This revolutionary approach can be complementary and may, in the future, replace tissue biopsy, which is still considered the gold standard for cancer diagnosis. “Classical” tissue biopsy is invasive, often cannot provide sufficient bioptic material for advanced screening, and can provide isolated information about disease evolution and heterogeneity. Recent literature highlighted how liquid biopsy is informative of proteomic, genomic, epigenetic, and metabolic alterations. These biomarkers can be detected and investigated using single-omic and, recently, in combination through multi-omic approaches. This review will provide an overview of the most suitable techniques to thoroughly characterize tumor biomarkers and their potential clinical applications, highlighting the importance of an integrated multi-omic, multi-analyte approach. Personalized medical investigations will soon allow patients to receive predictable prognostic evaluations, early disease diagnosis, and subsequent  In the last decades, the old “one-size-fits-all” approach in cancer treatment has been replaced by a personalized model in which therapeutic strategy is based on biological features of the patient’s disease . This apTo overcome these limitations, a non-invasive sampling approach known as liquid biopsy (LB) was developed . Based oTo better understand the mechanisms underlying different phenotypes during malignant transformation, investigating different omics, e.g., transcriptomics, epigenomics, proteomics and metabolomics, became necessary. Although LB represents a promising tool to monitor the dynamic evolution of cancer in a non-invasive way, its integration into clinical practice is hampered by the lack of reproducibility due to the absence of standardization across workflows . The maiIn this review we are going to explore how the main single omics are interrogated in LB, discussing multiple types of cancers and clinical applications. At the same time, we will explain how multidimensional analysis of different liquid components and integration of omics will lead to new biomarkers discovery for cancer management, and identification of therapeutic targets linked to cancer-specific molecular pathways.Described for the first time in 1869 by Ashworth, CTCs are tumor cells that detach from the main tumor masses and travel through the circulatory systems . It tookCTCs are related to tumor stage  but theiCTCs molecular phenotype has also a strong prognostic value: for instance patients with CTC expressing mesenchymal markers , stem maTo predict and monitor therapeutic responses, CTCs can be used in combination with serum biomarkers and imaging, as demonstrated by several clinical studies. For example, patients with Castration-Resistant Prostate Cancer (CRPC) expressing androgen receptor 7 splice variant (AR-V7) protein on CTCs had a better survival rate after taxane chemotherapy . Expressex vivo cultures have been suggested: these models represent an opportunity to identify drug susceptibility changes in patients as their tumors evolve , and methylation changes that provide valuable insight into disease status. There is an open debate in the scientific community about the use of ctDNA for early cancer detection . Grail iOver the last decade, the Food and Drug Administration (FDA) has approved a series of LB tests based on ctDNA. The Cobas EGFR Mutation Test v2  is a diagnostic test for EGFR tyrosine kinase inhibitor therapies in NSCLC, while Guardant360 CDx  and Liquid CDx , analyzing the complete tumor genomic profile, help clinicians to understand responsiveness to checkpoint inhibitors and targeted therapy. In the near future, it will be necessary to validate non-invasive methods to detect limited amounts of ctDNA to guide personalized therapeuticisions.Exosome-based oncology research has recently achieved impressive results by offering potential tools for the clinical management of the disease. Initially considered a cellular waste disposal system, they are important players in intercellular communication .They are small extracellular vesicles released from every type of cell which circulate stably in most biological fluids such as blood, urine, milk, saliva. They carry nucleic acids, proteins and lipids and can reach very distant cells influencing their biological functions .In cancer exosomes are involved in remodeling of the tumor microenvironment , formatiIn liquid biopsy, for their unique features, exosomes have complementary and potentially broader applications than CTCs and ctDNA. During exosome analysis, the real challenge to overcome is discriminating tumor exosomes from non-tumor extracellular vesicles still. A possible solution could be using highly specific analytical techniques such as mass spectrometry (MS), Flow Cytometry (FC) and High Throughput Sequencing (HTS). Big data analysis would then be capable of distinguishing tumor exosomes from the physiological material .To date, the majority of circulating nucleic acid studies in oncology concern ctDNA. However the spotlight has turned on cfRNA which has promised to improve cancer diagnosis and treatment . Like exIn the last years, several studies demonstrated that miRNA expression is dysregulated in cancer, and its signatures could be used for diagnosis, prognosis and therapeutic management of cancer . RecentlThe interaction between tumors and platelets was first observed in 1868 when Trousseau noted that spontaneous coagulation was common in cancer patients. A decade later, Billroth described that these clots contained cancer cells and for this reason could be agents of metastasis . Today wAll in all, several liquid biopsy markers have been characterized for their clinical relevance in informing about disease biology, treatment response and progression. Each of those makers has specific strengths and limitations, and it is apparent that their combined use could be instrumental in obtaining a comprehensive description of tumor features.For example, CTCs are informative of the tumor phenotype and genotype, representing a source of information about tumor heterogeneity and presence of molecular targets for therapy.CTC analysis anyway poses some limitations mostly related to their difficulty to be isolated due to their limiting number in the bloodstream. On the contrary, ctDNA is informative of tumor genotype, and also of its epigenetic profile  and is therefore useful to assess mutational profile, clonality, and tissue-of-origin. Anyway, some limits in sensitivity and specificity due to presence of other cfDNA sources and limited ctDNA quantities are still present.Relatively new markers such as exosomes and platelets are a great promise for liquid biopsy as they hold information about relevant tumor biomolecules  instrumental to retrieve information about tumor biology and microenvironment. Their combined analysis with more established markers such as CTC and ctDNA can help in obtaining a more comprehensive picture of tumor of origin, with clinical impact in diagnosis and monitoring.Tumor genetic profile is assessed in the clinical routine by molecular characterization of biopsy specimens. Due to the invasive nature of the procedure, limitations in longitudinal monitoring and intrinsic sampling biases, LB represents a valuable alternative tool to investigate the genomic landscape of tumors during time . The twoAvailable techniques to assess the genomic profile of ctDNA and CTCs can be divided into targeted and whole-genome, targeted approaches analyze the mutational profile of a subset of genes that have been linked to the pathogenesis of disease and include clinically actionable genes of interest. Such analysis can be performed using droplet digital PCR (ddPCR)  or Next (BEAMing)   exploitsBEAMing) .As for the NGS-based approaches, CAPP-Seq was first time reported to study ctDNA in NSCLC by Newman et al., proving high sensitivity and subsequently applied to other cancer types . This apTAm-Seq allows the amplification of specific regions of interest through the generation of short and overlapping fragments of about 150–200 bases, then indexed singularly and deep sequenced. This technique is particularly indicated for the detection of cancer mutations with an allele frequency up to 2%. Due to the short length of amplicons generated, the method is suitable for the analysis of highly fragmented ctDNA . The latFor CTCs analysis, due to the low input single cell DNA amount, whole genome amplification (WGA) is necessary to achieve enough genomic material suitable for library preparation and sequencing. The main WGA techniques utilized are: Degenerate Oligonucleotide-Primed (DOP-PCR) , MultiplDepending on the type of genomic alteration to be analyzed, e.g., CNVs, Insertion–deletion (indel) or single nucleotide variants (SNV), different metrics are required. For example, CNVs analysis requires high coverage uniformity, while for indels and SNV detection, low rate of amplification errors and allele dropout are particularly critical .Until now, most clinically validated LB tests are based on the assessment of actionable genomic alterations with the aim of supporting decisions about targeted cancer therapy and monitoring treatment response . Howeverhttps://grail.com) is developing a ctDNA-based multi-cancer screening test using advanced NGS approaches and Machine Learning (ML)  . Its firing (ML) . On the ing (ML) .Different studies exploit genomic approaches for the prognostic validation of circulating biomarkers. A large meta-study suggests that the presence of ctDNA KRAS mutations was associated with shorter OS in NSCLC . In anotTo address these challenges, Foundation Medicine has developed a new test based on circulating TMB which has already demonstrated clinical validity. Data published in Nature Medicine demonstrated that the blood-TMB (bTMB) test could predict response to atezolizumab in patients with previously treated NSCLC. Furthermore, the results show that bTMB may be an independent predictor of response compared to PD-L1 expression, as assessed by immunohistochemistry in patients who also had a tissue biopsy available . In otheThe use of genomic approach on ctDNA to identify resistance alterations to treatment still represents the only validated application in clinical practice. The ctDNA analysis of T790M-mutant NSCLC patients, resistant to first line EGFR Tyrosine Kinase Inhibitor (TKI) therapy, revealed different patterns of genetic alterations in patients with innate versus acquired resistance to Osimertinib . BRCA reTranscriptome analysis (both coding and non-coding RNAs) in LB represent a valuable source of biomarker for precocious identification, stratification and prediction of tumor outcomes . In partA more comprehensive view of circulating and CTC RNAs is provided by NGS-based approaches.Indeed, it