysfunction. DOX treatment led to a significant downregulation of cardiac levels of SIRT3, a deacetylase involved in mitochondrial protection, in WT mice, which was rescued by MST1 inhibition. Pharmacological inhibition of SIRT3 blunted the protective effects of MST1 inhibition, indicating that SIRT3 downregulation mediates the cytotoxic effects of MST1 activation in response to DOX treatment. Finally, we found a significant upregulation of MST1 and downregulation of SIRT3 levels in human myocardial tissue of cancer patients treated with DOX. In summary, MST1 contributes to DOX-induced cardiomyopathy through SIRT3 downregulation.

Retinoic acid and proteotoxic stress induce AML cell death overcoming stromal cell protection.

BACKGROUND: Acute myeloid leukemia (AML) patients bearing the ITD mutation in the tyrosine kinase receptor FLT3 (FLT3-ITD) present a poor prognosis and a high risk of relapse. FLT3-ITD is retained in the endoplasmic reticulum (ER) and generates intrinsic proteotoxic stress. We devised a strategy based on proteotoxic stress, generated by the combination of low doses of the differentiating agent retinoic acid (R), the proteasome inhibitor bortezomib (B), and the oxidative stress inducer arsenic trioxide (A). METHODS: We treated FLT3-ITD+ AML cells with low doses of the aforementioned drugs, used alone or in combinations and we investigated the induction of ER and oxidative stress. We then performed the same experiments in an in vitro co-culture system of FLT3-ITD+ AML cells and bone marrow stromal cells (BMSCs) to assess the protective role of the niche on AML blasts. Eventually, we tested the combination of drugs in an orthotopic murine model of human AML. RESULTS: The combination RBA exerts strong cytotoxic activity on FLT3-ITD+ AML cell lines and primary blasts isolated from patients, due to ER homeostasis imbalance and generation of oxidative stress. AML cells become completely resistant to the combination RBA when treated in co-culture with BMSCs. Nonetheless, we could overcome such protective effects by using high doses of ascorbic acid (Vitamin C) as an adjuvant. Importantly, the combination RBA plus ascorbic acid significantly prolongs the life span of a murine model of human FLT3-ITD+ AML without toxic effects. Furthermore, we show for the first time that the cross-talk between AML and BMSCs upon treatment involves disruption of the actin cytoskeleton and the actin cap, increased thickness of the nuclei, and relocalization of the transcriptional co-regulator YAP in the cytosol of the BMSCs. CONCLUSIONS: Our findings strengthen our previous work indicating induction of proteotoxic stress as a possible strategy in FLT3-ITD+ AML therapy and open to the possibility of identifying new therapeutic targets in the crosstalk between AML and BMSCs, involving mechanotransduction and YAP signaling.

Effects of Simulated Microgravity In Vitro on Human Metaphase II Oocytes: An Electron Microscopy-Based Study.

The Gravity Force to which living beings are subjected on Earth rules the functionality of most biological processes in many tissues. It has been reported that a situation of Microgravity (such as that occurring in space) causes negative effects on living beings. Astronauts returning from space shuttle missions or from the International Space Station have been diagnosed with various health problems, such as bone demineralization, muscle atrophy, cardiovascular deconditioning, and vestibular and sensory imbalance, including impaired visual acuity, altered metabolic and nutritional status, and immune system dysregulation. Microgravity has profound effects also on reproductive functions. Female astronauts, in fact, suppress their cycles during space travels, and effects at the cellular level in the early embryo development and on female gamete maturation have also been observed. The opportunities to use space flights to study the effects of gravity variations are limited because of the high costs and lack of repeatability of the experiments. For these reasons, the use of microgravity simulators for studying, at the cellular level, the effects, such as those, obtained during/after a spatial trip, are developed to confirm that these models can be used in the study of body responses under conditions different from those found in a unitary Gravity environment (1 g). In view of this, this study aimed to investigate in vitro the effects of simulated microgravity on the ultrastructural features of human metaphase II oocytes using a Random Positioning Machine (RPM). We demonstrated for the first time, by Transmission Electron Microscopy analysis, that microgravity might compromise oocyte quality by affecting not only the localization of mitochondria and cortical granules due to a possible alteration of the cytoskeleton but also the function of mitochondria and endoplasmic reticulum since in RPM oocytes we observed a switch in the morphology of smooth endoplasmic reticulum (SER) and associated mitochondria from mitochondria-SER aggregates to mitochondria-vesicle complexes. We concluded that microgravity might negatively affect oocyte quality by interfering in vitro with the normal sequence of morphodynamic events essential for acquiring and maintaining a proper competence to fertilization in human oocytes.

