Morphological Biomarkers Research Articles

Predicting biochemical recurrence of prostate cancer with artificial intelligence.

BackgroundThe first sign of metastatic prostate cancer after radical prostatectomy is rising PSA levels in the blood, termed biochemical recurrence. The prediction of recurrence relies mainly on the morphological assessment of prostate cancer using the Gleason grading system. However, in this system, within-grade morphological patterns and subtle histopathological features are currently omitted, leaving a significant amount of prognostic potential unexplored.MethodsTo discover additional prognostic information using artificial intelligence, we trained a deep learning system to predict biochemical recurrence from tissue in H&E-stained microarray cores directly. We developed a morphological biomarker using convolutional neural networks leveraging a nested case-control study of 685 patients and validated on an independent cohort of 204 patients. We use concept-based explainability methods to interpret the learned tissue patterns.ResultsThe biomarker provides a strong correlation with biochemical recurrence in two sets (n = 182 and n = 204) from separate institutions. Concept-based explanations provided tissue patterns interpretable by pathologists.ConclusionsThese results show that the model finds predictive power in the tissue beyond the morphological ISUP grading.

Morphological Biomarkers in the Amygdala and Hippocampus of Children and Adults at High Familial Risk for Depression.

Major Depressive Disorder (MDD) is highly familial, and the hippocampus and amygdala are important in the pathophysiology of MDD. Whether morphological markers of risk for familial depression are present in the hippocampus or amygdala is unknown. We imaged the brains of 148 individuals, aged 6 to 54 years, who were members of a three-generation family cohort study and who were at either high or low familial risk for MDD. We compared surface morphological features of the hippocampus and amygdala across risk groups and assessed their associations with depression severity. High- compared with low-risk individuals had inward deformations of the head of both hippocampi and the medial surface of the left amygdala. The hippocampus findings persisted in analyses that included only those participants who had never had MDD, suggesting that these are true endophenotypic biomarkers for familial MDD. Posterior extension of the inward deformations was associated with more severe depressive symptoms, suggesting that a greater spatial extent of this biomarker may contribute to the transition from risk to the overt expression of symptoms. Significant associations of these biomarkers with corresponding biomarkers for cortical thickness suggest that these markers are components of a distributed cortico-limbic network of familial vulnerability to MDD.

Brain morphology and underlying genetic correlates in schizophrenia

Schizophrenia (SZ) is a psychiatric disorder associated with brain and neurodevelopmental alterations that affects nearly 1% of the worldwide population. SZ is a complex disorder, polygenic and multifactorial, and despite large research efforts, its etiopathophysiology is still largely unknown. In this study, we assessed whether polymorphic variation in genes of the Sonic Hedgehog signaling pathway were associated with brain shape variation and contributed to differences between individuals diagnosed with SZ and healthy controls (HC). We genotyped three SNPs in SUFU, SHH and GLI3 genes (SUFU‐rs10786679, SHH‐rs10949808, GLI3‐rs3735361) in a sample of 308 subjects (113 patients with SZ and 195 HC), and quantified brain shape differences in a subsample of 100 subjects using the cartesian coordinates of brain landmarks recorded on head magnetic resonance scans.The genetic association analyses revealed that the T allele of the SHH‐rs10949808 was significantly associated with the risk for SZ, in both allelic (P=0.02586) and recessive (p=0.0312) models. Geometric Morphometric analyses showed that brain shape was significantly different between individuals diagnosed with SZ and HC. Discriminant functions correctly classified 72% of HC and 60% of patients based on brain morphology. Procrustes‐ANOVA analysis detected that diagnosis was a significant factor (P=0.001) and explained 5.11% of brain morphological variance; whereas any SNP presented significant effects in brain shape variance when analysed separately. However, when the three SNPs were combined into a single variable quantifying the number of risk variants (0 to 3) carried by each person, the analyses showed that patients with 3 risk variants presented a significantly different brain morphology as compared those carrying 2 (P=0.0297) or none risk variants (P=0.0487). These findings underscore the role of neurodevelopment‐related genes and Sonic Hedgehog pathway in SZ and the need to account for the polygenic nature of this disorder to further understand its etiology. Combining brain and genetic data, along with other morphological biomarkers, may open new lines of research in psychotic disorders.

