Proangiogenic properties of complement protein C1q can contribute to endometriosis.

Endometriosis (EM) is defined as the engraftment and proliferation of functional endometrial-like tissue outside the uterine cavity, leading to a chronic inflammatory condition. While the precise etiology of EM remains elusive, recent studies have highlighted the crucial involvement of a dysregulated immune system. The complement system is one of the predominantly altered immune pathways in EM. Owing to its involvement in the process of angiogenesis, here, we have examined the possible role of the first recognition molecule of the complement classical pathway, C1q. C1q plays seminal roles in several physiological and pathological processes independent of complement activation, including tumor growth, placentation, wound healing, and angiogenesis. Gene expression analysis using the publicly available data revealed that C1q is expressed at higher levels in EM lesions compared to their healthy counterparts. Immunohistochemical analysis confirmed the presence of C1q protein, being localized around the blood vessels in the EM lesions. CD68+ macrophages are the likely producer of C1q in the EM lesions since cultured EM cells did not produce C1q in vitro. To explore the underlying reasons for increased C1q expression in EM, we focused on its established pro-angiogenic role. Employing various angiogenesis assays on primary endothelial endometriotic cells, such as migration, proliferation, and tube formation assays, we observed a robust proangiogenic effect induced by C1q on endothelial cells in the context of EM. C1q promoted angiogenesis in endothelial cells isolated from EM lesions (as well as healthy ovary that is also rich in C1q). Interestingly, endothelial cells from EM lesions seem to overexpress the receptor for the globular heads of C1q (gC1qR), a putative C1q receptor. Experiments with siRNA to silence gC1qR resulted in diminished capacity of C1q to perform its angiogenic functions, suggesting that C1q is likely to engage gC1qR in the pathophysiology of EM. gC1qR can be a potential therapeutic target in EM patients that will disrupt C1q-mediated proangiogenic activities in EM.

Morphological and lipid metabolism alterations in macrophages exposed to model environmental nanoplastics traced by high-resolution synchrotron techniques.

The release of nanoplastics (NPs) in the environment is a significant health concern for long-term exposed humans. Although their usage has certainly revolutionized several application fields, at nanometer size, NPs can easily interact at the cellular level, resulting in potential harmful effects. Micro/Nanoplastics (M/NPs) have a demonstrated impact on mammalian endocrine components, such as the thyroid, adrenal gland, testes, and ovaries, while more investigations on prenatal and postnatal exposure are urgently required. The number of literature studies on the NPs' presence in biological samples is increasing. However, only a few offer a close study on the model environmental NP-immune system interaction exploited by advanced microscopy techniques. The present study highlights substantial morphological and lipid metabolism alterations in human M1 macrophages exposed to labeled polypropylene and polyvinyl chloride nanoparticles (PP and PVC NPs) (20 µg/ml). The results are interpreted by advanced microscopy techniques combined with standard laboratory tests and fluorescence microscopy. We report the accurate detection of polymeric nanoparticles doped with cadmium selenide quantum dots (CdSe-QDs NPs) by following the Se (L line) X-ray fluorescence emission peak at higher sub-cellular resolution, compared to the supportive light fluorescence microscopy. In addition, scanning transmission X-ray microscopy (STXM) imaging successfully revealed morphological changes in NP-exposed macrophages, providing input for Fourier transform infrared (FTIR) spectroscopy analyses, which underlined the chemical modifications in macromolecular components, specifically in lipid response. The present evidence was confirmed by quantifying the lipid droplet (LD) contents in PP and PVC NPs-exposed macrophages (0-100 µg/ml) by Oil Red O staining. Hence, even at experimental NPs' concentrations and incubation time, they do not significantly affect cell viability; they cause an evident lipid metabolism impairment, a hallmark of phagocytosis and oxidative stress.

Upside-Down Preference in the Forskolin-Induced In Vitro Differentiation of 50B11 Sensory Neurons: A Morphological Investigation by Label-Free Non-Linear Microscopy.

