Evaluation of an artificial intelligence-facilitated sperm detection tool in azoospermic samples for use in ICSI.

RESEARCH QUESTION: Can artificial intelligence (AI) improve the efficiency and efficacy of sperm searches in azoospermic samples? DESIGN: This two-phase proof-of-concept study began with a training phase using eight azoospermic patients (>10,000 sperm images) to provide a variety of surgically collected samples for sperm morphology and debris variation to train a convolutional neural network to identify spermatozoa. Second, side-by-side testing was undertaken on two cohorts of non-obstructive azoospermia patient samples: an embryologist versus the AI identifying all the spermatozoa in the still images (cohort 1, n = 4), and a side-by-side test with a simulated clinical deployment of the AI model with an intracytoplasmic sperm injection microscope and the embryologist performing a search with and without the aid of the AI (cohort 2, n = 4). RESULTS: In cohort 1, the AI model showed an improvement in the time taken to identify all the spermatozoa per field of view (0.02 ± 0.30  ×  10-5s versus 36.10 ± 1.18s, P < 0.0001) and improved recall (91.95 ± 0.81% versus 86.52 ± 1.34%, P < 0.001) compared with an embryologist. From a total of 2660 spermatozoa to find in all the samples combined, 1937 were found by an embryologist and 1997 were found by the AI in less than 1000th of the time. In cohort 2, the AI-aided embryologist took significantly less time per droplet (98.90 ± 3.19 s versus 168.7 ± 7.84 s, P < 0.0001) and found 1396 spermatozoa, while 1274 were found without AI, although no significant difference was observed. CONCLUSIONS: AI-powered image analysis has the potential for seamless integration into laboratory workflows, to reduce the time to identify and isolate spermatozoa from surgical sperm samples from hours to minutes, thus increasing success rates from these treatments.

In situ characterization of the tumor microenvironment.

The development of new therapies for cancer is underpinned by an increasing need to comprehensively characterize the tumor microenvironment (TME). While traditional approaches have relied on bulk or single-cell approaches, these are limited in their ability to provide cellular context. Deconvolution of the complex TME is fundamental to understanding tumor dynamics and treatment resistance. Spatially resolved characterization of the TME is likely to provide greater insights into the cellular architecture, tumor-immune cell interactions, receptor-ligand interactions, and cell niches. In turn, these aid in dictating the optimal way in which to target each patient's individual cancer. In this review, we discuss a number of cutting-edge in situ spatial profiling methods giving us new insights into tumor biology.

Whole transcriptome profiling of placental pathobiology in SARS-CoV-2 pregnancies identifies placental dysfunction signatures.

Objectives: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) virus infection in pregnancy is associated with higher incidence of placental dysfunction, referred to by a few studies as a 'preeclampsia-like syndrome'. However, the mechanisms underpinning SARS-CoV-2-induced placental malfunction are still unclear. Here, we investigated whether the transcriptional architecture of the placenta is altered in response to SARS-CoV-2 infection. Methods: We utilised whole-transcriptome, digital spatial profiling, to examine gene expression patterns in placental tissues from participants who contracted SARS-CoV-2 in the third trimester of their pregnancy (n = 7) and those collected prior to the start of the coronavirus disease 2019 (COVID-19) pandemic (n = 9). Results: Through comprehensive spatial transcriptomic analyses of the trophoblast and villous core stromal cell subpopulations in the placenta, we identified SARS-CoV-2 to promote signatures associated with hypoxia and placental dysfunction. Notably, genes associated with vasodilation (NOS3), oxidative stress (GDF15, CRH) and preeclampsia (FLT1, EGFR, KISS1, PAPPA2) were enriched with SARS-CoV-2. Pathways related to increased nutrient uptake, vascular tension, hypertension and inflammation were also enriched in SARS-CoV-2 samples compared to uninfected controls. Conclusions: Our findings demonstrate the utility of spatially resolved transcriptomic analysis in defining the underlying pathogenic mechanisms of SARS-CoV-2 in pregnancy, particularly its role in placental dysfunction. Furthermore, this study highlights the significance of digital spatial profiling in mapping the intricate crosstalk between trophoblasts and villous core stromal cells, thus shedding light on pathways associated with placental dysfunction in pregnancies with SARS-CoV-2 infection.

Microfluidics facilitating the use of small extracellular vesicles in innovative approaches to male infertility.

