MicroRNAs as Prognostic Markers for Chondrogenic Differentiation Potential of Equine Mesenchymal Stromal Cells.

Mesenchymal stromal cells (MSCs) are a promising cell source for cartilage tissue regeneration in animals and humans but with large interdonor variation in their in vitro chondrogenic differentiation potential. Underlying molecular mechanisms responsible for culture-expanded MSC heterogeneity remain poorly understood. In this study, we sought to identify variations in microRNA (miRNA) signatures associated with cultured equine MSC chondrogenic differentiation potential from different donors. Neocartilage tissue generated from equine cord blood-derived MSCs was categorized as having either high or low chondrogenic potential (LCP) based on their histological appearance and quantification of glycosaminoglycan deposition. Using next-generation sequencing, we identified 30 differentially expressed miRNAs among undifferentiated MSC cultures that corresponded with their chondrogenic potential. Of note, MSCs with LCP upregulated miR-146a and miR-487b-3p, which was also observed by quantitative real-time polymerase chain reaction. Our findings suggest that miRNA profiling of equine MSC cultures may have prognostic value in selecting MSC donors with regard to their chondrogenic differentiation potential.

Obesity and PCOS radically alters the snRNA composition of follicular fluid extracellular vesicles.

Introduction: The ovarian follicle consists of the oocyte, somatic cells, and follicular fluid (FF). Proper signalling between these compartments is required for optimal folliculogenesis. The association between polycystic ovarian syndrome (PCOS) and extracellular vesicular small non-coding RNAs (snRNAs) signatures in follicular fluid (FF) and how this relates to adiposity is unknown. The purpose of this study was to determine whether FF extracellular vesicle (FFEV)-derived snRNAs are differentially expressed (DE) between PCOS and non-PCOS subjects; and if these differences are vesicle-specific and/or adiposity-dependent. Methods: FF and granulosa cells (GC) were collected from 35 patients matched by demographic and stimulation parameters. FFEVs were isolated and snRNA libraries were constructed, sequenced, and analyzed. Results: miRNAs were the most abundant biotype present, with specific enrichment in exosomes (EX), whereas in GCs long non-coding RNAs were the most abundant biotype. In obese PCOS vs. lean PCOS, pathway analysis revealed target genes involved in cell survival and apoptosis, leukocyte differentiation and migration, JAK/STAT, and MAPK signalling. In obese PCOS FFEVs were selectively enriched (FFEVs vs. GCs) for miRNAs targeting p53 signalling, cell survival and apoptosis, FOXO, Hippo, TNF, and MAPK signalling. Discussion: We provide comprehensive profiling of snRNAs in FFEVs and GCs of PCOS and non-PCOS patients, highlighting the effect of adiposity on these findings. We hypothesize that the selective packaging and release of miRNAs specifically targeting anti-apoptotic genes into the FF may be an attempt by the follicle to reduce the apoptotic pressure of the GCs and stave off premature apoptosis of the follicle observed in PCOS.

Human platelet lysates stimulate in vitro proliferation of human endometrial cells from patients with a history of recurrent implantation failure.

OBJECTIVE: To optimize and compare the isolation of platelet-rich plasma (PRP) and its cryopreserved derivative, platelet lysate (PL), to a commercial human platelet lysate (HPL) product PLUS and investigate their proliferative stimulation on primary human endometrial cells in vitro. DESIGN: Basic research. SETTING: Academic fertility center. PATIENT(S): Three healthy blood donors and eight patients with a history of recurrent implantation failure. INTERVENTIONS(S): Stimulated proliferation of isolated primary endometrial epithelial cells and endometrial stromal cells in vitro with autologous and nonautologous HPL (PLUS; Compass Biomedical). MAIN OUTCOME MEASURE(S): Platelet-derived growth factor BB homodimer protein content in isolated PRP/PL and commercial HPL and endometrial epithelial cell and endometrial stromal cell proliferation after 24- or 48-hour stimulation with PL (measured by metabolic activity and Ki67 expression). RESULT(S): To optimize and compare the isolation of autologous PRP/PL, three double-centrifugation protocols were assessed by flow cytometry for platelet yield (CD45-CD41+CD61+) and platelet-derived growth factor BB homodimer protein content by enzyme-linked immunosorbent assay. Cryopreserved PL, especially isolated by our fastest protocol, contained higher protein concentrations and, thus, was optimal for experimental flexibility compared with fresh PRP. The autologous and commercial PLs displayed comparable immune and growth factor content and stimulation of cell proliferation in vitro. CONCLUSION(S): Our results provide the groundwork for the isolation and use of HPL to stimulate endometrial growth. Furthermore, commercial PL consistently stimulated cell proliferation and may allow standardization of clinical treatment for recurrent implantation failure.