In this study, we revealed a peculiar morphological feature of 50B11 nociceptive sensory neurons in in vitro culture related to the forskolin-induced differentiation of these cells growing upside-down on cover glass supports. Multi-photon non-linear microscopy was applied to monitor increased neurite arborization and elongation. Under live and unstained conditions, second harmonic generation (SHG) microscopy could monitor microtubule organization inside the cells while also correlating with the detection of cellular multi-photon autofluorescence, probably derived from mitochondria metabolites. Although the differentiated cells of each compartment did not differ significantly in tubulin or multi-photon autofluorescence contents, the upturned neurons were more elongated, presenting a higher length/width cellular ratio and longer neurites, indicative of differentiated cells. SHG originating from the axons' microtubules represented a proper tool to study neurons' inverted culture in live conditions without exogenous staining. This work represents the first instance of examining neuronal cell lines growing and differentiated in an upside-down orientation, allowing a possible improvement of 50B11 as a model in physiology studies of sensory neurons in peripheric nervous system disease (e.g., Fabry disease, Friedreich ataxia, Charcot-Marie-Tooth, porphyria, type 1 diabetes, Guillain-Barré syndrome in children) and analgesic drug screening.

Impact of Sample Preparation Methods on Single-Cell X-ray Microscopy and Light Elemental Analysis Evaluated by Combined Low Energy X-ray Fluorescence, STXM and AFM.

BACKGROUND: Although X-ray fluorescence microscopy is becoming a widely used technique for single-cell analysis, sample preparation for this microscopy remains one of the main challenges in obtaining optimal conditions for the measurements in the X-ray regime. The information available to researchers on sample treatment is inadequate and unclear, sometimes leading to wasted time and jeopardizing the experiment's success. Many cell fixation methods have been described, but none of them have been systematically tested and declared the most suitable for synchrotron X-ray microscopy. METHODS: The HEC-1-A endometrial cells, human spermatozoa, and human embryonic kidney (HEK-293) cells were fixed with organic solvents and cross-linking methods: 70% ethanol, 3.7%, and 2% paraformaldehyde; in addition, HEK-293 cells were subjected to methanol/ C3H6O treatment and cryofixation. Fixation methods were compared by coupling low-energy X-ray fluorescence with scanning transmission X-ray microscopy and atomic force microscopy. RESULTS: Organic solvents lead to greater dehydration of cells, which has the most significant effect on the distribution and depletion of diffusion elements. Paraformaldehyde provides robust and reproducible data. Finally, the cryofixed cells provide the best morphology and element content results. CONCLUSION: Although cryofixation seems to be the most appropriate method as it allows for keeping cells closer to physiological conditions, it has some technical limitations. Paraformaldehyde, when used at the average concentration of 3.7%, is also an excellent alternative for X-ray microscopy.

Morphological and Chemical Investigation of Ovarian Structures in a Bovine Model by Contrast-Enhanced X-ray Imaging and Microscopy.

An improved understanding of an ovary's structures is highly desirable to support advances in folliculogenesis knowledge and reproductive medicine, with particular attention to fertility preservation options for prepubertal girls with malignant tumors. Although currently the golden standard for structural analysis is provided by combining histological sections, staining, and visible 2D microscopic inspection, synchrotron radiation phase-contrast microtomography is becoming a new challenge for three-dimensional studies at micrometric resolution. To this aim, the proper use of contrast agents can improve the visualization of internal structures in ovary tissues, which normally present a low radiopacity. In this study, we report a comparison of four staining protocols, based on iodine or tungsten containing agents, applied to bovine ovarian tissues fixed in Bouin's solution. The microtomography (microCT) analyses at two synchrotron facilities under different set-ups were performed at different energies in order to maximize the image contrast. While tungsten-based agents allow large structures to be well identified, Iodine ones better highlight smaller features, especially when acquired above the K-edge energy of the specific metal. Further scans performed at lower energy where the setup was optimized for overall quality and sensitivity from phase-contrast still provided highly resolved visualization of follicular and intrafollicular structures at different maturation stages, independent of the staining protocol. The analyses were complemented by X-ray Fluorescence mapping on 2D sections, showing that the tungsten-based agent has a higher penetration in this type of tissues.

