Pediatric obstructive sleep apnea (OSA) is a breathing disorder that alters heart rate variability (HRV) dynamics during sleep. HRV in children is commonly assessed through conventional spectral analysis. However, bispectral analysis provides both linearity and stationarity information and has not been applied to the assessment of HRV in pediatric OSA. Here, this work aimed to assess HRV using bispectral analysis in children with OSA for signal characterization and diagnostic purposes in two large pediatric databases (0–13 years). The first database (training set) was composed of 981 overnight ECG recordings obtained during polysomnography. The second database (test set) was a subset of the Childhood Adenotonsillectomy Trial database (757 children). We characterized three bispectral regions based on the classic HRV frequency ranges , as well as three OSA-specific frequency ranges obtained in recent studies . In each region, up to 14 bispectral features were computed. The fast correlation-based filter was applied to the features obtained from the classic and OSA-specific regions, showing complementary information regarding OSA alterations in HRV. This information was then used to train multi-layer perceptron (MLP) neural networks aimed at automatically detecting pediatric OSA using three clinically defined severity classifiers. Both classic and OSA-specific MLP models showed high and similar accuracy (Acc) and areas under the receiver operating characteristic curve (AUCs) for moderate  and severe  OSA levels. Thus, the current findings highlight the usefulness of bispectral analysis on HRV to characterize and diagnose pediatric OSA. Obstructive sleep apnea (OSA) is a common respiratory disorder affecting up to 5% of the general pediatric population . OSA is 2) or airflow, among others [2 signals. Thus, there exists a lack of studies performing machine learning techniques using other physiological signals as alternative approaches to the gold standard PSG [Nocturnal laboratory-based polysomnography (PSG) is considered the standard diagnosis technique for pediatric OSA. This test not only allows for the detection of the presence or absence of pediatric OSA but also enables estimates of OSA severity ,9. During others . Then, mg others . Among tg others ,10. Despg others ,12. Thesg others ,12,13. Ag others  analyzeddard PSG .Evaluation of the adverse cardiovascular implications of pediatric OSA has highlighted autonomic dysfunction as the main reason for cardiac alterations, since the autonomic nervous system (ANS) plays a major role in cardiovascular system regulation ,16,17. IOSA was initially described in adults, and its cardiac implications and effects on HRV have been extensively studied in recent decades ,40,41. NOf note, previous bispectral analysis study in adults focused on OSA , as wellBased on these considerations, we hypothesized that bispectral analysis of HRV contains novel and useful information to characterize and diagnose pediatric OSA. Consequently, the main objectives of this study were to perform, for the first time in the field of pediatric OSA, a characterization and evaluation of bispectral regions bounded with classic and OSA-specific HRV frequency ranges. Therefore, the main novelty of this study is the analysis of HRV bispectral regions defined by classic and OSA-specific frequency ranges to characterize and diagnose pediatric OSA. Furthermore, we propose a novel bispectral parameter, which is analyzed here for the first time.The population included in this study was the same as that in a previous work  and was In regard to the UofC dataset, the Ethics Committee of the UofC Medicine Comer Children’s Hospital approved the protocol , and the study was conducted in accordance with the Declaration of Helsinki. Additionally, before the acquisition of the ECGs during the PSG, informed consent from all children caretakers was acquired. For comparison purposes, the training–test division carried out was the same as that in our preceding work , meaningRegarding the CHAT set , information about the rationale, study design and methodological aspects can be perused in the published literature ,44. InitThe PSG studies from both databases were evaluated by pediatric sleep experts from the different centers who diagnosed the children following the scoring rules established by the American Academy of Sleep Medicine . SubsequIn order to conduct the HRV analysis, ECGs from both datasets were equally pre-processed . First, The methodology followed can be divided into three stages. First, we conducted a feature extraction stage to characterize each of the bispectral regions considered in the