Sperm are transcriptionally and translationally quiescent and, therefore, rely on the seminal plasma microenvironment for function, survival and fertilization of the oocyte in the oviduct. The male reproductive system influences sperm function via the binding and fusion of secreted epididymal (epididymosomes) and prostatic (prostasomes) small extracellular vesicles (S-EVs) that facilitate the transfer of proteins, lipids and nucleic acids to sperm. Seminal plasma S-EVs have important roles in sperm maturation, immune and oxidative stress protection, capacitation, fertilization and endometrial implantation and receptivity. Supplementing asthenozoospermic samples with normospermic-derived S-EVs can improve sperm motility and S-EV microRNAs can be used to predict non-obstructive azoospermia. Thus, S-EV influence on sperm physiology might have both therapeutic and diagnostic potential; however, the isolation of pure populations of S-EVs from bodily fluids with current conventional methods presents a substantial hurdle. Many conventional techniques lack accuracy, effectiveness, and practicality; yet microfluidic technology has the potential to simplify and improve S-EV isolation and detection.

Immunotherapeutic targets in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is one of the most common types of cancer in the world and has a 5-year survival rate of ~20%. Immunotherapies have shown promising results leading to durable responses, however, they are only effective for a subset of patients. To determine the best therapeutic approach, a thorough and in-depth profiling of the tumour microenvironment (TME) is required. The TME is a complex network of cell types that form an interconnected network, promoting tumour cell initiation, growth and dissemination. The stroma, immune cells and endothelial cells that comprise the TME generate a plethora of cytotoxic or cytoprotective signalling pathways. In this review, we discuss immunotherapeutic targets in NSCLC tumours and how the TME may influence patients' response to immunotherapy.

Corrigendum: The effects of COVID-19 on the placenta during pregnancy.

[This corrects the article DOI: 10.3389/fimmu.2021.743022.].

Developing Novel Fabrication and Optimisation Strategies on Aggregation-Induced Emission Nanoprobe/Polyvinyl Alcohol Hydrogels for Bio-Applications.

The current study describes a new technology, effective for readily preparing a fluorescent (FL) nanoprobe-based on hyperbranched polymer (HB) and aggregation-induced emission (AIE) fluorogen with high brightness to ultimately develop FL hydrogels. We prepared the AIE nanoprobe using a microfluidic platform to mix hyperbranched polymers (HB, generations 2, 3, and 4) with AIE (TPE-2BA) under shear stress and different rotation speeds (0-5 K RPM) and explored the FL properties of the AIE nanoprobe. Our results reveal that the use of HB generation 4 exhibits 30-times higher FL intensity compared to the AIE alone and is significantly brighter and more stable compared to those that are prepared using HB generations 3 and 2. In contrast to traditional methods, which are expensive and time-consuming and involve polymerization and post-functionalization to develop FL hyperbranched molecules, our proposed method offers a one-step method to prepare an AIE-HB nanoprobe with excellent FL characteristics. We employed the nanoprobe to fabricate fluorescent injectable bioadhesive gel and a hydrogel microchip based on polyvinyl alcohol (PVA). The addition of borax (50 mM) to the PVA + AIE nanoprobe results in the development of an injectable bioadhesive fluorescent gel with the ability to control AIEgen release for 300 min. When borax concentration increases two times (100 mM), the adhesion stress is more than two times bigger (7.1 mN/mm2) compared to that of gel alone (3.4 mN/mm2). Excellent dimensional stability and cell viability of the fluorescent microchip, along with its enhanced mechanical properties, proposes its potential applications in mechanobiology and understanding the impact of microstructure in cell studies.

The Effects of COVID-19 on the Placenta During Pregnancy.

Coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic. The virus primarily affects the lungs where it induces respiratory distress syndrome ranging from mild to acute, however, there is a growing body of evidence supporting its negative effects on other system organs that also carry the ACE2 receptor, such as the placenta. The majority of newborns delivered from SARS-CoV-2 positive mothers test negative following delivery, suggesting that there are protective mechanisms within the placenta. There appears to be a higher incidence of pregnancy-related complications in SARS-CoV-2 positive mothers, such as miscarriage, restricted fetal growth, or still-birth. In this review, we discuss the pathobiology of COVID-19 maternal infection and the potential adverse effects associated with viral infection, and the possibility of transplacental transmission.

Miniature auto-perfusion bioreactor system with spiral microfluidic cell retention device.