Human Neocortex Layer Features Evaluated by PIXE, STIM, and STXM Techniques.

The human neocortex has a cytoarchitecture composed of six layers with an intrinsic organization that relates to afferent and efferent pathways for a high functional specialization. Various histological, neurochemical, and connectional techniques have been used to study these cortical layers. Here, we explore the additional possibilities of swift ion beam and synchrotron radiation techniques to distinguish cellular layers based on the elemental distributions and areal density pattern in the human neocortex. Temporal cortex samples were obtained from two neurologically normal adult men (postmortem interval: 6-12 h). A cortical area of 500 × 500 µm2 was scanned by a 3 MeV proton beam for elemental composition and areal density measurements using particle induced x-ray emission (PIXE) and scanning transmission ion microscopy (STIM), respectively. Zinc showed higher values in cortical layers II and V, which needs a critical discussion. Furthermore, the areal density decreased in regions with a higher density of pyramidal neurons in layers III and V. Scanning transmission X-ray microscopy (STXM) revealed the cellular density with higher lateral resolution than STIM, but not enough to distinguish each cortical lamination border. Our data describe the practical results of these approaches employing both X-ray and ion-beam based techniques for the human cerebral cortex and its heterogeneous layers. These results add to the potential approaches and knowledge of the human neocortical gray matter in normal tissue to develop improvements and address further studies on pathological conditions.

Detention and mapping of iron and toxic environmental elements in human ovarian endometriosis: A suggested combined role.

BACKGROUND: Endometriosis is a disease affecting 10-15 % of women worldwide, consisting in the ectopic growth of endometrial cells outside the uterine cavity. Whist the pathogenetic mechanisms of endometriosis remain elusive and contemplating even environmental causes, iron deposits are common in endometrial lesions, indicating an altered iron metabolism at this level. This study was undertaken to reveal a possible relationship between iron dysmetabolism and accumulation of environmental metals. METHODS: By combining histological and histochemical analysis (H&E and Perl's staining) with µ- and nano- synchrotron-based (SR-based) X-ray Fluorescence (XRF) microscopy, we investigated the distribution of iron and other elements in the ovarian endometriomas of 12 endometriosis patients and in 7 healthy endometrium samples. RESULTS: XRF microscopy expanded the findings obtained by Perl's staining, revealing with an exceptional sensitivity intracellular features of iron accumulation in the epithelial endometrium, stroma and macrophages of the endometriotic lesions. XRF evidenced that iron was specifically accumulated in multiple micro aggregates, reaching concentrations up to 10-20 % p/p. Moreover, by XRF analysis we revealed for the first time the retention of a number of exogenous and potentially toxic metals such as Pb, Br, Ti, Al Cr, Si and Rb partially or totally co-localizing with iron. CONCLUSION: µXRF reveals accumulation and colocalization of iron and environmental metals in human ovarian endometriosis, suggesting a role in the pathogenesis of endometriosis.

Strategies and Perspectives for UV Resonance Raman Applicability in Clinical Analyses of Human Sperm RNA.

Developing a deeper knowledge about the impact of DNA and RNA epigenetic mutations on sperm production and fertilization performance is essential for selecting best quality samples in Assisted Reproductive Technologies (ART). Indeed, sperm RNAs adenine and guanine are likely to be methylated in low quality RNA sperm samples and their study requires the employment of techniques able to isolate high quality nucleic acids. UV resonance Raman spectroscopy represents a valuable tool that is able to monitor peculiar molecular modifications occurring predominantly in nucleic acids, being less sensitive to the presence of other biological compounds. In this work, we used an UV Resonance Raman (UVRR) setup coupled to a synchrotron radiation source tuned at 250 nm, in order to enhance sperm RNAs adenine and guanine vibrational signals, reducing also the impact of a fluorescence background typically occurring at lower energies. Despite that our protocol should be further optimized and further analyses are requested, our results support the concept that UVRR can be applied for setting inexpensive tools to be employed for semen quality assessment in ART.

SARS-CoV-2 Short-Time Infection Produces Relevant Cytopathic Effects in Vero E6 Cell Line.

Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) is mainly transmitted through respiratory droplets from positive subjects to susceptible hosts or by direct contact with an infected individual. Our study focuses on the in vitro minimal time of viral absorption as well as the minimal quantity of virus able to establish a persistent infection in Vero E6 cells. We observed that 1 min of in vitro virus exposure is sufficient to generate a cytopathic effect in cells after 7 days of infection, even at a multiplicity of infection (MOI) value of 0.01. Being aware that our findings have been obtained using an in vitro cellular model, we demonstrated that short-time exposures and low viral concentrations are able to cause infection, thus opening questions about the risk of SARS-CoV-2 transmissibility even following short contact times.

UV Resonance Raman explores protein structural modification upon fibrillation and ligand interaction.

Amyloids are proteinaceous deposits considered an underlying pathological hallmark of several degenerative diseases. The mechanism of amyloid formation and its inhibition still represent challenging issues, especially when protein structure cannot be investigated by classical biophysical techniques as for the intrinsically disordered proteins (IDPs). In this view, the need to find an alternative way for providing molecular and structural information regarding IDPs prompted us to set a novel, to our knowledge, approach focused on UV Resonance Raman (UVRR) spectroscopy. To test its applicability, we study the fibrillation of hen-egg white lysozyme (HEWL) and insulin as well as their interaction with resveratrol, employing also intrinsic fluorescence spectroscopy, Fourier transform infrared (FTIR) spectroscopy, and atomic force microscopy (AFM). The increasing of the ß-sheet structure content at the end of protein fibrillation probed by FTIR occurs simultaneously with a major solvent exposure of tryptophan (Trp) and tyrosine (Tyr) residues of HEWL and insulin, respectively, as revealed by UVRR and intrinsic fluorescence spectroscopy. However, because the latter technique is successfully used when proteins naturally contain Trp residues, it shows poor performances in the case of insulin, and the information regarding its tertiary structure is exclusively provided by UVRR spectroscopy. The presence of an increased concentration of resveratrol induces mild changes in the secondary structure of both protein fibrils while remodeling HEWL fibril length and promoting the formation of amorphous aggregates in the case of insulin. Although the intrinsic fluorescence spectra of proteins are hidden by resveratrol signal, UVRR Trp and Tyr bands are resonantly enhanced, showing a good sensitivity to the presence of resveratrol and marking a modification in the noncovalent interactions in which they are involved. Our findings demonstrate that UVRR is successfully employed in the study of aggregation-prone proteins and of their interaction with ligands, especially in the case of Trp-lacking proteins.

Megapixel scanning transmission soft X-ray microscopy imaging coupled with compressive sensing X-ray fluorescence for fast investigation of large biological tissues.

Soft X-ray microscopy coupled with low energy X-ray fluorescence is a powerful tool for investigating complex biological systems like cells and tissues. Due to certain characteristics of X-ray sources, sample stage motors, and detectors, the examination of large areas at high resolutions is very time consuming, often confining the analysis only to a restricted number of pre-selected representative regions. Here we propose and demonstrate a compressive sensing method that provides an alternative approach for overcoming such limitations and can be applied to different kinds of samples and other microscopy and analytical techniques.

Scanning Probe Microscopies: Imaging and Biomechanics in Reproductive Medicine Research.

Basic and translational research in reproductive medicine can provide new insights with the application of scanning probe microscopies, such as atomic force microscopy (AFM) and scanning near-field optical microscopy (SNOM). These microscopies, which provide images with spatial resolution well beyond the optical resolution limit, enable users to achieve detailed descriptions of cell topography, inner cellular structure organization, and arrangements of single or cluster membrane proteins. A peculiar characteristic of AFM operating in force spectroscopy mode is its inherent ability to measure the interaction forces between single proteins or cells, and to quantify the mechanical properties (i.e., elasticity, viscoelasticity, and viscosity) of cells and tissues. The knowledge of the cell ultrastructure, the macromolecule organization, the protein dynamics, the investigation of biological interaction forces, and the quantification of biomechanical features can be essential clues for identifying the molecular mechanisms that govern responses in living cells. This review highlights the main findings achieved by the use of AFM and SNOM in assisted reproductive research, such as the description of gamete morphology; the quantification of mechanical properties of gametes; the role of forces in embryo development; the significance of investigating single-molecule interaction forces; the characterization of disorders of the reproductive system; and the visualization of molecular organization. New perspectives of analysis opened up by applying these techniques and the translational impacts on reproductive medicine are discussed.