Autologous platelet-rich plasma improves the endometrial thickness and live birth rate in patients with recurrent implantation failure and thin endometrium.

PURPOSE: The aim of this study was to evaluate the effects of intrauterine platelet-rich plasma (PRP) infusion on endometrial thickness and pregnancy outcomes in a population of patients with either recurrent implantation failure (RIF), thin endometrium (TE), or both (RIF + TE) METHODS: This retrospective study included patients attending the CReATe Fertility Centre between October 2018 and July 2021 who received intrauterine PRP infusion to prepare the endometrium for frozen embryo transfer. PRP was prepared from 21 cc of whole blood using the 2-step centrifugation method to yield 0.5-0.75 cc of concentrated platelets. Endometrial thickness was measured before infusion and within 72 h after infusion. All embryos transferred were tested for genetic abnormalities using next-generation sequencing. RESULTS: A total of 85 patients, 133 cycles, and 211 infusions were included. The majority of patients (56.5%) were diagnosed with RIF, some with TE (27.0%), and the remainder with both RIF and TE (16.5%). The majority of patients received one PRP infusion per cycle (55%). The endometrial thickness significantly increased across all diagnoses with a significant increase of 1.0 mm (0.5-1.7), which was also significantly greater than in previous cycles. The clinical pregnancy rate per embryo transfer after intrauterine PRP infusion was significantly greater compared to previous cycles (37% vs 20%, odds ratio 2.2) as was the live birth rate (19% vs 2%, odds ratio 11.6). CONCLUSION: Our study suggests that PRP should be considered a noninvasive front-line therapy for improving endometrial thickness and implantation in patients with RIF, a TE, or both.

Bovine piRNA-like RNAs are associated with both transposable elements and mRNAs.

PIWI proteins and their associated piRNAs have been the focus of intensive research in the past decade; therefore, their participation in the maintenance of genomic integrity during spermatogenesis has been well established. Recent studies have suggested important roles for the PIWI/piRNA system outside of gametogenesis, based on the presence of piRNAs and PIWI proteins in several somatic tissues, cancers, and the early embryo. Here, we investigated the small RNA complement present in bovine gonads, gametes, and embryos through next-generation sequencing. A distinct piRNA population was present in the testis as expected. However, we also found a large population of slightly shorter, 24-27 nt piRNA-like RNA (pilRNAs) in pools of oocytes and zygotes. These oocyte and embryo pilRNAs exhibited many of the canonical characteristics of piRNAs including a 1U bias, the presence of a 'ping-pong' signature, genomic clustering, and transposable element targeting. Some of the major transposons targeted by oocyte and zygote pilRNA were from the LINE RTE and ERV1 classes. We also identified pools of pilRNA potentially derived from, or targeted at, specific mRNA sequences. We compared the frequency of these gene-associated pilRNAs to the fold change in the expression of respective mRNAs from two previously reported transcriptome datasets. We observed significant negative correlations between the number of pilRNAs targeting mRNAs, and their fold change in expression between the 4-8 cell and 8-16 cell stages. Together, these results represent one of the first characterizations of the PIWI/piRNA pathway in the translational bovine model, and in the novel context of embryogenesis.

Tuberculosis Diagnosis Delaying Treatment of Cancer: Experience From a New Oncology Unit in Blantyre, Malawi.