study. Second, we performed an automatic feature selection stage based on the fast correlation-based filter (FCBF) algorithm  considerThe estimation of power spectra has been one of the main tools for the analysis of biological signals for decades . This teX(f) is the Fourier transform of a signal, f1 and f2 are the frequency indices, and Nf is the Nyquist frequency. The resultant matrix reflects the phase coupling degree between frequency components for each pair f1,f2 [HOS are spectral representations of higher-order cumulants of a random process . In partir f1,f2 .f1, f2 and f3 frequencies and phase angles φ1, φ2 and φ3 is described as f3 = f1 + f2 and φ3 = φ1 + φ2 [As the bispectrum preserves both amplitude and phase information, it allows for assessment of interactions between signal patterns . Likewis φ1 + φ2 . Thus, i φ1 + φ2 .10 samples with 50% overlapping and an FFT of 211 samples. After bispectrum matrix computation, a normalization was applied by dividing each element of the matrix by the sum of all matrix elements as [BP is the total bispectral power, measured as the sum of all magnitudes of the bispectral matrix. This normalization was applied in order to ensure that all elements of the matrix were bounded between 0 and 1, reducing subject inter-variability due to physiological conditions other than OSA [In this work, the HRV bispectrum was estimated employing a Hamming window of 2ments as ,46(2)BNf1 ≤ f2 ≤ f1+ f2 ≤ Nf [The bispectral matrix, by definition, presents symmetric properties that render evaluation of a triangular non-redundant area sufficient for a full bispectrum characterization ,34. This f2 ≤ fN ,34. As i f2 ≤ fN ,34,35. T f2 ≤ fN . Past st f2 ≤ fN ,37,38, w f2 ≤ fN : BW1 , and three based on the HRV OSA-related frequency ranges . To provide an overview, In order to characterize the bispectral regions under study, we computed features based on four different approaches: bispectral region amplitude, bispectral region entropy, bispectral region moment and weighted center of bispectrum (WCOB) region features. Furthermore, we introduced a new bispectral feature in this study.f1 ≠ f2); therefore, these features were not computed over the BWRes region in this study.As explained in maxB), measured as the maximum magnitude value inside each of the regions considered [Maximum amplitude (nsidered :(3)Bmax=minB), measured as the minimum magnitude value inside each of the regions considered [Minimum amplitude (nsidered :(4)Bmin=totalB), measured as the sum of all magnitudes inside each of the regions considered [Total bispectral power (nsidered :(5)BtotaThis parameter allows measuring deviations from Gaussianity .Following a similar tendency to the spectral approach , as seve1BE), normalized squared bispectral entropy (2BE) and normalized cubed bispectral entropy (3BE). These parameters, based on Shannon’s entropy, quantify the irregularity of the bispectral distribution in each region and are computed as [p is the magnitude distribution of the region elements:Normalized bispectral entropy BEThe values of the bispectral entropies increase with the randomness of a process, meaning changes in the HRV irregularity as a result of OSA  would bePE), which quantifies the phase regularity of the region [PE, as with the bispectral entropies, is higher as the randomness of a process increases, meaning it would be zero for a harmonic, periodic and predictable process [PE computation is performed applying Shannon’s entropy to the normalized distribution of the region phase angles [φ is within the range of histogram bins φ is the phase angle of the region, and N is the bin number of the histogram.Phase entropy PEH1), sum of the logarithmic magnitude values of the diagonal of the region (H2) and first- and second-order spectral moments of the magnitude values of the diagonal elements of the region . These features were included as they allow characterizing the nonlinearity of the regions and are computed as follows [The sum of the logarithmic magnitude values of the region ( follows :(11)H1=∑Those children suffering from OSA would be expected to present an increased bispectral power concentration in the region defined by frequency ranges related to the occurrence of apneic events (BW2). This would mean an increase in the phase coupling between the frequency components of this region and, accordingly, higher nonlinearity ,46. Therf1m and f2m, which indicate the coupling focus of the region as a summary of the frequency interaction [WCOB allows reflecting the interaction of different frequency components through the assignment of a weight to each bispectral point of the region . The weieraction . Those