Blue photobiomodulation LED therapy impacts SARS-CoV-2 by limiting its replication in Vero cells.

The study of any intervention able to counteract SARS-CoV-2 pandemic is considerably envisaged. It was previously shown, in in vitro models of infections, that the LED blue light is able to decrease the viral load of HSV-1 and ZIKV. In our study, LED photobiomodulation therapy (PBMT) at blue wavelengths (450, 454 and 470 nm) was tested in an in vitro model of SARS-CoV-2 infection, employing three experimental settings: SARS-CoV-2 was irradiated and then transferred to cells; already infected cells were irradiated; cells were irradiated prior to infection. A decrement of the viral load was observed when previously infected cells were irradiated with all three tested wavelengths and relevant effects were registered especially at 48 hours post-infection, possibly suggesting that the blue light could interfere with the intracellular viral replication machinery. Our in vitro findings could represent the starting point for translational applications of PBMT as a supportive approach to fight SARS-CoV-2.

FTIR Spectroscopy to Reveal Lipid and Protein Changes Induced on Sperm by Capacitation: Bases for an Improvement of Sample Selection in ART.

Although being a crucial step for Assisted Reproduction Technologies (ART) success, to date sperm selection is based only on morphology, motility and concentration characteristics. Considering the many possible alterations, there is a great need for analytical approaches allowing more effective sperm selections. The use of Fourier Transform Infrared (FTIR) may represent an interesting possibility, being able to reveal many macromolecular changes in a single measurement in a nondestructive way. As a proof of concept, in this observational study, we used a FTIR approach to reveal features related to sperm quality and chemical changes promoted by in vitro capacitation. We found indication that α-helix content is increased in capacitated sperm, while high percentages of the ß-structures seem to correlate to poor-quality spermatozoa. The most interesting observation was related to the lipid composition, when measured as CH2/CH3 vibrations (ratio 2853/2870), which resulted in being strongly influenced by capacitation and well correlated with sperm motility. Interestingly, this ratio is higher than 1 in infertile samples, suggesting that motility is related to sperm membranes stiffness and lipid composition. Although further analyses are requested, our results support the concept that FTIR can be proposed as a new smart diagnostic tool for semen quality assessment in ART.

Renin Angiotensin System, COVID-19 and Male Fertility: Any Risk for Conceiving?

The current knowledge concerning the connection between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the renin-angiotensin system (RAS) system in the male reproductive apparatus is still limited, so dedicated studies are urgently required. Concerns about the male fertility consequences of SARS-CoV-2 infection have started to emerge, since epidemiologic studies observed that this coronavirus affects male patients more frequently and with increased severity, possibly because of the hormone-regulated expression of angiotensin-converting enzyme 2 (ACE2) receptor. A disturbance in fertility is also expected based on studies of the previous SARS-CoV infection, which targets the same ACE2 receptor when entering the host cells. In addition, bioinformatics analyses reveal the abundant expression of ACE2 receptor in the male reproductive tissues, particularly in the testis. It has been proposed that pharmacological intervention favoring the angiotensin-(1-7)/ACE2/Mas receptor pathway and increasing ACE2 expression and activity could greatly prevent inflammatory lesions in this area. Finally, in laboratories performing assisted reproductive technologies it is recommended that more attention should be paid not only to sperm quality but also to safety aspects. Data about the potential infectivity of seminal fluid are in fact conflicting and do not exclude risks for both personnel and patients. The potential infectivity of SARS-CoV-2 in reproductive male tissues should be strongly considered and further investigated for the proper management of in vitro fertilization procedures.

SARS-CoV-2 and the next generations: which impact on reproductive tissues?

Coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is a severe global pandemic, affecting mostly the respiratory system. Understandably, attention is also being directed towards the urogenital tract. In this work, expression patterns of various host molecules possibly involved in viral entry and replication were investigated in human female and male reproductive systems by inquiring online repositories, including the Human Protein Atlas, GTEx, FANTOM5. Our findings highlight that male reproductive tissues could be targeted by SARS-CoV-2, particularly the testis since it co-expresses the receptor (ACE2) and the protease (TMPRSS) needed for viral entry. We hypothesized that SARS-CoV-2 infection could have repercussions on the fertility status of male individuals Potential infectivity of SARS-CoV-2 in reproductive tissues should be considered in reproductive medicine and management of in vitro fertilization in present and future generations.

Investigation of genomic DNA methylation by ultraviolet resonant Raman spectroscopy.

Cytosine plays a preeminent role in DNA methylation, an epigenetic mechanism that regulates gene expression, the misregulation of which can lead to severe diseases. Several methods are nowadays employed for assessing the global DNA methylation levels, but none of them combines simplicity, high sensitivity, and low operating costs to be translated into clinical applications. Ultraviolet (UV) resonant Raman measurements at excitation wavelengths of 272 nm, 260 nm, 250 nm, and 228 nm have been carried out on isolated deoxynucleoside triphosphates (dNTPs), on a dNTP mixture as well as on genomic DNA (gDNA) samples, commercial from salmon sperm and non-commercial from B16 murine melanoma cell line. The 228 nm excitation wavelength was identified as the most suitable energy for enhancing cytosine signals over the other DNA bases. The UV Raman measurements performed at this excitation wavelength on hyper-methylated and hypo-methylated DNA from Jurkat leukemic T-cell line have revealed significant spectral differences with respect to gDNA isolated from salmon sperm and mouse melanoma B16 cells. This demonstrates how the proper choice of the excitation wavelength, combined with optimized extraction protocols, makes UV Raman spectroscopy a suitable technique for highlighting the chemical modifications undergone by cytosine nucleotides in gDNA upon hyper- and hypo-methylation events.

Synchrotron radiation soft X-ray microscopy and low energy X-ray fluorescence to reveal elemental changes in spermatozoa treated with photobiomodulation therapy.

Male infertility is a worldwide clinical issue that increases the number of couples resorting to assisted reproductive technologies (ARTs) to achieve pregnancy. Photobiomodulation therapy (PBMT) is a promising technique that can biostimulate cells and tissues and it is currently successfully employed to enhance the sperm motility in vitro. Nevertheless, its use has been so far restricted to the research field. In the present work, we exploited two PBMT protocols at an 800 nm wavelength on sperm derived from infertile individuals, detecting an increase in sperm motility 1 hour after irradiation. Moreover, in order to add new information about the molecular effect of PBMT, the content of some light elements was evaluated using high resolution X-ray fluorescence imaging. Interestingly, an increase in Na content was detected in the irradiated samples, possibly suggesting a role of this element in sperm motility; indeed, a low Na content was previously correlated with a poor sperm quality, low semen volume, and modest fertilization rate. Amplifying the knowledge of PBMT in the ART field will expedite the translational potentiality of the PBMT use in clinical settings.

Photobiomodulation therapy for male infertility.

Male infertility is a worldwide critical condition that affects about the 7.5% of males in Europe leading to an increment of the couples referring to reproductive medicine units to achieve pregnancy. Moreover, in the recent years, an increased number of patients have required to freeze their gametes in order to preserve their fertility. Photobiomodulation (PBM) therapy is a potential treatment that has been used for different clinical application basically aimed at biostimulating cells and tissues. Here, we report a deep overview of the published studies, focusing on PBM mechanism of action, with the aim of expanding the knowledge in the field of laser light for a rational utilization of irradiation in the clinical practice. In the field of reproductive science, PBM was employed to increment spermatozoa's metabolism, motility, and viability, due to its beneficial action on mitochondria, leading to an activation of the mitochondrial respiratory chain and to the ATP production. This treatment can be particularly useful to avoid the use of chemicals in the spermatozoa culture medium as well as to promote the spermatozoa survival and movement especially after thawing or in largely immotile sperm samples.