Sorcin promotes migration in cancer and regulates the EGF-dependent EGFR signaling pathways.

The epidermal growth factor receptor (EGFR) is one of the main tumor drivers and is an important therapeutic target for many cancers. Calcium is important in EGFR signaling pathways. Sorcin is one of the most important calcium sensor proteins, overexpressed in many tumors, that promotes cell proliferation, migration, invasion, epithelial-to-mesenchymal transition, malignant progression and resistance to chemotherapeutic drugs. The present work elucidates a functional mechanism that links calcium homeostasis to EGFR signaling in cancer. Sorcin and EGFR expression are significantly correlated and associated with reduced overall survival in cancer patients. Mechanistically, Sorcin directly binds EGFR protein in a calcium-dependent fashion and regulates calcium (dys)homeostasis linked to EGF-dependent EGFR signaling. Moreover, Sorcin controls EGFR proteostasis and signaling and increases its phosphorylation, leading to increased EGF-dependent migration and invasion. Of note, silencing of Sorcin cooperates with EGFR inhibitors in the regulation of migration, highlighting calcium signaling pathway as an exploitable target to enhance the effectiveness of EGFR-targeting therapies.

A Different Exosome Secretion Pattern Characterizes Patient-Derived Colorectal Cancer Multicellular Spheroids and Their Mouse Xenografts.

Up-to-date in vitro and in vivo preclinical models expressing the patient-specific cancer lineage responsible for CRC and its metastatic behavior and responsiveness to therapy are needed. Exosomes' role in tumorigenesis and the metastatic process was demonstrated, and the material content and size of the exosomes are associated with a poor prognosis of CRC. Exosomes are generally imagined after their recovery from blood serum as isolated entities, and our work aims to investigate them "in situ" in their native environment by scanning and transmission electron microscopy to understand their secretion modalities. We studied CRC stem cells in patient-derived multicellular tumor spheroids (MTSs) and in their mouse xenograft to find possible differences in terms of exosome amount, size, and secretion site between in vitro and in vivo models. We observed that MTSs' exosome secretion patterns depend on their structural complexity: few-layer MTSs show a lesser exosome secretion, limited to the apical domain of cancer cells, secretion increases in multilayered MTSs, and it develops from apical and basolateral cancer cells domains. In xenograft models, exosome secretion occurs from all cancer cell domains, and it is quantitatively greater than that observed in MTSs. This difference in exosome secretion pattern between MTSs and xenografts may be due to the influence of surrounding non-tumor cells.

Biomimetic Keratin-Coated Gold Nanoparticles for Photo-Thermal Therapy in a 3D Bioprinted Glioblastoma Tumor Model.

Before entering human clinical studies to evaluate their safety and effectiveness, new drugs and novel medical treatments are subject to extensive animal testing that are expensive and time-consuming. By contrast, advanced technologies enable the development of animal-free models that allow the efficacy of innovative therapies to be studied without sacrificing animals, while providing helpful information and details. We report on the powerful combination of 3D bioprinting (3DB) and photo-thermal therapy (PTT) applications. To this end, we realize a 3DB construct consisting of glioblastoma U87-MG cells in a 3D geometry, incorporating biomimetic keratin-coated gold nanoparticles (Ker-AuNPs) as a photo-thermal agent. The resulting plasmonic 3DB structures exhibit a homogeneous cell distribution throughout the entire volume while promoting the localization of Ker-AuNPs within the cells. A 3D immunofluorescence assay and transmission electron microscopy (TEM) confirm the uniform distribution of fluorescent-labeled Ker-AuNPs in the volume and their capability to enter the cells. Laser-assisted (λ = 532 nm) PTT experiments demonstrate the extraordinary ability of Ker-AuNPs to generate heating, producing the highest temperature rise of about 16 °C in less than 2 min.

Green Synthesis of Silver Nanoparticles Using Spent Coffee Ground Extracts: Process Modelling and Optimization.