Medium perfusion is critical in maintaining high cell concentration in cultures. The conventional membrane filtration method for medium exchange has been challenged by the fouling and clogging of the membrane filters in long-term cultures. In this study, we present a miniature auto-perfusion system that can be operated inside a common-size laboratory incubator. The system is equipped with a spiral microfluidic chip for cell retention to replace conventional membrane filters, which fundamentally overcomes the clogging and fouling problem. We showed that the system supported continuous perfusion culture of Chinese hamster ovary (CHO) cells in suspension up to 14 days without cell retention chip replacement. Compared to daily manual medium change, 25% higher CHO cell concentration can be maintained at an average auto-perfusion rate of 196 ml/day in spinner flask at 70 ml working volume (2.8 VVD). The auto-perfusion system also resulted in better cell quality at high concentrations, in terms of higher viability, more uniform and regular morphology, and fewer aggregates. We also demonstrated the potential application of the system for culturing mesenchymal stem cells on microcarriers. This miniature auto-perfusion system provides an excellent solution to maintain cell-favorable conditions and high cell concentration in small-scale cultures for research and clinical uses.

Characterizing the effect of substrate stiffness on the extravasation potential of breast cancer cells using a 3D microfluidic model.

Different biochemical and biomechanical cues from tumor microenvironment affect the extravasation of cancer cells to distant organs; among them, the mechanical signals are poorly understood. Although the effect of substrate stiffness on the primary migration of cancer cells has been previously probed, its role in regulating the extravasation ability of cancer cells is still vague. Herein, we used a microfluidic device to mimic the extravasation of tumor cells in a 3D microenvironment containing cancer cells, endothelial cells, and the biological matrix. The microfluidic-based extravasation model was utilized to probe the effect of substrate stiffness on the invasion ability of breast cancer cells. MCF7 and MDA-MB-231 cancer cells were cultured among substrates with different stiffness which followed by monitoring their extravasation capability through the microfluidic device. Our results demonstrated that acidic collagen at a concentration of 2.5 mg/ml promotes migration of cancer cells. Additionally, the substrate softening resulted in up to 46% reduction in the invasion of breast cancer cells. The substrate softening not only affected the number of extravasated cells but also reduced their migration distance up to 53%. We further investigated the secreted level of matrix metalloproteinase 9 (MMP9) and identified that there is a positive correlation between substrate stiffening, MMP9 concentration, and extravasation of cancer cells. These findings suggest that the substrate stiffness mediates the cancer cells extravasation in a microfluidic model. Changes in MMP9 level could be one of the possible underlying mechanisms which need more investigations to be addressed thoroughly.

Mesenchymal stem cells induce PD-L1 expression through the secretion of CCL5 in breast cancer cells.

Various factors in the tumor microenvironment (TME) regulate the expression of PD-L1 in cancer cells. In TME, mesenchymal stem cells (MSCs) play a crucial role in tumor progression, metastasis, and drug resistance. Emerging evidence suggests that MSCs can modulate the immune-suppression capacity of TME through the stimulation of PD-L1 expression in various cancers; nonetheless, their role in the induction of PD-L1 in breast cancer remained elusive. Here, we assessed the potential of MSCs in the stimulation of PD-L1 expression in a low PD-L1 breast cancer cell line and explored its associated cytokine. We assessed the expression of MSCs-related genes and their correlation with PD-L1 across 1826 breast cancer patients from the METABRIC cohort. After culturing an ER+/differentiated/low PD-L1 breast cancer cells with MSCs conditioned-medium (MSC-CM) in a microfluidic device, a variety of in-vitro assays was carried out to determine the role of MSC-CM in breast cancer cells' phenotype plasticity, invasion, and its effects on induction of PD-L1 expression. In-silico analysis showed a positive association between MSCs-related genes and PD-L1 expression in various types of breast cancer. Through functional assays, we revealed that MSC-CM not only prompts a phenotype switch but also stimulates PD-L1 expression at the protein level through secretion of various cytokines, especially CCL5. Treatment of MSCs with cytokine inhibitor pirfenidone showed a significant reduction in the secretion of CCL5 and consequently, expression of PD-L1 in breast cancer cells. We concluded that MSCs-derived CCL5 may act as a PD-L1 stimulator in breast cancer.

Understanding the tumor microenvironment for effective immunotherapy.

Advances in immunotherapy have led to durable and long-term benefits in a subset of patients across a number of solid tumor types. Understanding of the subsets of patients that respond to immune checkpoint inhibitors at the cellular level, and in the context of their tumor microenvironment (TME) is becoming increasingly important. The TME is composed of a heterogeneous milieu of tumor and immune cells. The immune landscape of the TME can inhibit or promote tumor initiation and progression; thus, a deeper understanding of tumor immunity is necessary to develop immunotherapeutic strategies. Recent developments have focused on characterizing the TME immune contexture (type, density, and function) to discover mechanisms and biomarkers that may predict treatment outcomes. This has, in part, been powered by advancements in spatial characterization technologies. In this review article, we address the role of specific immune cells within the TME at various stages of tumor progression and how the immune contexture determinants affecting tumor growth are used therapeutically.