Upper airways dysmorphology and obstructive sleeping apnea in Down syndrome

Down syndrome (DS) is a complex genetic disorder caused by trisomy of chromosome 21 that alters the development of multiple systems, including craniofacial dysmorphologies. Mandibular hypoplasia, macroglossia and midfacial flatness are associated with higher prevalence of malocclusion and mouth breathing in DS. In this study, we assessed the morphology of the upper airways and tested whether alterations in the size and shape of the upper airways were associated with Obstructive Sleep Apnea (OSA), which compromises the quality of life in individuals with DS.To quantify differences in volume and shape of the upper airways, we obtained 3D reconstructions from head magnetic resonances scans in a cross‐sectional sample of 246 adult individuals, including 159 control euploid individuals and 87 individuals diagnosed with DS. Polysomnography tests were available in a subsample of individuals with DS, and AHI Index scores representing the number of apnea and hypopnea events per hour of sleep were used to indicate the severity of sleep apnea.Geometric Morphometric analyses based on the 3D coordinates of 15 anatomical landmarks located on the surface of the upper airways showed significant shape differences between DS and control individuals (P‐value=0.001), involving a retraction of the whole upper airways structure and a change in the angular inclination of the oropharynx region in individuals with DS. Volumetric analyses confirmed a significant 40% reduction in total upper airway volume in DS (P‐value<0.0001). Multivariate regression analysis showed that AHI scores explained 10.1% of upper airways morphological variation in DS, with a significant positive correlation between the shape and volume of the upper airways and the severity of OSA. Our study suggests that morphological biomarkers assessed from non‐invasive magnetic resonance images can be useful in diagnosing OSA, a sleep disturbance with high prevalence in DS that if treated correctly, can improve the quality of life of people with DS and prevent the appearance of early dementia.

Inter-auricular distance: A morphological biomarker for ethylene production in the boot and coincidental grain loss of rice plant

Inter-auricular distance (IAD) has been often used for physical calibration of defects in panicle emergence of rice plants induced by stress-prone environments or cytoplasmic male sterility genetic factors slackening male gametogenesis. Information is scant on the physiological relationship inherent between IAD, panicle enclosure, and spikelet filling despite the wide phenotypic diversity of rice cultural types. In the present study, IAD extension and grain filling pattern in the panicle of the main shoot of rice cultivars contrasting for spikelet sterility were assessed concerning physiological factors like assimilate homeostasis, ethylene evolution, and expression of ethylene signal transducers genes at the anthesis stage of development. The panicle retention time was longer and the concentration and expression of ethylene and its transducer protein genes respectively, were higher in high sterile cultivars compared to low sterile ones. Spikelets subjected to a high concentration of ethylene for a longer time interval filled poorly because of infringement of starch synthesis in the post-anthesis stage of development. Alternatively, faster release of panicle by enhanced IAD extension shortened the exposure time and marginalized inhibitory effects of ethylene on grain filling. Rapid IAD extension and attenuation of boot ethylene production could be beneficial for male gametogenesis and grain filling.

Is precision medicine for colorectal liver metastases still a utopia? New perspectives by modern biomarkers, radiomics, and artificial intelligence.

The management of patients with liver metastases from colorectal cancer is still debated. Several therapeutic options and treatment strategies are available for an extremely heterogeneous clinical scenario. Adequate prediction of patients’ outcomes and of the effectiveness of chemotherapy and loco-regional treatments are crucial to reach a precision medicine approach. This has been an unmet need for a long time, but recent studies have opened new perspectives. New morphological biomarkers have been identified. The dynamic evaluation of the metastases across a time interval, with or without chemotherapy, provided a reliable assessment of the tumor biology. Genetics have been explored and, thanks to their strong association with prognosis, have the potential to drive treatment planning. The liver-tumor interface has been identified as one of the main determinants of tumor progression, and its components, in particular the immune infiltrate, are the focus of major research. Image mining and analyses provided new insights on tumor biology and are expected to have a relevant impact on clinical practice. Artificial intelligence is a further step forward. The present paper depicts the evolution of clinical decision-making for patients affected by colorectal liver metastases, facing modern biomarkers and innovative opportunities that will characterize the evolution of clinical research and practice in the next few years.