is possible to profile mRNAs using methods that enrich poly(A) RNAs, or selectively reverse transcribe them. In case of non-coding RNAs, sequencing libraries can be obtained from total RNA as well as after enrichment for short transcripts.To profile the reduced quantity of RNA present in CTCs, several single cell analysis methods have been developed, e.g., Smart-Seq , CEL-seqOver time, it became clear that direct analysis of circulating RNAs could help better understand the evolutionary dynamics of cancer, by providing useful insights for developing personalized approaches for tumor diagnosis and therapy . SeveralFurthermore, Sabato and colleagues recently identified specific upregulated and downregulated circulating plasma EV linked to microRNA expression in metastatic melanoma patients compared to healthy donors. They bioinformatically identified 4 pEV-microRNAs able to distinguish metastatic patients from healthy controls with a high diagnostic potential . In addiIt has been shown that CTC-derived cell cultures and xenograft models could more accurately define cell clones with an initial metastatic long-term potential . AnalyziElsewhere, CTCs isolated from metastatic gastric cancer (GC) patients presented upregulation of genes involved in platelet adhesion. The transcriptomic profile of CTCs demonstrated the important contribution of platelets in EMT progression and acquisition of chemoresistance . Taken tEpigenetic alterations include DNA methylation, histone modification and chromatin accessibility to transcription factors. By determining gene expression patterns, those modifications shape cellular phenotype. Alterations resulting in an overly permissive or overly restrictive epigenetic regulation can lead to the generation of malignant subclones responsible for tumor progression and therapeutic resistance . For thiDNA methylation is the most studied epigenetic modification. Silencing of tumor suppressor genes by extensive promoter hypermethylation has been observed in several cancers . DNA metThe bisulfite conversion is the gold standard in 5-metil-cytosine (5 mC) detection and it’s based on chemical modification of unmethylated cytosines. Comparison of bisulfite-converted and reference unconverted sequences allows the identification of methylated cytosine with a single base resolution. Bisulfite conversion can be exploited for targeted analysis, when followed by Methylation-Specific PCR (MSP) or genome-wide characterization by means of Whole-Genome Bisulfite Sequencing (WGBS-seq), Reduced-Representation Bisulfite Sequencing (RRBS-seq)  methylatAs a general limitation associated with use of bisulfite is the damaging effect of chemical treatment on DNA, which is associated with material loss and requirement of moderately high starting material , bisulfiOther bisulfite-free assays are based on affinity enrichment either using anti-methylcytosine antibodies (meDIP-seq)  and low In addition to 5mC, 5-hydroxymethylcytosine (5hmC) has recently proven to be a regulatory modification associated with transcriptional activation  and seveFinally, a recently developed technological approach for detecting cancer-specific methylation and cancer-associated fragmentation signatures, without disrupting bisulfite conversion, is native Nanopore sequencing . In thisAside from DNA methylation, chromatin accessibility and histone modifications are becoming increasingly studied in parallel with the development of new technologies for their detection in the context of liquid biopsy. Open chromatin regions undergo higher fragmentation than compacted portions which are protected by nucleosomes. Those accessible regions are associated with transcription and have been associated with a typical fragmentation pattern related to the tissue-of-origin .As reference chromatin accessibility profiles for many cancer types are now available , computaIt has been observed that cancer-derived cfDNAbe more variable in length than cfDNA from non-cancer cells, due to the altered chromatin accessibility and associated nucleosome positioning. This feature of cfDNA has been exploited in the combined detection of fragmentation patterns and genetic markers resulting in improved sensitivity in cfDNA analysis . This apin vivo and of cell-free DNA nucleosomes (NucPosDB) has been recently released . Immunoprecipitation of cell-free fragments followed by low-depth NGS sequencing (cfChIP-seq) has been shown to inform about cell type and program-specific expression patterns  Sadeh e. In CC preleased . It has Alteration of epigenetic markers in liquid biopsy has shown to be clinically meaningful although it is still affected by limited availability of standardized tests .Most promising diagnostic applications are based on cfDNA methylation analysis, especially with custom panels, which better balance sensitivity and test cost. The PanSeer assay was developed for interrogation of cancer-specific DNA methylation signatures from peripheral blood . This asBeside those pan-cancer panels, several tumor-specific methylation patterns have been identified such as in BC in which the analysis of cfDNA methylation signature improves early detection of BC compared to mammography . WhereasEpigenetic markers monitoring has also proven useful for outcome prediction and longitudinal monitoring of minimal residual disease in different cancer types. Interrogation of a panel of ctDNA methylation markers in post-surgery colon cancer patients has been shown to be predictive of recurrence with good accuracy . SimilarMethylation markers are not only significant for prognosis, but also for predicting therapeutic response. Indeed, in patients with NSCLC, increased RARB2 methylation in cfDNA after chemotherapy and tumor resection was associated with disease recurrence at 9 months  as well Paired analysis of another nine-gene promoter methylation panel in ctDNA and CTC of a different NSCLC cohort, analyzed before osimertinib treatment and during progression, revealed inconsistency between CTC and ctDNA profiles. However, in both cases Progression Disease (PD) was associated with increased methylation levels with respect to baseline samples. Kaplan-Meier analysis revealed correlation between earlier PD and methylation status of at least one panel gene . As thosProteomic analysis of LB represents a minimally invasive and repeatable way to accomplish a broad range of milestones such as: identification of targets to direct new treatments, development and validation of biomarkers to allow early detection of diseases, design of proteomic signatures as predictive models for cancer diagnosis and prognosis . Using tA traditional method to validate protein biomarkers is using enzyme linked immunosorbent assays (ELISA), but this approach is time consuming, expensive and depends on the availability of existent antibodies pairs for every target protein. A method which does not rely on existing validated antibodies is MS . To analA complementary analysis of both plasma and urine from the same patient allows a thorough examination since urine is a filtrate product of blood . As a reAlthough extremely informative, MS results are often validated using more conventional techniques such as ELISA and Western Blotting (WB), which can be still useful to identify a few target proteins in low abundance, but without giving information about the protein expression levels . NeverthDifferently to standard ELISA, multiplex ELISA can detect and process up to 1,000 human proteins, allowing us to develop a multiplex bioassay and add more candidate proteins into a panel of interest . Howeverad hoc microwells all steps of WB are performed, minimizing cell loss and maximizing protein concentration , which comes from the fusion of two technologies, FC and MS. MC allows in-depth analysis of homogenous cell populations, providing measurement of over 40 parameters at single cell resolution  from a lNoteworthy, a technology that could be used for rapidly investigating surface protein expression in CTCs is the DEPArray system . Already known for its ability to isolate single, viable rare cells using dielectrophoretic principles combined with an image-based selection, DEPArray technology has gradually gained clinical relevance during the lastade .It has been decades that the diagnostic, prognostic and treatment monitoring value of serum and urine tumor markers have been proved. Serum PSA levels for diagnosing prostate cancer in men, cancer antigen 15-3 (CA15-3) for breast cancer and cancer antigen 19-9 (CA19-9) for pancreatic cancer are just a few examples of how medicine has applied soluble protein dosage to diagnose cancer to date . HoweverPlasma and urine have been the most characterized biological source of data by far, however some biomarkers are massively diluted and alternative more concentrated clinical samples have been investigated recently. Among the broad set of biological fluids considered valuable sources for biomarker discovery, nipple aspirate fluid (NAF) is gaining an emerging role for BC screening in young women at high risk. Indeed, not only mammography has low specificity in differentiating between benign and malignant growth, but also in this scenario this test lacks accuracy due to young breast density. Sadr-ul Shaheed and collaborators  investigSimilarly, Gabriel Vales et al. have recently validated 20 proteins using quantitative multiplex ELISA array starting from vitreous samples while looking for uveal melanoma biomarkers associated with metastatic risk . In thisIf soluble proteins’ dosage is effortless because they are easily accessible, we cannot state the same for surface, intracellular and particles’ proteins. Over the past decade, EV and particles EV (EVP), which include small exosomes, large exosomes and exomeres  have gaiA recent multicentric study has elegantly investigated the proteomic profile of EVPs in 426 human samples from tissues, plasma and other bodily fluids, related to adult  and pediatric cancers (neuroblastoma and osteosarcoma) . Using MAs for many exploratory studies, Hoshino’s research group interrogated publicly available protein databases to find proteome quantitative data and then make a list of tumor specific EVP proteins of interest . After hSimilarly, Yunee Kim and his collaborators used targeted proteomics combined with computational biology to define proteomic signatures for prostate cancer from urines collected by men with extra prostatic and organ-confined prostate cancer, in a 74-patient cohort. Since protein signatures, rather than individual soluble proteins, allow us to accurately discriminate patient groups, they tried to distinguish pT2 stage from pT3 stage tumors, before radical prostatectomy, for potentially modifying and personalizing patient treatment . SelecteRecently, FC-based technologies have