Background-free fibre optic Brillouin probe for remote mapping of micromechanics.

Brillouin imaging (BI) has become a valuable tool for micromechanical material characterisation, thanks to extensive progress in instrumentation in the last few decades. This powerful technique is contactless and label-free, thus making it especially suitable for biomedical applications. Nonetheless, to fully harness the non-contact and non-destructive nature of BI, transformational changes in instrumentation are still needed to extend the technology's utility into the domain of in vivo and in situ operation, which we foresee to be particularly crucial for wide spread usage of BI, e.g. in medical diagnostics and pathology screening. This work addresses this challenge by presenting the first demonstration of a fibre-optic Brillouin probe, capable of mapping the micromechanical properties of a tissue-mimicking phantom. This is achieved through combination of miniaturised optical design, advanced hollow-core fibre fabrication and high-resolution 3D printing. Our prototype probe is compact, background-free and possesses the highest collection efficiency to date, thus providing the foundation of a fibre-based Brillouin device for remote, in situ measurements in challenging and otherwise difficult-to-reach environments in biomedical, material science and industrial applications.

A miniaturized piezoresistive flow sensor for real-time monitoring of intravenous infusion.

Drug overdose (DO) is considered one of the current issues of intravenous (IV) infusion particularly resulting in serious injuries and deaths. Malfunction of infusion pumps is reported as the main cause of the drug overdose. Live monitoring and flow rate calculation by health professionals have been practicing to avoid DO. However, human errors and miscalculations are inevitable. A secondary measurement tool is required to avoid the risk of OD when infusion pump malfunctions cannot be detected immediately. Here, inspired by nature, we developed a real-time monitoring device through which an administrator can review, evaluate, and modify the IV infusion process. Our flow sensor possesses an erected polymer hair cell on a multi-layered silicon base forming from a patterned gold strained gauge layer on a piezoresistive liquid crystal polymer (LCP) membrane. Gold strain gauges on an LCP membrane have been used instead of a piezoresistive silicon membrane as the sensing element. The combination of gold strain gauges and LCP membrane provides better sensitivity than a piezoresistive silicon membrane of the same dimensions and thickness. We also miniaturized our biocompatible sensor such that it can be possible to install it inside the IV tube in contact with the liquid providing an in-suite online flow monitoring. The proposed LCP membrane sensor is compared with two commercially available IV sensors to validate its flow sensing ability. The experimental results demonstrate that the proposed sensor provides a low threshold detection limit of 5 mL/hr, which betters the performance of other commercial sensors at low flow rates.

The Prognostic Role of Circulating Tumor Cells (CTCs) in Lung Cancer.

Lung cancer affects over 1. 8 million people worldwide and is the leading cause of cancer related mortality globally. Currently, diagnosis of lung cancer involves a combination of imaging and invasive biopsies to confirm histopathology. Non-invasive diagnostic techniques under investigation include "liquid biopsies" through a simple blood draw to develop predictive and prognostic biomarkers. A better understanding of circulating tumor cell (CTC) dissemination mechanisms offers promising potential for the development of techniques to assist in the diagnosis of lung cancer. Enumeration and characterization of CTCs has the potential to act as a prognostic biomarker and to identify novel drug targets for a precision medicine approach to lung cancer care. This review will focus on the current status of CTCs and their potential diagnostic and prognostic utility in this setting.

Selective separation of microalgae cells using inertial microfluidics.

Microalgae represent the most promising new source of biomass for the world's growing demands. However, the biomass productivity and quality is significantly decreased by the presence of bacteria or other invading microalgae species in the cultures. We therefore report a low-cost spiral-microchannel that can effectively separate and purify Tetraselmis suecica (lipid-rich microalgae) cultures from Phaeodactylum tricornutum (invasive diatom). Fluorescent polystyrene-microbeads of 6 µm and 10 µm diameters were first used as surrogate particles to optimize the microchannel design by mimicking the microalgae cell behaviour. Using the optimum flowrate, up to 95% of the P. tricornutum cells were separated from the culture without affecting the cell viability. This study shows, for the first time, the potential of inertial microfluidics to sort microalgae species with minimal size difference. Additionally, this approach can also be applied as a pre-sorting technique for water quality analysis.

A Collective Route to Head and Neck Cancer Metastasis.