Large amounts of spent coffee grounds (SCGs) are produced annually worldwide. SCGs contain high levels of phenolics and other bioactive compounds that make them a potential source of reducing and stabilizing agents for the synthesis of metal nanoparticles. This study investigates the use of SCG extracts as a green strategy to produce silver nanoparticles (AgNPs). SCG extracts were obtained using aqueous ethanol as the solvent and then contacted with a silver nitrate solution under the selected conditions. A central composite design coupled with response surface methodology was used to evaluate the effects of solvent composition (C = 30-70% v/v), silver-to-phenolic ratio (R = 3-7 mol/mol), temperature (T = 25-55 °C) and pH (10-12) on the production of AgNPs. Characterization of AgNPs by DLS, TEM and XRD techniques showed that they were highly crystalline with a narrow size distribution. Under optimal reaction conditions, AgNPs with an average size of about 10 nm and a zeta potential of -30.5 to -20.7 mV were obtained. Overall, the results of this study indicate that SCGs are a promising material for the green synthesis of small-sized and stable AgNPs.

Human platelet lysate-derived extracellular vesicles enhance angiogenesis through miR-126.

OBJECTIVES: Extracellular vesicles (EVs) are key biological mediators of several physiological functions within the cell microenvironment. Platelets are the most abundant source of EVs in the blood. Similarly, platelet lysate (PL), the best platelet derivative and angiogenic performer for regenerative purposes, is enriched of EVs, but their role is still too poorly discovered to be suitably exploited. Here, we explored the contribution of the EVs in PL, by investigating the angiogenic features extrapolated from that possessed by PL. METHODS: We tested angiogenic ability and molecular cargo in 3D bioprinted models and by RNA sequencing analysis of PL-derived EVs. RESULTS: A subset of small vesicles is highly represented in PL. The EVs do not retain aggregation ability, preserving a low redox state in human umbilical vein endothelial cells (HUVECs) and increasing the angiogenic tubularly-like structures in 3D endothelial bioprinted constructs. EVs resembled the miRNome profile of PL, mainly enriched with small RNAs and a high amount of miR-126, the most abundant angiogenic miRNA in platelets. The transfer of miR-126 by EVs in HUVEC after the in vitro inhibition of the endogenous form, restored angiogenesis, without involving VEGF as a downstream target in this system. CONCLUSION: PL is a biological source of available EVs with angiogenic effects involving a miRNAs-based cargo. These properties can be exploited for targeted molecular/biological manipulation of PL, by potentially developing a product exclusively manufactured of EVs.

New Insights into Intestinal Permeability in Irritable Bowel Syndrome-Like Disorders: Histological and Ultrastructural Findings of Duodenal Biopsies.

BACKGROUND AND AIM: Diarrhea, abdominal pain, and bloating are frequent in irritable bowel syndrome (IBS)-like disorders, although little is known about their intestinal ultrastructural alterations. The aim of the present study was to study duodenal biopsies from IBS-like patients to find ultrastructural alterations. MATERIALS AND METHODS: Study design: descriptive comparative pilot study. Thirty outpatients (9 male and 21 female; median age 37.7 years; range, 20 to 65 years) complaining of IBS-like symptoms were enrolled between January 2015 to May 2019 and were divided into 6 groups, each equally consisting of 5 patients: (A) untreated celiac disease (uCD); (B) treated celiac disease (tCD); (C) wheat allergy (WA); (D) Non-celiac gluten sensitivity (NCGS); (E) Nickel allergic contact mucositis (Ni ACM); (F) controls affected by GERD. Transmission electron microscopy (TEM) morphological characteristics were: microvilli length, intermicrovillar distance, junctional complexes (JC) gap width, autophagic bodies, apoptosis, altered mitochondria, lipid/chylomicron droplets, and mast cells. Regarding JC, we focused on tight junctions (TJ), adherens junctions (AJ), and desmosomes. RESULTS: Major alterations in microvilli length and intermicrovillar distance have been observed in the subjects affected by uCD. Microvilli of tCD patients showed marked recovery after adequate GFD, although not comparable to controls. Intermediate microvillar alterations were instead observed in NCGS and Ni ACM, while characteristics of WA subjects appeared more similar to tCD. Regarding JC, TJ did not show significant differences between all groups studied, including controls. The AJ were significantly more dilated in all groups compared to controls, while no significant differences were found between the pathological groups. The distance between desmosomes was greater in uCD, NCGS, and Ni ACM than in tCD, WA, and controls. Finally, intracellular alterations have been detected in most of the groups studied although they seemed more unspecific. CONCLUSIONS: TEM analysis confirmed damages to the intestinal barrier and defense mechanisms by enterocytes in IBS-like patients, probably linked to low-grade inflammation or adverse reactions triggered by food allergens, heavy metals, or other unknown. On the other hand, our study needs confirmation and further investigations with larger populations to facilitate diagnosis, therapy, and prevention of IBS-like disorders in the future.