been used and adapted to detect multiple markers at a single cell level too. In this regard, CTCs from osteosarcoma patient blood samples were characterized by Shulin Li’s research group a couple of years ago . After hSince the discovery of the famous Warburg effect , severalMetabolomics focuses on the profiling of small intracellular or free metabolites  in bodily fluids including blood, urine, CSF and saliva . MetabolMetabolomics is mainly used for early cancer detection and biomarkers discovery . UchiyamInteresting results have been obtained from studies of metabolomic composition of urine(u)-EV of prostate cancer patients . AccordiIncreased evidence has demonstrated the influence of microbiota in human malignancies including cancer . MicrobiIn conclusion, implementation of analytical techniques and validation of algorithms for analyzing metabolomics data are still needed to let metabolomics take its rightful place among precision oncology omics.The reduced amount of target molecules in LB affects the capability to detect low-frequency genomic variations. To increase detection sensitivity, a panel of genes rather than the whole genome can be sequenced numerous times, even though this leads to higher false positivity risk. To solve this issue, advanced bioinformatic tools or machine learning algorithms are needed to reduce false positive results and eliminate background noise.Tools such as the popular IchorCNA , have beSimilar approaches can be used to detect CTC genome alterations. Once CTC are isolated from biological fluids their DNA is amplified and sequenced by LP or ultra LP-WGS. At a coverage of 1X or 0.1X heterogeneity that might arise due to small genomic aberrations such as SNVs and short indels will be missed. Several computational tools are available for CNV analysis using bulk sequencing data including CNVkit , ControlWhen the goal is to analyze large datasets, more computationally efficient strategies are required. AneuFinder  and SCOPRecent research suggests that cfDNA fragmentation patterns can provide additional information beyond the genetic analysis of somatic mutations and copy-number abnormalities. Indeed, DNA fragmentation from dying tumor cells seems not randomly distributed but also determined by the DNA sequence: it appears to reflect the chromatin structure and epigenetic states of the cells, from which DNA fragments derive . Penederhttps://www.proteinatlas.org): a repository of information about mRNA and protein expression across several healthy tissues and cancers, and the Genotype-Tissue Expression (GTEx) project .Among the positive outcomes of multi-omic investigations there is the construction of useful databases, built to help researchers delineate which proteins are expressed in specific tissues in physiological and pathological conditions. Examples are computational resources such as the Human Protein Atlas  and ICGA (https://dcc.icgc.org/) have been created to collect all omics data in an orderly manner. Since there is an urgent need to connect multi-level information concerning molecular signatures and the phenotypic manifestation of different cancer types, AI and ML approaches have been proposed to transform big-sized complex data into evidence-based medicalisions. Although there is growing evidence demonstrating the potential of ML to improve the performance of various LB tests, their integration into the clinical workflow represents the main challenge of the upcoming years (On the one hand, multi omic analysis seeks to fullyipher the complexity of cancer heterogeneity, in contrast it generates a huge amount of data. Omic data can “apparently” be very different from each other as numerous biological and non-biological variables can influence their production. The choice of patients, the type of protocol adopted to perform the analysis, or the personal experience of the operator can greatly affect the final data. Over the years different repositories like TCGA (ng years .


The need to advance cryogenic technologies for fertility preservation is becoming increasingly critical across a variety of fields. In humans, these techniques are important not only to address fertility problems, but for men and women with medical concerns or treatments affecting reproductive potential, or those wanting to delay child-bearing years. In species of agricultural value, similar tools can be applied to assist in production efficiency, whereas, in endangered animals, the goal of maintaining genetic diversity in small populations may only be possible through the application of gamete and gonadal tissue cryopreservation. This Research Topic highlights the challenges and areas of advancement in the field of cryopreservation for fertility preservation .Antonouli et al., the cryopreservation protocols most commonly used in human fertility preservation for each cell/tissue type are detailed, including describing the growing preference for vitrification methods for oocyte and in vitro-derived embryo cryopreservation, comparing and contrasting recently developed tools for open (direct liquid nitrogen contact) vs. closed vitrification systems, and discussing the concerns for patient accessibility to necessary fertility preservation options and their variable regulation across countries.Currently, the two most commonly utilized methods for preservation using cryogenics are slow freezing, in which cells are progressively cooled to −196°C, either controlled via a programmable freezer, or uncontrolled in a container with an internal cooling rate of ~1°C/min, and vitrification, a rapid process in which the liquid phase of a cell or tissue solidifies into a glassy phase without ice crystal formation. Both techniques are regularly applied in human clinics. In the review paper by Swelum et al., Japanese quail egg yolk (higher water and select monounsaturated fatty acids and trace elements content) was compared with the gold-standard chicken egg yolk (higher protein and poly-unsaturated fatty acid content) and a mixture of the two for their ability to support ram sperm cryopreservation. They found that the chicken egg yolk-based semen extender maintained the highest levels of sperm survival, total motility, and plasma membrane and DNA integrity, whereas sperm morphology and certain motility parameters were better supported by the quail egg yolk-based extender. These variances were attributed to differences in fatty acid, protein, fat, moisture, and ash content in quail vs. chicken yolks, and represent insight into the relative contributions of the broadly-used but not fully understood egg yolk cryo-diluent.Despite their breadth of use in human clinics, no cryopreservation protocol is fully without its challenges. Potential cell damage may occur due to ice formation, cryoprotectant toxicity, and/or thermal, oxidative, and osmotic stress to the cells. Further, cryopreservation conditions which are successful in humans may have very different outcomes when utilized in other species. This includes ram sperm, which are particularly sensitive to cold-shock due to their low membrane cholesterol:phospholipid ratio. In the research by Ji et al., the authors assessed the influence of melatonin supplementation on combating the oxidative stress of cryopreservation in sheep morulae. While vitrification significantly altered several gene expression pathways compared with fresh controls, the authors demonstrated that within-group variation was higher than treatment-based variation, suggesting that individual embryos responded differently to cryopreservation. They also found that melatonin supplementation in vitrified ovine morulae upregulated several key embryonic development-associated genes in the transforming growth factor β and forkhead box subfamily pathways, as well as supporting XPA expression, part of the Nucleotide excision repair system for DNA damage, following vitrification.Focusing on embryos, in the research by Chen et al., the technical challenges presented by cryopreservation can be enhanced in tissues and organs, as the samples in question represent heterogeneous cell populations with complex architecture and cell-cell interactions, while also facing the additional task of needing rapid and even heat transfer across a large mass. Despite this, gonadal tissue cryopreservation efforts are rapidly expanding for both human and endangered species work, as options to collect mature gametes are often limited . The research described by Hartzler et al. and Peris-Frau et al. evaluated which of these methods better supported the survival of gonadal tissues for wildlife species, in ovarian and testicular tissues, respectively. In both studies, slow freezing of gonadal tissues was compared with needle-immersed vitrification, in which tissues are threaded onto a small-gauge needle for cryoprotectant equilibration and plunged directly into liquid nitrogen. Hartzler et al. demonstrated that African painted dog ovarian tissue maintained a higher density of morphologically normal ovarian follicles in needle-immersed vitrification compared with slow freezing, but minimal differences in apoptosis via gene expression markers or TUNEL stain for DNA integrity were observed among cryopreservation groups.As reviewed by Peris-Frau et al. determined that carnivore testicular round cells (spermatogonia through to early spermatids) had higher viability and DNA integrity in needle-immersed vitrification-treatments compared with slow freezing. However, most of the 33 species assessed in the study—including artiodactyls, carnivores, and primates—elongated testicular cells (elongated spermatids and spermatozoa) were better preserved under slow freezing conditions in testicular tissue, with only rodents displaying no significant difference among the two cryopreservation treatments. Together, these studies advance our understanding of the species- and tissue-specific needs for gonadal tissue cryopreservation for species conservation. Nevertheless, new advances in low-toxicity cryoprotectants, CPA delivery/perfusion methods, and novel nano-warming techniques hold promise for gonadal tissue cryopreservation efforts, as reviewed by Chen et al.. Moreover, the use of cryopreserved-warmed gonadal tissue either via tissue transplantation or in vitro culture reviewed by Antonouli et al. represents growing areas of research to both utilize preserved tissues for fertility restoration, and also to improve our understanding of the mechanisms of cryo-damage such that we may continue to develop tools to address them moving forward.Highlighting the challenges both of cell heterogeneity in organs and species-specific differences in cryo-sensitivity, BM: Visualization, Writing—review and editing. JS-F: Writing—review and editing. CS-Ú: Writing—review and editing. JN: Writing—original draft.