In Vitro Evaluation of the Cytotoxic Potential of Environmental Contaminant Mixtures Present in Water for Human Use

Chemicals from Pharmaceuticals and Personal Care Products (PPCPs) have become much more prevalent in the environment in recent years. The effects of these substances on human health and the environment are frequently debatable because they typically have poorly understood mechanisms of toxic action. For this reason, we set out to evaluate a binary mixture consisting of butylhydroxyanisole (BHA) and propylparaben (PPB), two approved additives that have been found in the environment and have a range of health-related effects. Based on our prior research, we chose an experimental model called Vero cells, kidney fibroblasts from the African green monkey ( Chlorocebus aethiops), with high sensitivity in toxicology studies and ideal characteristics for the analysis of chemical compounds’ mechanisms of action. The experimental design includes a battery of tests using many complimentary biochemical and morphological biomarkers, the usefulness of which we have previously established. The outcomes demonstrated that the mixtures of BHA and PPB cause significant functional alterations brought on by osmotic imbalance, which are connected to irregularities in cell cycle progression, increases in ploidy, which included cell cycle imbalances as well as increases in proliferation. On the other hand, we have been able to show that the quantitative estimate of the anticipated cellular responses generally does not adjust precisely to the observed effect through the analysis of the prediction of the combined effect using mathematical models. However, once the individual compounds’ respective mechanisms of action have been established, the toxicity caused by the mixtures can be qualitatively predicted.

Large-scale quantification of human osteocyte lacunar morphological biomarkers as assessed by ultra-high-resolution desktop micro-computed tomography.

Ultra-high-resolution imaging of the osteocyte lacuno-canalicular network (LCN) three-dimensionally (3D) in a high-throughput fashion has greatly improved the morphological knowledge about the constituent structures - positioning them as potential biomarkers. Technologies such as serial focused ion beam/scanning electron microscopy (FIB/SEM) and confocal scanning laser microscopy (CLSM) can image in extremely high resolution, yet only capture a small number of lacunae. Synchrotron radiation computed tomography (SR-CT) can image with both high resolution and high throughput but has a limited availability. Desktop micro-computed tomography (micro-CT) provides an attractive balance: high-throughput imaging on the micron level without the restrictions of SR-CT availability. In this study, accuracy, reproducibility, and sensitivity of large-scale quantification of human osteocyte lacunar morphometries were assessed by ultra-high-resolution desktop micro-computed tomography. For this purpose, thirty-one transiliac human bone biopsies containing trabecular and cortical regions were imaged using ultra-high-resolution desktop micro-CT at a nominal isotropic voxel resolution of 1.2 µm. The resulting 3D images were segmented, component labeled, and the following morphometric parameters of 7.71 million lacunae were measured: Lacunar number (Lc.N), density (Lc.N/BV), porosity (Lc.TV/BV), volume (Lc.V), surface area (Lc.S), surface area to volume ratio (Lc.S/Lc.V), stretch (Lc.St), oblateness (Lc.Ob), sphericity (Lc.Sr), equancy (Lc.Eq), and angle (Lc.θ). Accuracy was quantified by comparing automated lacunar identification to manual identification. Mean true positive rate (TPR), false positive rate (FPR), and false negative rate (FNR) were 89.0%, 3.4%, and 11.0%, respectively. Regarding the reproducibility of lacunar morphometry from repeated measurements, precision errors were low (0.2-3.0%) and intraclass correlation coefficients were high (0.960-0.999). Significant differences between cortical and trabecular regions (p<0.001) existed for Lc.N/BV, Lc.TV/BV, local lacunar surface area (<Lc.S>), and local lacunar volume (<Lc.V>), all of which demonstrate the sensitivity of the method and are possible biomarker candidates. This study provides the foundation required for future large-scale morphometric studies using ultra-high-resolution desktop micro-CT and high-throughput analysis of millions of osteocyte lacunae in human bone samples.

Identification of the Prodromal Symptoms and Pre-Ataxic Stage in Cerebellar Disorders: The Next Challenge.

Cerebellar ataxias (CAs) manifest with a combination of motor incoordination, cognitive, affective and recently identified social symptoms. Novel therapies aim to stop the progression of the subgroup of the degenerative ataxias, or even to cure the disease with a functional and anatomical restoration of the cerebellar circuitry in the near future. The goal of stopping the progression of the disease is particularly relevant if applied at a very early stage of the disease, when the cerebellar reserve is only slightly impaired. Therefore, the search of the prodromal phase or pre-ataxic stage of CAs represents a very important challenge for the scientific community. The identification of pre-manifest individuals and the recruitment of individuals at risk has become a key-challenge to address neuroprotective therapies. The feasibility is high due to the recent progress in the biological and morphological biomarkers of CAs.