Intramedullary Spinal Cord Metastasis Mimicking Astrocytoma: A Rare Case Report.

Intramedullary spinal cord metastases (ISCMs) are infrequent lesions. Their incidence is estimated to range from 0.9 to 2.1%, found in autopsies of cancer patients. However, as the life expectancy of malignant tumour patients constantly increases, the reported incidences of ISCMs are consequently rising. This report presents a case of the misdiagnosis of an anaplastic astrocytoma type of tumour due to its similarities to small-cell neuroendocrine carcinoma. Therefore, we would like to underline the importance of further investigation that could assist and support the surgeon in the making of the differential diagnosis. We present the clinical case of a 73-year-old woman with a solitary intramedullary spinal cord metastasis as the initial manifestation of a carcinoid type of tumour. The patient was admitted to our department while presenting a rapid onset of paraparesis. Magnetic resonance imaging was performed, which showed an intramedullary mass at the C2-C6 vertebral level with a heterogeneous contrast enhancement. In light of these findings, the patient underwent surgery for a partial tumour resection. The lesion resulted in being a small-cell neuroendocrine type of carcinoma. This peculiar type of tumour presents similar radiological characteristics to the anaplastic astrocytoma type, which is why our diagnostical mismatch occurred. This is the report of a rare case of solitary intramedullary spinal cord metastasis, which is the result of an initial presentation of a lung small-cell neuroendocrine type of carcinoma. We conclude that ISCMs should be regularly considered as a part of the differential diagnosis of intramedullary lesions, especially in the case of a rapid onset and deterioration of neurological symptoms.

The Ultrastructural Analysis of Human Colorectal Cancer Stem Cell-Derived Spheroids and Their Mouse Xenograft Shows That the Same Cells Types Have Different Ratios.

Spheroids from primary colorectal cancer cells and their mice xenografts have emerged as useful preclinical models for cancer research as they replicate tumor features more faithfully as compared to cell lines. While 3D models provide a reliable system for drug discovery and testing, their structural complexity represents a challenge and their structure-function relationships are only partly understood. Here, we present a comparative ultrastructural and flow citometric analysis of patient colorectal cancer-derived spheroids and their mice xenografts. Ultrastructural observations highlighted that multicellular spheroids and their xenografts contain the same cancer cell types but with different ratios, specifically multicellular spheroids were enriched in cells with a stem-like phenotype, while xenografts had an increased amount of lipid droplets-containing cells. The flow cytometric analysis for stem cell marker and activity showed enrichment of stem-like cells presence and activity in spheroids while xenografts had the inverse response. Our results evidence the effects on cancer cells of different in vitro and in vivo microenvironments. Those differences have to be paid into account in designing innovative experimental models for personalized drug testing.

Biomimetic keratin gold nanoparticle-mediated in vitro photothermal therapy on glioblastoma multiforme.

Aim: To realize and characterize a new generation of keratin-coated gold nanoparticles (Ker-AuNPs) as highly efficient photosensitive nanosized therapeutics for plasmonic photothermal (PPT) therapy. Materials & methods: The chemical, physical, morphological and photothermal properties of Ker-AuNPs are investigated using dynamic light scattering, ζ-potential, UV-Visible, Fourier transform infrared spectroscopy, x-ray photoelectron spectroscopy, transmission electron microscopy and high-resolution thermography. In vitro experiments are performed on a human glioblastoma cell line (i.e., U87-MG), using viability assays, transmission electron microscopy, fluorescence microscopy, cytometric analyses and PPT experiments. Results: Experiments confirm the excellent biocompatibility of Ker-AuNPs, their efficient cellular uptake and localized photothermal heating capabilities. Conclusion: The reported structural and functional properties pointed out these Ker-AuNPs as a promising new tool in the field of biocompatible photothermal agents for PPT treatments against cancer-related diseases.