ceraction (15)f1m=f1m and f2m values implying an activity shift towards lower frequencies [WCOB parameters are associated with bispectral peak values, with decreases in quencies . Hence, DiagRP), computed as the sum of the bispectral amplitudes of the diagonal elements of the region, after a normalization applied over the whole diagonal. This novel parameter evaluates the relative bispectral magnitude value inside the diagonal of the region with respect to the complete bispectral diagonal magnitude:Diag is the diagonal of the bispectral matrix, and DP is the diagonal power, measured as the sum of all amplitudes of the Diag elements.The relative power of the diagonal  is a parameter to be optimized. To deal with overfitting, we also introduced a regularization parameter (λ) in the tuning of the network weights, which were randomly initialized [Similar to previous pediatric OSA diagnosis studies, we conducted the classification stage using MLP neural networks ,46,51. Ttialized .HN and λ) was performed, again, by means of 1000 bootstrap replicates from the training dataset, but different from the replicates employed in the feature selection stage. We computed Cohen’s kappa (k) for each H/Nλ combination and selected those values where k was maximum [The optimization of the hidden layer design parameters  between bispectral features from OSA severity groups through the non-parametric Kruskal–Wallis test. To provide a visual representation of these differences, along with the distribution followed by the features in each severity group, boxplots for each selected feature were also constructed.In the training set, the features included in this study did not pass normality and homoscedasticity tests. For this reason, we assessed statistically significant differences (Sρ), controlling the possible effect of age. The polysomnographic indices, related to OSA, as well as sleep structure and quality, were the same as those in [Subsequently, we conducted a correlation analysis. To this effect, relationships between the features selected and some polysomnographic indices were evaluated using Spearman’s partial correlation coefficient , specificity (Sp), accuracy (Acc) and area under the receiver operating characteristic curve (AUC).Classic) was formed by three features, one of each region: VLF_f2m, 2LF_BE and HF_PE. Regarding the OSA-specific region set (Specific) was composed of four features selected more than 500 times: DiagBW2_RP, 1BW2_BE, minBWRes_B and 3BWRes_BE. None of the BW1 region features considered were selected over 500 times.We conducted two feature selection processes. As gion set b, the oplassic and BISPSpecific subsets, respectively. The p-value resulting from the Kruskal–Wallis test is also depicted in these figures. It can be appreciated that, in the BISPClassic subset, VLF_f2m experienced an increase with OSA severity, while a decrease in 2LF_BE and HF_PE occurred as the OSA severity increases. For the features included in BISPSpecific, there was a clear rise in the DiagBW2_RP values, along with a slight increase in 3BWRes_BE with OSA severity. In contrast, the 1BW2_BE values experienced a decrease with the disease. minBWRes_B was the only parameter showing an unclear tendency among the severity groups, which led it to be the only one that did not show statistically significant differences. The remaining six parameters showed statistically significant differences among the four OSA severity groups .lassic features as input , and three models with BISPSpecific as input features . For each model, HN varied from 2 to 20 in steps of 1, and from 22 to 50 in steps of 2. Similarly, λ varied from 0.5 to 10 in steps of 0.5. Each H/Nλ pair resulted in an averaged k through 1000 bootstrap replicates of the training set; therefore, we selected the H/Nλ combination with the higher averaged k. HN = 2 and λ = 5 were the optimized design parameters selected in four out of six MLP models: MLP1Classic, MLP5Classic, MLP5Specific and MLP10Specific. For the MLP10Classic model, the optimized design parameters were HN = 34 and λ = 5. Finally, the optimized parameters in MLP1Specific were HN = 38 and λ = 5.After extraction of the optimum feature subsets, six MLP models were optimized: three models with BISPcSρ| obtained were generally low, some of the correlations evaluated were statistically significant . VLF_f2m and DiagBW2_RP showed similar behaviors, with a statistically significant positive Sρ with the four respiratory indices  and TAI, as well as a negative Sρ with %REM. In the opposite way, 1BW2_BE obtained a negative Sρ with the four respiratory indices and TAI. 2LF_BE also reached a statistically significant