What do we still need to learn on digitally assessed biomarkers?

What do we still need to learn on digitally assessed biomarkers?

PassFlow: a multimodal workflow for predicting deep brain stimulation outcomes.

Deep Brain Stimulation (DBS) is a proven therapy for Parkinson's Disease (PD), frequently resulting in an enhancement of motor function. Nonetheless, several undesirable side effects can occur after DBS, which can worsen the quality of life of the patient. Thus, the clinical team has to carefully select patients on whom to perform DBS. Over the past decade, there have been some attempts to relate pre-operative data and DBS clinical outcomes, with most focused on the motor symptomatology. In this paper, we propose a machine learning-based method able to predict a large number of DBS clinical outcomes for PD. We propose a multimodal pipeline, referred to as PassFlow, which predicts 84 clinical post-operative clinical scores. PassFlow is composed of an artificial neural network to compress clinical information, an image processing method from the state-of-the-art to extract morphological biomarkers our of T1 imaging, and an SVM to perform the regressions. We validated PassFlow on 196 PD patients who undergone a DBS. PassFlow showed correlation coefficients as high as 0.71 and were able to significantly predict 63 out of the 84 scores, outperforming a comparative linear method. The number of metrics that are predicted with this pre-operative information was also found to be correlated with the number of patients with this information available, indicating that the PassFlow method is still actively learning. We presented a novel, machine learning-based pipeline to predict a variety of post-operative clinical outcomes of DBS for PD patients. PassFlow took into account various bio-markers, arising from different data modalities, showing high correlation coefficients for some scores from pre-operative data only. It indicates that many clinical outcomes of DBS can be predicted agnostic to the specific simulation parameters, as PassFlow has been validated without such stimulation-related information.

Habitat differences affect the nuclear morphology of the erythrocytes and the hepatic melanin in Leptodactylus fuscus (Anura) in the Brazilian Cerrado savanna.

The sensitivity of anuran to the effects of habitat destruction and contamination has led to a preoccupying global decline in their populations. Morphological biomarkers such as micronuclei and other erythrocyte nuclear abnormalities (ENAs), as well as the occurrence of hepatic melanin, can be used to evaluate the effects of habitat impacts. In the present study, these two parameters were combined for the in situ assessment of the effects of soybean cultivation on the grassfrog, Leptodactylus fuscus. Specimens were also collected from a protected area to provide a reference site (non-agricultural environment). The frequency of some of the nuclear abnormalities in the animals from the soybean plantation was much higher than that recorded at the reference site, in particular micronuclei, which were 3.6 times more frequent in the plantation, lobulated nuclei (3.4 times more frequent), and reniform nuclei, which were four times more common than at the reference site. The combined analysis of all the ENAs together also revealed a frequency approximately 1.4 times higher in the animals from the soybean plantation, in comparison with the protected area. Smaller areas of hepatic melanin were observed in the specimens from the soybean plantation. These results provide further evidence of the sensitivity of anurans to habitat impacts and indicate that animals found in soybean plantations are susceptible to systematic alterations of their cells.

FP07.04 Predictive Efficacy of Morphological Biomarkers Based on Digital Pathology for ICI Therapy of Non-Small Cell Lung Cancer

FP07.04 Predictive Efficacy of Morphological Biomarkers Based on Digital Pathology for ICI Therapy of Non-Small Cell Lung Cancer

Ecotoxicological evaluation of water from the Sorocaba River using an integrated analysis of biochemical and morphological biomarkers in bullfrog tadpoles, Lithobates catesbeianus ( )