Does in vitro application of pentoxifylline have beneficial effects in assisted male reproduction?

Application of nonspecific phosphodiesterases inhibitors, such as pentoxifylline (PTX), is a strategy utilised to aid sperm selection from immotile sperm samples prior to ICSI. No extensive studies have yet been performed to verify the safety of the clinical outcomes of ICSI after PTX administration. In this article, we summarise the data reported in the literature that assess the implication of in vitro usage of PTX on sperm parameters, as well as clinical outcomes during assisted male reproduction programme.

LINC00174 is a novel prognostic factor in thymic epithelial tumors involved in cell migration and lipid metabolism.

Long non-coding RNAs are emerging as new molecular players involved in many biological processes, such as proliferation, apoptosis, cell cycle, migration, and differentiation. Their aberrant expression has been reported in variety of diseases. The aim of this study is the identification and functional characterization of clinically relevant lncRNAs responsible for the inhibition of miR-145-5p, a key tumor suppressor in thymic epithelial tumors (TETs). Starting from gene expression analysis by microarray in a cohort of fresh frozen thymic tumors and normal tissues, we identified LINC00174 as upregulated in TET. Interestingly, LINC00174 expression is positively correlated with a 5-genes signature in TETs. Survival analyses, performed on the TCGA dataset, showed that LINC00174 and its associated 5-genes signature are prognostic in TETs. Specifically, we show that LINC00174 favors the expression of SYBU, FEM1B, and SCD5 genes by sponging miR-145-5p, a well-known tumor suppressor microRNA downregulated in a variety of tumors, included TETs. Functionally, LINC00174 impacts on cell migration and lipid metabolism. Specifically, SCD5, one of the LINC00174-associated genes, is implicated in the control of lipid metabolism and promotes thymic cancer cells migration. Our study highlights that LINC00174 and its associated gene signature are relevant prognostic indicators in TETs. Of note, we here show that a key controller of lipid metabolism, SCD5, augments the migration ability of TET cells, creating a link between lipids and motility, and highlighting these pathways as relevant targets for the development of novel therapeutic approaches for TET.

Histomorphology and immunohistochemical patterns in degenerative disc disease and clinical-radiological correlations: a prospective study.

PURPOSE: Degenerative disc disease (DDD) is a common condition causing low-back pain, disability and, eventually, neurological symptoms. This investigation aimed to investigate intervertebral disc DDD-related changes, evaluating histomorphology and cytokines secretion, and their clinical-radiological correlations. METHODS: This is a monocentric prospective observational study. A cohort of patients who underwent microdiscectomy for DDD, from June 2018 to January 2019, were enrolled. Discs samples were examined for histomorphology, chondrons count, immunohistochemistry for Hif-1α, Nf200 and Egr-1. Demographical and clinical data were also collected. RESULTS: Twenty patients were finally included. MRI evaluation showed a Modic I alteration in nine patients and a Modic II in 11. The disability grade was low-moderate (ODI score was ≤ 40%) in eight patients and high (ODI score > 40%) in 12. The Modic I was associated with a low-moderate disability in two (22%) patients and to a high disability in seven (88%) (p < 0.01). In Modic I group and in ODI > 40% groups, there were a significative higher mean disability grade 48.4 (± 8.3)%, number of chondrons per section, cells per chondron, Nf200+ nerve fibers and Hif-1α expression, compared with Modic II and ODI ≤ 40% groups, respectively. There were no differences in terms of Egr-1 expression. CONCLUSIONS: The discs with Modic I MRI signal could represent potential targets for medical treatments, whereas Modic II seems to be a more likely point of no return in a degenerative process. Therefore, further investigations are to better investigate inflammatory pathways and degenerative mechanisms in DDD.

Type of protein supplement in cryopreservation solutions impacts on the degree of ultrastructural damage in frozen-thawed human oocytes.