Thedevelopment of biocompatible coatings and the availability of differentcalibration approaches enable the in vivo samplingof exogenous and endogenous compounds from the living plants and animalswithout the need for tissue collection. In addition, new geometriessuch as thin-film coatings, needle-trap devices, recession needles,coated tips, and blades have increased the sensitivity and robustnessof in vivo sampling. In this paper, we detail thefundamentals of in vivo SPME, including the variousextraction modes, coating geometries, calibration methods, and dataanalysis methods that are commonly employed. We also discuss recentapplications of in vivo SPME in environmental studiesand in the analysis of pollutants in plant and animal tissues, aswell as in human saliva, breath, and skin analysis. As we show, in vivo SPME has tremendous potential for the targeted anduntargeted screening of small molecules in living organisms for environmentalmonitoring applications.Solid-phase microextraction(SPME) is a well-established sample-preparationtechnique for environmental studies. The application of SPME has extendedfrom the headspace extraction of volatile compounds to the captureof active components in living organisms via the direct immersionof SPME probes into the tissue ( However,such studies require a feasible analytical method that facilitatesthroughput/efficient isolation and quenching of exogenous and endogenouscompounds of interest along with precise analysis.Theanalysis of contaminants in environmental samples is the firststep in evaluating the negative impact of such compounds in an ecosystemand the living organisms within it. 5 SPME combinessampling, cleanup, and preconcentration into a single step and canbe coupled with modern instruments, such as gas chromatography–massspectrometry (GC-MS)6 and liquid chromatography–massspectrometry (LC-MS).7 Recently, researchershave enabled fast and high-throughput analysis in pharmaceutical studiesvia the direct coupling of SPME devices to MS.8 SPME is a nonexhaustive extraction method, which means that it onlyextracts a free fraction of the analyte from the sample matrix. Thisfeature enables the measurement and discussion of the bioavailabilityof various environmental contaminants.9Solid-phasemicroextraction (SPME) was introduced in 1989 as afast, simple, and green sample-preparation technique that can be usedwith a wide range of samples.12 In addition, the availability of different extraction phases makesit possible to use SPME devices to extract pollutants with a rangeof different physicochemical properties, such as pharmaceutical andpersonal care products (PPCPs), pesticides, and metal organic compounds,among others.15 It is generally thought that the free concentrationof a given compound is a stronger indication of its bioavailabilitycompared to its total concentration.16 Byextracting a small portion of the free fraction of analytes (negligibledepletion), SPME enables the direct measurement of the free concentrationof certain contaminants in environmental samples. Specifically, SPMEcan be used to monitor dynamic biological processes in living systems,and it also makes it possible to perform repeated extractions froma single spot on a given sample. Furthermore, SPME has also been optimizedfor on-site environmental analysis with microinstruments, such asfield-water sampling.17 Other extractionmethods such as liquid–liquid extraction (LLE) or solid-phaseextraction (SPE) can also be applied in in vivo or ex vivo studies; however, those techniques could not bedirectly used for in vivo sampling performed withoutthe collection of tissue biopsy or biofluid. In addition, other in vivo sampling methods such as microdialysis (MD) showa different coverage of metabolites than in vivo SPMEdoes, and this is because MD primarily facilitates the extractionof polar species while SPME covers semihydrophobic and hydrophobiccompounds as reported for the sampling of brain tissue.18SPME was initially introduced as novel extraction strategyin environmentalstudies, but it has since become a leading method in this scientificfield. Currently, SPME—and headspace-solid-phase microextraction(HS-SPME) in particular—is widely employed in analyses of volatileorganic compounds (VOCs) and semivolatile organic compounds (SVOCs).in vivo environmentalstudies to monitor changes in small endogenous molecules as well asto directly monitor pollutants in living organisms.19 Additionally, SPME is a nonexhaustive and nonlethal samplingmethod, which has enabled its use for measuring the free concentrationof drugs in solid tissue, both via a series of laboratory experimentsand in silico using a mathematical model developedin COMSOL Multiphysics.20 The invivo application of SPME in environmental studies allowsresearchers to dynamically analyze the distribution, accumulation,and depreciation of single or multiple molecules, as it enables therepeated sampling of the same tissue in a subject.8In vivo SPME sampling can also captureshort-lived and unstable metabolites by stabilizing highly reactivesmall molecules, which may undergo degradation during sample handlingand storage in typical ex vivo studies.21 This is a significant feature, as such metabolitesmay provide additional information about metabolic changes, for instance,in exposome-wide association studies (EWAS).22SPME has been applied in in vivo SPME technique,24 this reviewexamines how in vivo SPME has been applied in environmentalstudies over the past 5 years. We begin by providing a brief overviewof the fundamentals of in vivo SPME and some relatedtechniques before moving on to a more detailed discussion of its mostimportant aspects, namely, the development of in vivo SPME devices, calibration methods, and data analysis. Next, we conducta review of the main applications of this technique in environmentalstudies monitoring plant, animal, and human systems. Finally, thispaper concludes by considering future possibilities for SPME and itspotential in environmental studies.Whereas previous reviews have mainly focused on the developmentof the 22.125 Moreover, cold fiber SPME (CF-SPME)—which entailsheating the sample matrix and cooling the fiber coating simultaneously—canbe used to increase sensitivity to VOCs released from soil and sedimentsamples.27 This technique implies the need for a systemto control the SPME fiber’s temperature in order to capturetrace level molecules released by a living system. Furthermore, theuse of SPME-Arrows can offer improved sensitivity and mechanical robustnessfor air-phase extraction, as they contain larger volumes of sorbentthan standard HS-SPME fibers.28Small molecules in environmental samples can bedistributed or released into the sample’s gas, liquid, andsolid phase. HS-SPME is a commonly used approach for capturing thefree concentration of pollutants in the gaseous phase. In this extractionmode, an SPME fiber is exposed to the air phase above samples to extractvolatilized compounds. In contrast, needle-trap devices (NTD) enableexhaustive extraction by using small needles containing a packed sorbentbed to trap both fluid-borne analytes and particles. Both HS-SPMEfibers and NTDs can be directly coupled to GC injection systems, whereinthe analytes adsorbed from the air phase are released for instrumentalanalysis. In this case, NTDs and SPME fibers can be used in concertto detect both the free and total concentrations of pollutants inthe air phase, thus providing a comprehensive assessment of the risksassociated with certain environmental processes.29 For instance, in-tube SPME has been appliedin environmental studies for its ability to directly measure nonvolatilecompounds in aqueous matrices.30 Needleswith a recession notch coated with an extraction phase can protectagainst mechanical damage and have been applied in untargeted analysesusing living fish.31 Space-resolved SPMEwith discontinued coatings can be used to perform simultaneous extractionsfrom different tissue in vivo.32 Coated tips and minitips, which were developed based onthe a coated tip apex (1 mm), allow the extraction of small moleculesfrom limited sample amounts (<10uL), such as blood from mice.34 Such designs also allow direct coupling with MS for high-throughputanalysis and can be applied for nontarget analysis—featureswhich are highly beneficial in exposome studies. In SPME, larger surfaceareas will result in improved extraction efficiency and sensitivity.35 Thin-film SPME (TFME) offers a larger surfacearea to extraction phase volume ratio, which means that more of thedevice is coming into contact with the sample. Depending on the researchobjectives, the TFME membrane coatings can be modified for gas chromatographyor liquid chromatography. In addition, TFME and CF-SPME can also becombined to further enhance method sensitivity for specific applications,such as measuring fragrance compounds in the air.36 Coated blade spray (CBS) is another geometry that allowssample preparation to be directly connected to MS, and it has alsobeen used in environmental analyses.37 Furthermore,the analysis of nonvolatile pollutants via GC has been enabled bythe development of an on-fiber derivatization technique that combinesthe derivatization reaction and extraction steps during SPME.38 Notably, simulation-based geometry optimizationcan also be used to design new SPME devices based on the aims of theresearch and the sample matrix.39in vivo studies.Onthe other hand, direct immersion SPME (DI-SPME) is the major extractionmode for liquid, solid, and semisolid environmental samples. In DI-SPME,SPME devices coated with an extraction phase are immersed into thesamples for a fixed time; the analytes are then desorbed from theextraction phase by directly coupling the device to mass spectrometry(MS) or by applying an optimized solvent, which is subsequently subjectedto LC-MS analysis. The application scope of DI-SPME has graduallybroadened with the development of new SPME device geometries.in vivo samplingvia coating optimization. For in vivo studies, theextraction phase coating should be safe and biocompatible with theorganism being studied. Since a pure PDMS extraction phase possessesantifouling properties, PDMS-coated SPME fibers, such as PDMS/DVB/PDMS,are able to provide stable extraction performance after over 100 extraction/desorptioncycles.40 SPME coatings that use polytetrafluoroethyleneamorphous fluoroplastics (PTFE AF 2400) as a