Lithobates catesbeianus tadpoles were exposed for 96 h to water from two sites of the Sorocaba River (summer and winter), Ibiúna (PI) and Itupararanga reservoir (PIR) that contained metals. In the liver, in PI, the glutathione peroxidase (GPx) decreased, and the glutathione S-transferase (GST) and carbonyl proteins (PCO) increased. In PIR, the glutathione reduced (GSH) increased, while there was a decrease in catalase (CAT), GPx, GST, PCO, and superoxide dismutase (SOD). In winter, GPx and GST increased in both points. Regarding the kidneys, lipoperoxidation (LPO) levels and GST decreased, while GSH increased in the summer. In the winter, LPO increased in PI. In the muscle, in the summer, there was an increase in GSH and GST and change in PCO. In the winter, the levels of PCO increased and CAT decreased in PIR. The area and volume of the hepatocyte and nucleus area increased in the summer and decreased in the winter. Hepatic melanin decreased in the summer after exposure to PIR water. There were the systemic effects of Sorocaba River water exposure at different times of the year with alterations in biomarkers at different levels, in which kidney shows highest Integrated Response of Biomarkers (IBR) value followed by liver and muscle. Biochemical biomarkers were more sensitive than morphological ones. The more sensitive biochemical markers were MT, PCO, GST and LPO. These effects confirm the hypothesis of metabolic alteration in bullfrog tadpoles by the Sorocaba River water.

Diagnostics of Amyotrophic Lateral Sclerosis: Up to Date.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by gradual loss of upper and lower motor neurons and their pathways, usually without affecting the extraocular and sphincter muscles. The cause of the disease is not yet known. It is a chain of subsequent events, ending in programmed cell death in selective neuronal subpopulations. The prognosis for survival is rather short with a median of 2 to 4 years. Survival may be prolonged based on prompt diagnosis, ALS subtype and proper management with supportive treatment (tracheostomy, gastrostomy, etc.). According to the clinical picture, the typical form of ALS with upper and lower motoneuron involvement and progressive bulbar paralysis with bulbar muscle involvement is observed. The ALS form with progressive muscle atrophy, where only the lower motoneuron is affected, and primary lateral sclerosis with only upper motoneuron damage are rare. Familiar forms of ALS (FALS) associated with specific genes (the most common is C9orf72) have been discovered. FALS is usually associated with dementia (frontotemporal lobar dementia, FTLD), behavioral disorders, cognitive dysfunction and impairment of executive functions. The diagnosis of ALS is determined by excluding other conditions and utilizing clinical examinations, laboratory and genetic tests and nerve conduction/needle electromyography studies (EMG). Needle EMG records abnormal activities at rest and looks for neurogenic patterns during muscle contraction. Motor evoked potentials after transcranial magnetic stimulation remain the test of choice to identify impairment of upper motor neurons. New biochemical, neurophysiological and morphological biomarkers are extensively studied as early diagnostic and prognostic factors and have implications for clinical trials, research and drug development.

Genotoxic and melanic alterations in Lithobates catesbeianus (anura) tadpoles exposed to fipronil insecticide.

The aim of this study was to evaluate the genotoxic and morphological systemic effects of both an acute and a chronic exposure of bullfrog tadpoles to fipronil. Lithobates catesbeianus tadpoles had morphological biomarkers (skin, liver, and blood) analyzed at Gosner stages 36-38, when exposed to four different concentrations of Regent® 800 WG (80% fipronil): 0.00 (control), 0.04, 0.08, 0.4 mg/L, and four experimental times: 4, 8, 12, and 16 days. Body darkness responded directly to the treatment and exposure time. There was a treatment-dependent decrease in darkness of heads and tails. In relation to the biometric analysis, fipronil induced a decrease in the individual weight and liver mass at the end of the experiments, whereas the hepatosomatic index did not vary according to the treatment. For the exposed animals and for the control group, the area of hepatic melanin increased as exposure time increased. Fipronil has genotoxic effects on L. catesbeianus tadpoles even after short exposure times (e.g., 4 and 8 days), and the main nuclear abnormality is in the anucleate cells. A relevant correlation was observed between genotoxic biomarkers and cutaneous and internal melanin. The frequency of nuclear abnormalities is inversely correlated both with the hepatic melanin area and with the cutaneous melanin of animals. Fipronil has distinct systemic effects on tadpoles based on its concentration, as well as on its exposure time. Such alterations (pigmentation level and rate of erythrocyte abnormality) result in morphological and physiological effects, which may compromise the behavior and survival of the anurans.

Reviewing applications of structural and functional MRI for bipolar disorder.