Protein sources used as supplements of IVF culture media are known to have several implications for the function and stability of embryo culture environment. In fact, they i) transport biologically active molecules ii) chelate heavy metals, iii) regulate media pH, iii) scavenge reactive oxygen species (ROS) and iv) attenuate osmotic stress to which cells are exposed in sub-optimal culture conditions. Instead, their specific relevance to the formulation of cryopreservation solutions used for gamete and embryo cryopreservation remains uncertain. In the present work, we tested the hypothesis that different protein supplements present in cryopreservation solutions, serum or plasma protein solution (PPS), or different concentrations of the same supplement (serum), are associated with different types and/or magnitude of cryopreservation-derived cell damage. To this end, using cryopreservation solutions containing serum or PPS, donated supernumerary human mature oocytes were frozen-thawed by slow freezing and compared with fresh controls. Ultrastructural markers of oocyte quality were adopted as objective measure to assess possible damage from cryopreservation. The study results indicate that the adoption of serum minimises cell damage induced by cryopreservation. Indeed, typical hallmarks of cryodamage in human oocytes, i.e. loss of cortical granules, zona pellucida hardening and above all vacuolization, were largely reduced in oocytes cryopreserved with solutions containing serum, especially if used a higher concentration. This suggest that oocyte cryopreservation still has significant margins of improvement that may derive also from composition of cryopreservation media.

SEM BSE 3D Image Analysis of Human Incus Bone Affected by Cholesteatoma Ascribes to Osteoclasts the Bone Erosion and VpSEM dEDX Analysis Reveals New Bone Formation.

Bone erosion is considered a typical characteristic of advanced or complicated cholesteatoma (CHO), although it is still a matter of debate if bone erosion is due to osteoclast action, being the specific literature controversial. The purpose of this study was to apply a novel scanning characterization approach, the BSE 3D image analysis, to study the pathological erosion on the surface of human incus bone involved by CHO, in order to definitely assess the eventual osteoclastic resorptive action. To do this, a comparison of BSE 3D image of resorption lacunae (resorption pits) from osteoporotic human femur neck (indubitably of osteoclastic origin) with that of the incus was performed. Surface parameters (area, mean depth, and volume) were calculated by the software Hitachi MountainsMap© from BSE 3D-reconstructed images; results were then statistically analyzed by SPSS statistical software. Our findings showed that no significant differences exist between the two groups. This quantitative approach implements the morphological characterization, allowing us to state that surface erosion of the incus is due to osteoclast action. Moreover, our observation and processing image workflow are the first in the literature showing the presence not only of bone erosion but also of matrix vesicles releasing their content on collagen bundles and self-immuring osteocytes, all markers of new bone formation on incus bone surface. On the basis of recent literature, it has been hypothesized that inflammatory environment induced by CHO may trigger the osteoclast activity, eliciting bone erosion. The observed new bone formation probably takes place at a slower rate in respect to the normal bone turnover, and the process is uncoupled (as recently demonstrated for several inflammatory diseases that promote bone loss) thus resulting in an overall bone loss. Novel scanning characterization approaches used in this study allowed for the first time the 3D imaging of incus bone erosion and its quantitative measurement, opening a new era of quantitative SEM morphology.

Age related changes seen in human cornea in formalin fixed sections and on biomicroscopy in living subjects: A comparison.

The purpose of our experimental research was to assess the effects of aging on the main corneal structures in healthy corneas. Small, human cornea samples were collected from 20 Caucasian subjects during surgery for traumatic lesions to the eye. Ten subjects were adults (mean age 28 years) and 10 were elderly (mean age 76 years). Morphological analysis was carried out using light microscopy and electron microscopy. Another 40 patients (20 young: mean age < 30 years; 20 elderly: mean age > 70 years) were studied in vivo by confocal microscopy. The resulting images were analyzed qualitatively, quantitatively, and statistically. The basic light microscope revealed a decrease in endothelial cell density with age accompanied by an increase in endothelial cell size. Transmission electron microscopy revealed a corneal thinning and a decrease in the number of corneal stromal cells. A marked decrease in stromal nerve fibers was observed in the older subjects compared to the younger ones. Variable pressure scanning electron microscopy (VP-SEM) was used to make surface morphological observations and to determine the chemical composition of in vivo hydrated human corneas. Our results showed the effects of aging on normal corneal morphology highlighting the structural diversity of the corneal layers and revealing an age-related reduction in nerve fibers, thus explaining the decreased corneal sensitivity that may be observed in the elderly. Clin. Anat. 33:245-256, 2020. © 2019 Wiley Periodicals, Inc.