Distant metastasis (DM) from head and neck cancers (HNC) portends a poor patient prognosis. Despite its important biological role, little is known about the cells which seed these DM. Circulating tumour cells (CTCs) represent a transient cancer cell population, which circulate in HNC patients' peripheral blood and seed at distant sites. Capture and analysis of CTCs offers insights into tumour metastasis and can facilitate treatment strategies. Whilst the data on singular CTCs have shown clinical significance, the role of CTC clusters in metastasis remains limited. In this pilot study, we assessed 60 treatment naïve HNC patients for CTCs with disease ranging from early to advanced stages, for CTC clusters utilizing spiral CTC enrichment technology. Single CTCs were isolated in 18/60-30% (Ranging from Stage I-IV), CTC clusters in 15/60-25% (exclusively Stage IV) with 3/15-20% of CTC clusters also containing leukocytes. The presence of CTC clusters associated with the development of distant metastatic disease(P = 0.0313). This study demonstrates that CTC clusters are found in locally advanced patients, and this may be an important prognostic marker. In vivo and in vitro studies are warranted to determine the role of these CTC clusters, in particular, whether leukocyte involvement in CTC clusters has clinical relevance.

Isolation and detection of circulating tumour cells from metastatic melanoma patients using a slanted spiral microfluidic device.

Circulating Tumour Cells (CTCs) are promising cancer biomarkers. Several methods have been developed to isolate CTCs from blood samples. However, the isolation of melanoma CTCs is very challenging as a result of their extraordinary heterogeneity, which has hindered their biological and clinical study. Thus, methods that isolate CTCs based on their physical properties, rather than surface marker expression, such as microfluidic devices, are greatly needed in melanoma. Here, we assessed the ability of the slanted spiral microfluidic device to isolate melanoma CTCs via label-free enrichment. We demonstrated that this device yields recovery rates of spiked melanoma cells of over 80% and 55%, after one or two rounds of enrichment, respectively. Concurrently, a two to three log reduction of white blood cells was achieved with one or two rounds of enrichment, respectively. We characterised the isolated CTCs using multimarker flow cytometry, immunocytochemistry and gene expression. The results demonstrated that CTCs from metastatic melanoma patients were highly heterogeneous and commonly expressed stem-like markers such as PAX3 and ABCB5. The implementation of the slanted microfluidic device for melanoma CTC isolation enables further understanding of the biology of melanoma metastasis for biomarker development and to inform future treatment approaches.

PD-L1 expressing circulating tumour cells in head and neck cancers.

BACKGROUND: Blockade of the PD-1/PD-L1 immune checkpoint pathway is emerging as a promising immunotherapeutic approach for the management and treatment of head and neck cancer patients who do not respond to 1st/2nd line therapy. However, as checkpoint inhibitors are cost intensive, identifying patients who would most likely benefit from anti PD-L1 therapy is required. Developing a non-invasive technique would be of major benefit to the patient and to the health care system. CASE PRESENTATION: We report the case of a 56 year old man affected by a supraglottic squamous cell carcinoma (SCC). A CT scan showed a 20 mm right jugulodigastric node and suspicious lung lesions. The lung lesion was biopsied and confirmed to be consistent with SCC. The patient was offered palliative chemotherapy. At the time of presentation, a blood sample was taken for circulating tumour cell (CTC) analysis. The dissemination of cancer was confirmed by the detection of CTCs in the peripheral blood of the patient, measured by the CellSearch System (Janssen Diagnostics). Using marker-independent, low-shear spiral microfluidic technology combined with immunocytochemistry, CTC clusters were found in this patient at the same time point, expressing PD-L1. CONCLUSION: This report highlights the potential use of CTCs to identify patients which might respond to anti PD-L1 therapy.

Enrichment of circulating head and neck tumour cells using spiral microfluidic technology.

Whilst locoregional control of head and neck cancers (HNCs) has improved over the last four decades, long-term survival has remained largely unchanged. A possible reason for this is that the rate of distant metastasis has not changed. Such disseminated disease is reflected in measurable levels of cancer cells in the blood of HNC patients, referred to as circulating tumour cells (CTCs). Numerous marker-independent techniques have been developed for CTC isolation and detection. Recently, microfluidics-based platforms have come to the fore to avoid molecular bias. In this pilot, proof of concept study, we evaluated the use of the spiral microfluidic chip for CTC enrichment and subsequent detection in HNC patients. CTCs were detected in 13/24 (54%) HNC patients, representing both early to late stages of disease. Importantly, in 7/13 CTC-positive patients, CTC clusters were observed. This is the first study to use spiral microfluidics technology for CTC enrichment in HNC.