Bipolar disorders (BDs) represent one of the leading causes of disability and morbidity globally. The use of functional magnetic resonance imaging (fMRI) is being increasingly studied as a tool to improve the diagnosis and treatment of BDs. While morphological biomarkers can be identified through the use of structural magnetic resonance imaging (sMRI), recent studies have demonstrated that varying degrees of both structural and functional impairments indicate differing bipolar subtypes. Within fMRI, resting-state fMRI has specifically drawn increased interest for its capability to detect different neuronal activation patterns compared to task-based fMRI. This study aims to review recently published literature regarding the use of fMRI to investigate structural-functional relationships in BD diagnosis and specifically resting-state fMRI to provide an opinion on fMRI's modern clinical application. All sources in this literature review were collected through searches on both PubMed and Google Scholar databases for terms such as 'resting-state fMRI' and 'functional neuroimaging biomarkers of bipolar disorder'. While there are promising results supporting the use of fMRI for improving differential accuracy and establishing clinically relevant biomarkers, additional evidence will be required before fMRI is considered a dependable component of the overall BD diagnostic process.

Role of glucose in the repair of cell membrane damage during squeeze distortion of erythrocytes in microfluidic capillaries.

The rapid development of portable precision detection methods and the crisis of insufficient blood supply worldwide has led scientists to study mechanical visualization features beyond the biochemical properties of erythrocytes. Combined evaluation of currently known biochemical biomarkers and mechanical morphological biomarkers will become the mainstream of single-cell detection in the future. To explore the mechanical morphology of erythrocytes, a microfluidic capillary system was constructed in vitro, with flow velocity and glucose concentration as the main variables, and the morphology and ability of erythrocytes to recover from deformation as the main objects of analysis. We showed the mechanical distortion of erythrocytes under various experimental conditions. Our results showed that glucose plays important roles in improving the ability of erythrocytes to recover from deformation and in repairing the damage caused to the cell membrane during the repeated squeeze process. These protective effects were also confirmed in in vivo experiments. Our results provide visual detection markers for single-cell chips and may be useful for future studies in cell aging.

Comparative toxicity assessment of in situ burn residues to initial and dispersed heavy fuel oil using zebrafish embryos as test organisms

In situ burning (ISB) is discussed to be one of the most suitable response strategies to combat oil spills in extreme conditions. After burning, a highly viscous and sticky residue is left and may over time pose a risk of exposing aquatic biota to toxic oil compounds. Scientific information about the impact of burn residues on the environment is scarce. In this context, a comprehensive ISB field experiment with approx. 1000L IFO 180 was conducted in a fjord in Greenland. The present study investigated the toxicity of collected ISB residues to early life stages of zebrafish (Danio rerio) as a model for potentially exposed pelagic organisms. The toxicity of ISB residues on zebrafish embryos was compared with the toxicity of the initial (unweathered) IFO 180 and chemically dispersed IFO 180. Morphological malformations, hatching success, swimming behavior, and biomarkers for exposure (CYP1A activity, AChE inhibition) were evaluated in order to cover the toxic response on different biological organization levels. Across all endpoints, ISB residues did not induce greater toxicity in zebrafish embryos compared with the initial oil. The application of a chemical dispersant increased the acute toxicity most likely due to a higher bioavailability of dissolved and particulate oil components. The results provide insight into the adverse effects of ISB residues on sensitive life stages of fish in comparison with chemical dispersant application.

Effects on Ultrastructure, Composition and Specific Surface Area of the Gills of Odontesthes bonariensisUnder Subchronic Glyphosate-Based Herbicide Exposure.

Gills represent one of the major sites of gas exchange of fish, consequently they are in continuous close contact with the aquatic environment and its pollutants. In the present study the effects on gills of pejerrey fish, Odontesthes bonariensis, under glyphosate-based herbicide subchronic exposure were analyzed. Adult animals were exposed to sublethal concentrations of a glyphosate-based commercial formulation (1 and 10 PMG mg L-1, PMG: glyphosate active ingredient) for 15 days, while control group was maintained in rearing water. Ultrastructural changes in gills were observed by scanning electron microscopy (SEM). The composition of the surface epithelium and specific surface area were determined by energy dispersive spectroscopy (EDS) and N2 (g) adsorption-desorption isotherms, respectively. The herbicide exposure induced severe alterations in gill ultrastructure, as shown in the SEM micrographs. Accordingly, an increase in surface area of the gills of exposed animals was determined. These results support that gills parameters of freshwater fish are sensitive morphological biomarkers for glyphosate exposure.