PURPOSE: Malawi is a low-income country in sub-Saharan Africa with limited health care infrastructure and high prevalance of HIV and tuberculosis. This study aims to determine the characteristics of patients presenting to Queen Elizabeth Central Hospital Oncology Unit, Blantyre, Malawi, who had been treated for tuberculosis before they were diagnosed with cancer. METHODS: Clinical data on all patients presenting to the oncology unit at Queen Elizabeth Central Hospital from 2010 to 2014 after a prior diagnosis of tuberculosis were prospectively recorded, and a descriptive analysis was undertaken. RESULTS: Thirty-four patients who had been treated for tuberculosis before being diagnosed with cancer were identified between 2010 and 2014, which represents approximately 1% of new referrals to the oncology unit. Forty-one percent of patients were HIV positive. Mean duration of tuberculosis treatment before presentation to the oncology unit was 3.6 months. The most common clinical presentation was a neck mass or generalized lymphadenopathy. Lymphoma was the most common malignancy that was subsequently diagnosed in 23 patients. CONCLUSION: Misdiagnosis of cancer as tuberculosis is a significant clinical problem in Malawi. This study underlines the importance of closely monitoring the response to tuberculosis treatment, being aware of the possibility of a cancer diagnosis, and seeking a biopsy early if cancer is suspected.

Spatial frequency analysis of high-density lipoprotein and iron-oxide nanoparticle transmission electron microscope image structure for pattern recognition in heterogeneous fields.

The optical spatial frequencies of tumor interstitial fluid (TIF) are investigated. As a concentrated colloidal suspension of interacting native nanoparticles, the TIF can develop internal ordering under shear stress that may hinder delivery of antitumor agents within tumors. A systematic method is presented to characterize the TIF nanometer-scale microstructure in a model suspension of superparamagnetic iron-oxide nanoparticles and reconstituted high-density lipoprotein by Fourier spatial frequency (FSF) analysis so as to differentiate between jammed and fluid structural features in static transmission electron microscope images. The FSF method addresses one obstacle faced in achieving quantitative dosimetry to neoplastic tissue, that of detecting these nanoscale barriers to transport, such as would occur in the extravascular space immediately surrounding target cells.

Spatial frequency analysis of anisotropic drug transport in tumor samples.

Directional Fourier spatial frequency analysis was used on standard histological sections to identify salient directional bias in the spatial frequencies of stromal and epithelial patterns within tumor tissue. This directional bias is shown to be correlated to the pathway of reduced fluorescent tracer transport. Optical images of tumor specimens contain a complex distribution of randomly oriented aperiodic features used for neoplastic grading that varies with tumor type, size, and morphology. The internal organization of these patterns in frequency space is shown to provide a precise fingerprint of the extracellular matrix complexity, which is well known to be related to the movement of drugs and nanoparticles into the parenchyma, thereby identifying the characteristic spatial frequencies of regions that inhibit drug transport. The innovative computational methodology and tissue validation techniques presented here provide a tool for future investigation of drug and particle transport in tumor tissues, and could potentially be used a priori to identify barriers to transport, and to analyze real-time monitoring of transport with respect to therapeutic intervention.

Gold nanocrystal labeling allows low-density lipoprotein imaging from the subcellular to macroscopic level.

Low-density lipoprotein (LDL) plays a critical role in cholesterol transport and is closely linked to the progression of several diseases. This motivates the development of methods to study LDL behavior from the microscopic to whole-body level. We have developed an approach to efficiently load LDL with a range of diagnostically active nanocrystals or hydrophobic agents. We performed focused experiments on LDL labeled with gold nanocrystals (Au-LDL). The labeling procedure had minimal effect on LDL size, morphology, or composition. Biological function was found to be maintained from both in vitro and in vivo experiments. Tumor-bearing mice were injected intravenously with LDL, DiR-LDL, Au-LDL, or a gold-loaded nanoemulsion. LDL accumulation in the tumors was detected with whole-body imaging methods, such as computed tomography (CT), spectral CT, and fluorescence imaging. Cellular localization was studied with transmission electron microscopy and fluorescence techniques. This LDL labeling procedure should permit the study of lipoprotein biointeractions in unprecedented detail.