particle binder—whichcan hold hydrophilic–lipophilic balance (HLB) adsorptive particlescovering compounds with a broad range of hydrophobicities—havealso frequently used for in vivo analysis.41 Other biocompatible coatings, such as C18, mixed C18 (mixed-mode) and cation-exchange particles,polydopamine (PDA), polymeric ionic liquids (PILs), and polyacrylonitrile(PAN), are also suitable for in vivo studies.42 Furthermore, the introduction of nanomaterialsand electrosorption makes it possible to shorten the extraction processwith in vivo SPME fibers to around 1 min.43 Besides assembling multiple SPME fibers withdifferent coatings as a “swab” for in vivo saliva analysis, the chemical coverage of these coatings can beextended to cover different environmental pollutants or endogenousmetabolites.44 In addition, coatings composedof modern components, such as nanomaterials, metal–organicframeworks (MOFs), and molecularly imprinted polymer (MIPs), haveenhanced the durability, sensitivity, and specificity of invivo SPME studies.8SPME can also be optimized for 45 and CBS47 have increased thethroughput of the regular analytical workflow. Furthermore, a mechanicallyrobust SPME sampler composed of six stainless-steel bolts coated witha layer of HLB/PAN particles was designed for the on-site extractionof untargeted pollutants in environmental waters.48 Moreover, the development of drone-based TF-SPME watersampling49 and USB-powered CBS50 with a transportable MS device has also extendedthe scope of on-site analysis and could be used in future in vivo environmental studies.On-sitebiomonitoring and high-throughput analysis have also benefitedfrom constant developments and improvements to SPME. For instance,the development of the microfluidic open interface (MOI)2.251 In SPME,the microextraction of a given compound can be performed after equilibriumhas been achieved between the concentration of the target compoundin the sample matrix and the extraction phase (SPME coating). Theamount of the compound captured by the extraction phase can be expressedasne is theamount extracted, C0 is the initial concentrationof the target analyte in the sample, Vs is the sample volume, Vf is the volumeof extraction phase, and Kfs is the distributioncoefficient of the analyte between the extraction phase and the samplematrix. However, in most SPME applications, the volume of the samplematrix is much larger than the volume of the SPME coating (Vs ≫ KfsVf); thus, invivo SPME study, it is recommended to generate the compound’sextraction time profile in advance to select the appropriate calibrationmethods. External mass transfer would impact the calibration approach,especially when the sample matrix is complex. For example, the tissueof the living organism would contain both the solid phase and liquidphase. The Kfs would change due to theexternal mass transfer among two different phases. In this case, loadinginternal standards on the fiber could be a convenient option for methodcalibration. More diffusion-based calibration methods can be foundin previous work.52The quantitative analysisof compounds extracted via SPME differs from typical exhaustive extractionanalysis. As such, various calibration methods have been developedto quantify the concentrations of target analytes extracted from biosamplesusing SPME extraction phases.54 Such kinetic calibration models require isotope-labeled standardsto be preloaded onto the fiber. During microextraction, the labeledstandards are desorbed into the sample matrix and the analytes areextracted into the SPME coatings, and the free concentration of analytesis then calculated based on the amount of remaining labeled standardsand the extracted compounds. In this case, the losing percentage ofisotope labeled standards on the fiber will be equal to the increasingpercentage of analyst on the fiber:Q as the amount of remaining isotope labeled standards on the fiberafter a fixed time, q0 as the originalisotope labeled standard loading on the fiber, and n as the amount of analyte on the fiber. Then the amounts in the samplescan be calculated by in vivo studies dueto the introduction of exogenous compounds  into the living tissue or biofluids.However, other calibrationmethods can be used for compounds thatrequire longer times to reach equilibrium, for example, on-fiber kineticcalibration and sampling rate calibration.invivo studies. This method is based on the pre-equilibriumstage of a fast microextraction, wherein the amounts of extractedanalytes follow linear response patterns ,numbers, identified compounds, or internal standards to compare differentextraction methods.57 If internal standardsare selected for the in vivo SPME workflow, theirproperties, such as cLogP, should be within the range of regular environmentalpollutants.582.359 However,for in vivo studies, the sample-preparation protocolshould minimally impact both the regular functioning of the livingorganism being studied and the ability to perform dynamic sampling.As shown in in vivo SPME analytical protocol, as this helps to ensurethe living system under investigation is protected from contamination.For in vivo SPME sampling devices, the solid extractionphase should be introduced into the living tissue using a hypodermicneedle or directly placed in the biomatrix for the extraction. Aftera fixed time, the extraction phase is removed from the living systemand the analytes are desorbed into an appropriate mixture of solvents,which is followed by instrumental analysis. In vivo SPME should use biocompatible coatings, and several parameters affectingextraction efficiency, including precondition time, extraction time,and desorption time, should be optimized for certain analytes. Moreover,SPME can be directly coupled to MS to provide close-to-real-time informationabout metabolic changes, as well as their levels, in the analyzedmatrices.A typical environmental sample-preparationprotocol includes samplecollection, homogenization, extraction of small molecules, and cleanupprior to instrumental analysis.in vivo SPMEstudies fall into one of two major classes: (1) investigations ofthe content and behavior of environmental pollutants and (2) analysesof how environmental contaminants impact the functioning of livingorganisms (metabolomic profiling). Both classes involve the targetedand untargeted analysis of exogenous and endogenous compounds, and in vivo SPME devices are capable of extracting these moleculesat the same time. In the first approach, the environmental behaviorof exogenous compounds includes the targeted analysis of certain compoundsand their known metabolites as well as not yet discovered productsof their transformations. In the second approach, the environmentalanalysis within the living organisms comprises the extraction of knownendogenous compounds as well as unknown metabolites. The invivo SPME workflow can be optimized for certain compoundsduring targeted analysis; however, advanced data analysis is particularlyimportant in untargeted analyses, as it facilitates the extractionof reliable in vivo bioinformation.Depending on the research objective, in vivo SPME sampling isthe coverage of analytes by SPME coatings. In vivo study usually needs to calibrate multiple compounds such as exposurecompounds, their biotransformation products and/or various metabolitesin the environmental analysis. Those analytes show different Kfs and a fixed time extraction process willcapture compounds in different stages of reaching the equilibrium among studied groups, a relative quantificationbased on the response of known and unknown compounds can be performedwithout standards. However, a calibration based on compounds withsimilar physicochemical properties or synthesized standards shouldbe performed to validate the findings after metabolomics data analysis.Such validation is typically missing in in vivo studiesdue to either the availability of standards or the complexity of experiments.In most in vivo studies, targeted compounds for methoddevelopment are used, which however limits its application for othercompounds captured by SPME fibers. In this case, in vivo analytical method development should consider the simultaneous calibrationof compounds with different physicochemical properties such as polarityor log Kow.One ofthe issues of riumsee 2. Oneofin vivo SPME analytical workflowis the impact of sample storage on thebiochemical profile and composition of the analytes. For example, ex vivo SPME analysis of fish muscle samples that had beenstored for 1 year revealed a 10-fold decrease in the number of detectedmolecular features compared to in vivo SPME performedon living fish.21 Thus, in vivo SPME extracts should be analyzed in real time to avoid the detrimentalimpacts of storage. As confirmed in a recent study, instrumental analysisimmediately following in vivo SPME sampling and areverse time series experimental design should be the preferred approachesfor the metabolomic profiling of unstable compounds, while storingSPME fibers or the desorption solution for 1 month will not affectthe metabolic profile. At the reaction level, metabolites involvedin homologous series with butylation reactions were shown to be themost stable during storage.60Another importantconsideration in the 2.4in vivo SPME and regular SPME is nearly the same.However, the workflow for the two types of SPME analysis differs significantlyfor untargeted analysis, such as LC-MS-based analysis. At present,untargeted analysis, or nontargeted analysis, is the preferred methodfor screening harmful compounds or finding linkages between environmentalexposure and endogenous metabolites.61 Asshown in Fortargeted analyses, the data analysis procedurefor in vivo studies.62 Findings have demonstrated that in vivo SPME is able to capture active compounds that might disappear aftereven 1 day of storage at −80 °C;60 however, such short-lived compounds may never be reported or fullyinvestigated for biological functions. In this case, in vivo study usually only reports a few known compounds with availablestandards, some tentative compounds with database entries, and a lotof totally unknown features from instruments. Biological functionaldiscussions of in vivo studies exclusively focuson known compounds, which omits any discussion of their origins andrelations with other compounds.19The annotation of small molecules from living samples remains achallenge for in vivo SPME-based studies.63 Under this approach, the mass-to-charge ratiosof unknown compounds are coupled with fragmental ions for identification.64 Furthermore, the distances between the mass-to-chargeratios may reveal certain types of reactions or structures, whilethe data mining of known reactions can serve to validate such phenomena.65 For instance, a paired-mass distance (PMD) of2.02 Da is always associated with a double-bond broken process. Theextension of reaction/structure directed analysis, namely, PMD-basedreactomics, further provides the qualification and quantificationmethods for PMD analysis.65 In this case,evaluating changes in certain PMDs enables researchers to determinethe reaction level changes within the sample67 and skip the annotation of specific compounds.In addition, such reactomics analysis can also be used to generate in vivo metabolic pathways composed of connections amongmetabolites based on high-frequency PMDs. As shown in in vivo SPME studies at the reaction level, which is helpful in discoveringnew pathways of pollution or influenced endogenous compounds.To address this issue, reaction/structure directed analysis wasdeveloped for 3in vivo SPME has been employedin numerous studies to analyze environmental contaminants in complexmatrices with results proving its ability to provide useful informationabout the dynamics of the studied ecosystems.24 Chemicals such as persistent organic pollutants (POPs), endocrine-disruptingcompounds (EDCs), pesticides, PPCPs, disinfection byproducts (DBPs),and heavy metals can be released into environmental matrices suchas soil, air, water, and sediments. As a result, plants, animals,and humans may be exposed to these compounds, either through directcontact with environmental matrices or via the food chain. The aforementionedemerging contaminants can accumulate and exhibit toxicity in livingorganisms, particularly with respect to their influence on differentcellular processes at the genomic, proteomic, and metabolomic levels.As such, it is critical to investigate or monitor the behavior, levels,and distribution of these compounds in living systems. Fortunately,tools such as in vivo SPME are capable of extractingsuch exogenous compounds quickly and efficiently, and the developmentof a commercially available SPME fiber for in vivo studies has led to it becoming the preferred technology in an increasingnumber of environmental studies. Furthermore, in vivo SPME has been introduced as a novel technology for simultaneouslyextracting a wide range of small molecules directly from living systems,which has been key in developing a more robust understanding of howexogenous substances impact the functioning of living organisms. Thefindings of various metabolomic studies have confirmed the feasibilityof using SPME devices to extract and stabilize endogenous molecules—especiallyhighly reactive metabolites or intermediates of biochemical processes—directlyfrom living systems. in vivo SPMEin environmental science over the past 5 years.In recentyears, 3.1in vivo SPME isnow usually implemented to track the absorption, transmission, anddistribution behaviors of certain exogenous compounds and their metabolitesor for the analysis of metabolic pathways (untargeted metabolomics)affected by environmental contaminants. DI-SPME has also been usedto investigate the distribution of pharmaceuticals, phytohormones,organophosphorus pesticides, and organochlorine in living plants,with extractions being performed by placing the SPME fibers insideprepunched holes on the foliage.73 Furthermore, several environmental properties, suchas distribution concentration factor (DCF), have been calculated toimprove our understanding of molecular behavior in living plant sap.In addition, PMDS-coated SPME fibers have been implemented to reducethe matrix fouling during in vivo sampling of fatrich matrices, such as avocado.84 In anotherrecent study, researchers developed a water-swelling sampling probeto detect neonicotinoids in plants. This fiber decreased the limitsof detection for neonicotinoids by introducing a water-swelling structure,thus providing better performance compared to commercially available in vivo SPME fibers.72Plants are constantly exposed tovarious environmental pollutantsvia the air, water, soil, and sediments. Initially, SPME was introducedas a HS-SPME mode in plant-related research, mainly to extract flavorsreleased by the plants. However, in vivo SPME in plant metabolomicsis another research trend in environmental studies. Musteata et al. evaluated the performance of in vivo SPME in Amazonian plants, with results showing that it was ableto detect a number of unique compounds.68 Risticevic et al. utilized in vivo SPME and two-dimensional gas chromatography-time-of-flight massspectrometry (GCxGC-ToFMS) to investigate changes in the “Honeycrisp”apple metabolome profile during maturation.74 Their findings showed that several metabolites and chemical classeswere upregulated during ripening and that the in vivo SPME device was able to successfully extract and detect amaryllidaceaealkaloids as a bioactive metabolite in the analyzed apples. Such compoundshave not been previously reported due to changes in metabolite compositionduring sample preparation .The application of in vivo environmental studies on plants, tissues, and organs.This approach is especially valuable in metabolomics studies thataim to evaluate the influence of exposure to contaminants beyond theirdistribution within plants. Nevertheless, new coatings or device geometriesare still needed to improve SPME’s overall performance in plantmetabolomics, as is the development of novel methodologies that accountfor the complex matrix effects related to the sampling of living plants.DI-SPME isbecoming the preferred mode of extraction for 3.2in vivo animal studies are more challenging, particularlywith respect to performing extractions. Typically, animal studiesare based on the withdrawal of biosamples, such as blood or tissuebiopsy, which introduces extra influences that may be harmful to theorganism. As a minimally invasive sampling technique, in vivo SPME has been applied to monitor the distribution and metabolicprocesses of certain exogenous  and endogenous compounds in living animals.4 Similar to in vivo plant studies, SPME devices used in animal studiesmust be designed to ensure that the developed method is easy, fast,and feasible for sampling. For HS-SPME, a regular SPME device canbe used to extract analytes from collected biofluids. However, in vivo tissue or blood sampling—which is where DI-SPMEis usually applied—requires a device with low invasiveness,such as the use of an SPME syringe to deliver the SPME fiber intothe living system. Notably, the needle of the SPME syringe featuresa recession notch that is coated with the extraction phase, whichallows the delivery device and extraction device to be integratedas one.31 Finally, TF-SPME blades can alsobe used in animal studies, as they offer enhanced sensitivity dueto their larger surface area compared to SPME fibers.22Unlike plant studies, where the living organism is largely stationary, In vivo SPME animal studies canbe divided into two categories: laboratory studies and field (on-site)sampling. Conventional sampling methods can be damaging to livingtissue, such as brain tissue; however, the use of biocompatible SPMEfibers can minimize the negative effects of sampling and allow researchersto monitor dynamic molecular-level changes in response to certainstimuli or exposures in such sensitive tissue. SPME was initiallyapplied in animals to monitor the pharmacokinetics of toluene in thebrains of free-moving mice in order to examine the neural system damagecaused by exposure to this compound.85 Theresults revealed a peak concentration of toluene in the hippocampuswithin 30 min and depletion after 90 min. In another study, an in-depthprofiling of 52 oxylipins, which at very low concentrations are lipidmediators of important brain processes, was also carried out in vivo by inserting SPME fibers into the brain of freelymoving rats.81 In another study, deep brainstimulation processes were monitored in rats to track metabolite-levelalterations, with findings revealing significant changes in the aminoacid, citrulline, as well as in various phospho- and glycosphingolipids.80 In addition, in vivo samplingof rat liver with custom-made MIP-SPME fibers was successfully appliedto monitor luteolin and its metabolites.7986 In this study, different sectionsof sponges were sampled, and the SPME devices were successfully usedto isolate different signaling molecules and organic pollutants, suchas monocyclic aromatic hydrocarbons (MAHs) and polycyclic aromatichydrocarbons (PAHs), among others absorbed from the surrounding waterthat may accumulate in the sponge and affect its metabolism.Other exposure studies monitoring emerging environmental pollutionhave been conducted on-site with living aquatic animals. SPME’sability to extract a wide range of metabolites has also been exploitedto analyze the composition of small molecules present in the spongeholobiont.in vivo animalmodel to study the exposure effects of pollutants present in water.For this purpose, in vivo SPME technology has beenapplied in EWAS in combination with metabolomics studies in orderto track the behavior of certain toxins and monitor biochemical responsesafter exposure to those factors, mainly in an aquatic environment.Several quantitative in vivo SPME analyses of a widerange of pharmaceuticals showed that their concentrations in fishare related to exposure concentrations in effluents. In vivo SPME has also been used in wild fish to monitor emerging contaminantsin natural fish habitats.87 Roszkowska et al. employed a case-control in vivo SPMEexperimental design to analyze the changes in the fish muscle metabolomefollowing exposure to benzo[a]pyrene. Their findingsrevealed that the levels of selected amino acids, lipids, and componentsof osmotic regulation changed in fish muscles after exposure to thistoxin.82 Moreover, a new SPME device withelectrosorption enhancement—namely, a novel, custom-made CNT@PPY@pNEfiber—was employed to monitor ionized acidic pharmaceuticalsin fish.43 The results of this study showedthat the new electrosorption-enhanced fiber was able to provide ultrafastsampling (1 min) and continuous monitoring.Living fish are commonly used as an In vivo SPME can also be applied to monitor howenvironmental pollutants impact the functioning of living systemsin animals. In a recently published work, the metabolites and potentialcontaminants of 60 white suckers (Catastomus commersonii) in the oil sands development region and outside the deposit region(pulp and paper mill discharge region) were assessed.4 To this end, SPME probes were placed in the dorsal-epaxialmuscles to facilitate the extraction of various organic pollutants,such as aliphatic and aromatic hydrocarbons, pesticides, PPCPs, andpetroleum-related compounds. At the same time, endogenous metabolitessuch as eicosanoids, linoleic acids, and fat-soluble vitamins werealso extracted by the SPME device, revealing significant changes inthe biochemical profiles of the components of the white suckers’skeletal muscle tissue. Exposome studies, along with the metabolomicprofiling of endogenous tissue components, enable multifactorial explorationsof the cause–effect relationship between exogenous and endogenousmolecules in living organisms. This is due to the fact that in vivo SPME extracts both endogenous and exogenous compounds,which makes it possible to simultaneously monitor the compositionand effects of these pollutants, including their toxicity on the cellularmetabolome of living animals.3.3invivo sampling of plants and animals, in vivo studies with humans require even more careful evaluationof the analytical workflow, particularly with respect to extractions.HS-SPME has been used as a diagnostic tool to detect ethanol, acetone,and isoprene in human breath samples.89 In addition,needle-trap device (NTD) technology coupled with thermal-desorptionphotoionization time-of-flight mass spectrometry (TD-PI-TOFMS) hasalso been used for in vivo breath sampling of smokersand nonsmokers; the results of this study showed that the device wasable to detect xenobiotic substances such as benzene, toluene, styrene,and ethylbenzene in the breath samples collected from the smokers.90 Traditional methods for breath or odor analysisneed to use a Tedlar bag,91 and the samplingprocess is usually separated with extraction. However, SPME and relatedtechniques can perform sampling and extraction at the same time witha portable designUnlike in vivo SPME. A review of the literatureand an open-source saliva-metabolome database revealed the existenceof at least 14 metabolic pathways in the human saliva exposome, includingamino acid metabolism, TCA cycles, gluconeogenesis, glutathione metabolism,pantothenate and CoA biosynthesis, and butanoate metabolism. Mixed-modeSPME fibers have also been used to extract metabolites from saliva,92 and, more recently, researchers have attemptedto perform extractions from saliva via TFME.76 To this end, a TFME device was directly placed in the mouth of humansubjects for a 5 min extraction, followed by analysis. The analysisshowed that TFME enabled the quantification of 49 prohibited substancesand provided limits of quantification (LOQs) ranging between 0.004and 0.98 ng/mL.93 Other extraction methodssuch as LLE can also be applied for exhaustive extraction of salivasamples,94 while in vivo SPME can capture the free concentration of certain compounds asit extracts via negligible depletion.Saliva is another complex human matrix thatcan be used for invivo skin analysis of human scent profiles,95 and TFME membranes could be applied directly on the skinto detect semi- and low-volatility compounds in vivo.96 In addition, in vivo SPME has been used to monitor the delivery and impact of doxorubicinon the profile and composition of metabolites in human lungs duringthe chemoperfusion process.97 This approachcan also be implemented to evaluate the delivery of xenobiotics fromthe environment and their impact on the human metabolome. In anotherstudy, in vivo SPME fibers were used to extract metabolitesfrom 33 fresh and 87 frozen human muscle samples.98 The results of these assays showed a shift from the utilizationof carbohydrates to the use of lipids for energy production in malignanthyperthermia susceptible individuals.Skin odor may also containinformation about environmental exposuresand endogenous metabolites. HS-SPME has been applied for in vivo remains a challenge.However, wearable devices, such as necklaces or pins, may be a promisingoption for the dynamic monitoring of exposures. Indeed, Smith et al. were able to successfully monitor ketamine usinga necklace that had been outfitted with an SPME device.99 Furthermore, it may be possible to create wearable in vivo sampling devices by coating the surface of the deviceswith biocompatible materials or even using 3D printing to customizethe design of such SPME devices.100 Suchinnovations would enable the use of in vivo SPMEto collect the environmental information for a person, which wouldin turn allow the construction of a personalized exposure history.101Due to multiple ethicaland legal regulations, the use of SPMEdevices for the real-time monitoring of exogenous and endogenous compoundsin living human systems 4in vivo SPME in various environmentalstudies focusing on the living system of plants, animals, and humans.In addition to providing comparable performance to traditional extractionmethods, SPME offers a miniaturized, minimally invasive extractionmethod that can provide accurate and reliable results in invivo applications aimed at tracking the biological fate ofspecific exogenous compounds and their metabolites as well as enablingthe direct evaluation of metabolic profile changes in the living organs/tissuesbeing studied.The development of new biocompatible coatings and SPME device geometrieshas enabled the use of in vivo SPME technologystill suffers from some limitations. For instance, specific in vivoSPME fibers used for the method development are homemade, which limitsits wider availability; however, several works have detailed stepsto prepare in vivo SPME devices41 and also some of them have been commercialized as shown in in vivo studies.Those limitations share similar scenarios for metabolomics studiesand can be improved with the development of other disciplines in thisarea, such MS-based instrumentation or data analysis. Moreover, methoddevelopment of in vivo SPME is still focused on specificcompound(s) or animal/plant models, and rarely applications can beperformed on human samples mainly due to ethical issues and law regulations.At the current stage, in vivo SPME techniques in environmental monitoring.Laboratory studies examining the effects of toxicant(s) usually provideimportant information about the fate and impact of single contaminantson living systems, but they may not comprehensively address concernsregarding the exposome’s total impact on the functioning oforganisms. In this respect, nontargeted analysis would also be helpfulin identifying novel biomarkers of exposure, and, when conducted via in vivo SPME, they could extend our chemical knowledge regardingactive or short-lived environmental components. In addition, the lackof an active compounds database for environmental studies continuesto contribute to a bottleneck in data analysis. However, approachessuch as reactomics might allow researchers to skip the annotationof the identified features in untargeted toxicological and metabolomicsstudies. The incorporation of multidisciplinary knowledge can contributeto the success of in vivo SPME sampling projects,as it can encourage the integration of the most advanced techniquesin order to solve complex environmental scientific problems. In thefuture, such an interdisciplinary approach could provide crucial informationthat will enable a better understanding of the persistence and effectsof pollutants in the environment.In the future, more exploratory and longitudinal studies trackingthe fate of environmental pollutants and their impact on biochemicalprofiles of living organisms should be conducted. At present, moststudies consist of laboratory-based evaluations of sampling methods;thus, more field studies are needed to identify other unique featuresof


Transmission mode is a key factor that influences host–parasite coevolution. Vector-borne pathogens are among the most important disease agents for humans and wildlife due to their broad distribution, high diversity, prevalence and lethality. They comprise some of the most important and widespread human pathogens, such as yellow fever, leishmania and malaria. Vector-borne parasites  follow unique transmission routes towards their vertebrate hosts. Consequently, each part of this tri-partite  interaction can influence co- and counter-evolutionary pressures among antagonists. This mode of transmission may favour the evolution of greater virulence to the vertebrate host; however, pathogen–vector interactions can also have a broad spectrum of fitness costs to the insect vector. To complete their life cycle, vector-borne pathogens must overcome immune responses from 2 unrelated organisms, since they can activate responses in both vertebrate and invertebrate hosts, possibly creating a trade-off between investments against both types of immunity. Here, we assess how dipteran vector-borne transmission shapes the evolution of hosts, vectors and the pathogens themselves. Hosts, vectors and pathogens co-evolve together in a constant antagonistic arms race with each participant's primary goal being to maximize its performance and fitness. Here, we explore the evolutionary pressures and consequences of the use of dipteran vectors for the pathogens, hosts and the vectors themselves, hereby unifying these 3 components in a common framework. By applying this framework, we aim to identify potential gains and deleterious effects of co- and counter-evolution among the host–vector–pathogen triad to highlight trends in host–vector–pathogen evolution. We focus mostly on vertebrate hosts, such as mammals and birds, which sustain pathogen development and transmission et al., et al., et al., 2) has been recognized as one of the most important olfactory cues for host-seeking behaviour  or tolerance (i.e. host capability to reduce pathogenic effects of infection without controlling pathogen load/burden) depending on the cost of infection  are likely to increase mortality in vectors, whereas Bunyaviruses vertically transmitted within Aedes mosquitoes from females to their progeny do not induce mortality in the vector  gut  of oocysts per mosquito  infection, which in turn will be more likely to infect another vertebrate host  or by cell lyses as ookinetes cross the midgut  circulating in host blood  and scientific advances (e.g. vaccines and the development of antiparasitic medications) can directly alter vector-borne pathogen evolution by changing the taxonomic composition and abundance of mosquito communities  and, due to their undoubtedly faster evolutionary rates compared to both hosts and vectors, parasites are unlikely to be overtaken naturally by either their vertebrate or vector hosts in this tripartite coevolutionary arms race. Thus, scientists should consider the evolutionary context encompassing hosts, vectors, pathogens and their microbiome to create new effective pathways for treatments and preventive interventions (see Here, we summarized the main evolutionary pressures faced by hosts, vectors and pathogens associated with vector-borne transmission see . Pathogeions see , which c


Vitiligo is a disorder characterized by loss of epidermal melanocytes, resulting in depigmented macules and patches. While the relationship between ocular pathology and vitiligo has been demonstrated in conditions such as Vogt-Koyanagi-Harada and Alezzandrini syndromes, the ocular associations of non-syndromic vitiligo are incompletely understood. We conducted a systematic review to comprehensively describe the structural and functional changes seen in the eyes of patients with vitiligo, to identify patients at heightened risk for ocular disease, and to provide an approach to management of ocular manifestations of vitiligo. Overall, the strongest link between vitiligo and ocular pathology seems to lie with dry eye disease and pigmentary abnormalities of various ocular structures, especially the retinal pigment epithelium. Normal-tension glaucoma may also be more prevalent in the vitiligo population. The available literature did not provide conclusive evidence for increased risk of cataracts or uveitis. Aside from the impact of symptomatic dry eye disease, it seems unlikely that there are significant functional consequences of these ocular manifestations such as impaired visual acuity or visual fields. Vitiligo is an acquired, autoimmune and chronic disorder that is characterized by skin and hair depigmentation secondary to epidermal melanocyte destruction. Importantly, melanocytes – neural-crest-derived, melanin-producing cells – are found in a variety of anatomic locations other than the skin, including mucosa, cardiac valves , 2, the The aim of this systematic review (SR) is to comprehensively describe the structural and functional changes identified in the eyes of patients with vitiligo and to provide an approach to management of ocular manifestations in vitiligo.This review was registered with Prospero (CRD42021233327). A medical librarian (J.P.), with training in SR methodology, searched MEDLINE, EMBASE, and the Cochrane Library for articles published up until February 5, 2021. The following subject terms and keywords were used: vitiligo, eye, ocular, and vision. During review, eligible studies included patients with vitiligo and ocular manifestation(s). Case reports, case series, and reviews were excluded. Randomized controlled trials, prospective analyses, retrospective analyses, case control, cohort, cross-sectional, and non-controlled before-and-after studies published in peer-reviewed journals in the English language were included. Gray literature was also included.A pair of independent reviewers  independently screened all abstracts. When primary reviewers could not reach a consensus, a third independent reviewer (R.K.) adjudicated the results. For abstracts identified as potentially relevant, two authors  independently reviewed full-text articles, determining final inclusion by consensus. When consensus could not be reached, a third independent reviewer (R.K.) adjudicated the results. Two authors  extracted study characteristics  from the final list of included studies. The authors independently abstracted the data and then reviewed and confirmed the accuracy of the others’ work.A total of 1715 unique records were identified , several studies examined the relative presence of iris atrophy and/or pigmentary abnormalities (such as heterochromia and hypopigmentation) in vitiligo , 22–24. No studies found a statistically significant difference in intraocular pressure (IOP) between patients with vitiligo and healthy controls , 20, 36.Nevertheless, one group compared 49 patients with vitiligo to 20 healthy controls and identified a rate of 18.4% of normal-tension glaucoma (NTG) among those with vitiligo versus 0% in the control group [Rogosić et al. found an extremely high prevalence of primary open angle glaucoma (POAG)  among patients with vitiligo referred for eye exams . There wKhurrum et al. studied 90 patients with periocular and/or facial vitiligo with 90 age-matched healthy controls and detected no difference in glaucoma prevalence between those with and without vitiligo. Both patients who developed glaucoma used long-term periorbital topical corticosteroids, but the majority of patients who used long-term topical corticosteroids on the eyelids/periorbital region did not develop glaucoma  . HoweverPerhaps because of the symptomatology of Vogt-Koyanagi-Harada and Alezzandrini syndrome, many of the included studies investigated the possible link between vitiligo and uveitis. Overall, the studies found little evidence of increased rates of uveitis among those with non-syndromic vitiligo, as illustrated by no significant difference in prevalence of ocular inflammation between vitiligo and control groups –24. A siTwo studies investigated the prevalence of vitiligo in patients with idiopathic uveitis, observing that vitiliginous lesions are more common in those presenting to ophthalmology clinic with symptomatic uveitis than in the general population , 30. CleSubstantial evidence linking vitiligo to changes in the retina and choroid exists. Two studies found a higher rate of prominent choroidal pattern (aka “tigroid fundus”) in vitiligo compared to healthy controls , 16. SimThe trend of evidence strongly suggests that there is increased retinal and choroidal pigmentary abnormalities in vitiligo. But, as most of these pigmentary changes and areas of atrophy are peripheral, it remains unclear if there is any associated functional consequence. As previously mentioned, there does not appear to be any damage to the ellipsoid zone or atrophy of the central macula on OCT . HoweverNonetheless, patients  with decAside from the well-established connection between the vitiliginous pigmentary changes and ocular inflammation that can coexist in Vogt-Koyanagi-Harada (VKH) and Alezzandrini syndromes, the link between non-syndromic vitiligo and ocular inflammation is not well understood.Vitiligo can affect skin, hair, and mucous membranes anywhere on the body, but the most common subtypes—generalized and acrofacial vitiligo—share a predilection for the face. Thus, patients with vitiligo experience periocular pigmentary changes and poliosis more often than the general population. There is some level of evidence – though far from definitive – indicating that those with periocular depigmentation are at greater risk of certain structural ocular abnormalities than the vitiligo population in general. It would be prudent to pay close attention to symptoms of dry eyes and intraocular pigmentary changes in patients with periocular vitiligo.functional consequence of structural abnormalities, with the OSDI results indicating symptomatically significant pathology [Overall, the strongest link between vitiligo and ophthalmic pathology seems to lie with tear film abnormalities and ocular surface disease. Though DED is a multifactorial and a common entity in the general population, it is known to have an underlying inflammatory component, suggesting a possible etiologic link with the inflammatory nature of vitiligo . Additioathology .Scant evidence exists associating vitiligo to the presence of cataracts. A single study identified three patients with vitiligo in their mid-30’s who developed posterior subcapsular cataracts. However, the authors posit that these were most likely secondary to a remote inflammatory event . NeverthThe prevalence of glaucoma in individuals under age 55 is rare (< 1%). This SR did identify a few studies that suggest a significantly higher rate of glaucoma in patients with vitiligo, specifically the normal-tension form of glaucoma, which is not associated with elevated intraocular pressures. The rates of glaucoma were significantly higher in patients older than 55 years and those with greater duration of time since diagnosis of vitiligo . AmongstThe relationship between vitiligo and uveitis is disputed and is based on the current understanding of VKH symptomatology. Some hypothesize that VKH lies at the extreme end of the ocular inflammatory spectrum, with ocular manifestations of non-syndromic vitiligo lying at a less extreme end. VKH is characterized pathologically by diffuse thickening within the uveal tract secondary to non-necrotizing granulomatous inflammation, with inflammatory cells found in close proximity to melanocytes. Vitiliginous skin is identified in less than one-quarter of patients with VKH . OverallDespite some evidence of abnormal ERG responses, data regarding good visual acuity and perimetry suggests that the retinal and choroidal findings have limited to no impact on visual function. Histopathologic changes include degeneration of photoreceptor cells and disruption of the outer segment/ RPE interdigitation similar to RP, a finding that has also been identified in vitiligo mutant mice. Tang et al. reported abnormal flash ERG findings, including delayed a- and b-waves, that correlated to histopathologic abnormalities such as short and disoriented rod outer segments leading to retinal separation from the pigment epithelium . In a MiLimitations relevant to this study include the review of articles exclusively in the English language. Additionally, techniques used in various manuscripts can be particularly user-dependent  and therefore results from these studies should be interpreted with caution.In summary, it appears that patients with vitiligo are at higher risk for DED and for ocular pigmentary abnormalities. However, outside of the potential impact of dry eyes, it does not seem likely that there are any functional consequences of these ocular manifestations .