negative Sρ with AHI, OAHI and TAI. DiagBW2_RP reached the highest absolute correlation values among almost all of these statistically significant correlations, only being equaled by the Sρ reached between VLF_f2m and OAHI. None of the selected BWRes features nor HF_PE obtained statistically significant correlations with any of the polysomnographic indices considered in this study.The results of the correlation study are shown in DiagBW2_RP obtained the highest results in terms of Acc and AUC. For the 5 e/h threshold, again, DiagBW2_RP showed a higher Acc than the other features selected, being only slightly surpassed by VLF_f2m in terms of AUC. When considering 10 e/h as severity cutoff for binary classification, 2LF_BE was the feature showing the higher Acc, but with a lower AUC and a more unbalanced Se/Sp pair than DiagBW2_RP.Regarding the individual performance, in the 1 e/h threshold, Classic model was the only one that achieved a higher diagnostic performance than the OSA-specific models in terms of Acc and AUC at the cost of a strongly unbalanced Se/Sp pair and a very low Se value (43.5%).All the individual diagnostic yields were outperformed by the MLP models. The MLP models formed by the OSA-specific region features obtained the highest results of this study in terms of Acc and AUC in the 1 and 5 e/h thresholds. The MLP10In this work, bispectral analysis of HRV signals, a process performed for the first time in pediatric OSA research, was conducted herein. Features from bispectral HRV regions based on the classic and OSA-specific frequency ranges were extracted to assess their usefulness in the characterization and diagnosis of pediatric OSA. In both types of regions separately, the selected features showed their complementarity, and the models constructed achieved a high diagnostic performance. The OSA-specific region models generally outperformed the classic ones, highlighting the importance of these novel bispectral features in the study of pediatric OSA.VLF_f2m. The values of VLF_f2m increase with OSA severity, as depicted in The averaged normalized bispectrum in the training set shown in DiagBW2_RP was one of the two features selected. This parameter, as with VLF_f2m, showed an increasing tendency with OSA severity (DiagRP is intended to measure phase coupling between the harmonic components (f3 = 2f1 and φ3 = 2φ1) of the HRV. The increment in DiagBW2_RP with OSA severity would indicate increasing nonlinear interactions between those harmonics of OSA-affected children. Consequently, there is an increment in less random/more periodic harmonics in HRV signals inside the BW2 region due to apneic events. The increase in VLF_f2m and DiagBW2_RP with OSA severity is supported by the correlation study, showing statistically significant correlations with all respiratory indices, as well as with TAI. Furthermore, DiagBW2_RP generally obtained the highest individual diagnostic performance. Taken together, these facts highlight the importance of analyzing the BW2 bispectral region in the field of pediatric OSA, especially characterized through our new proposed parameter DiagRP.Inside this BW2 apneic-related region, severity a. As exp1BE inside the BW2 region, 2BE inside the LF region and 3BE inside BWRes. These parameters measure the irregularity of the HRV from the bispectral distribution in each region, with the inclusion of quadratic and cubic components scaling the differences in the bispectral amplitude [BW2_BE1, a decrease with severity was observed, reflecting a reduction in irregularity with apneic events in the HRV components linked to the frequencies of this region. As a result of apneic events, it can be appreciated that the bispectral amplitude in BW2 is more concentrated at low frequencies in the severe group  and a higher AUC (0.627 versus 0.600). In the 5e/h threshold, the Acc obtained by MLP5Specific and MLP5Classic was 81.0% in both cases, but with the specific model, showing a more balanced Se/Sp, a higher AUC was found (0.774 versus 0.791). Finally, in the 10e/h severity cutoff, MLP10Classic obtained the highest Acc and AUC from the study. However, the Se/Sp pair was strongly unbalanced, with a very low Se value of 43.5%. Nevertheless, the MLP10Specific model, at the cost of a very slight reduction in terms of Acc and AUC , resulted in a more balanced Se/Sp pair (66.7%/91.6%). These results reinforce the conclusion from our previous work [In this study, the information extracted from the bispectral HRV regions obtained an overall high diagnostic performance both in the classic and OSA-specific bispectral region models in the test set. The results obtained in the MLP models outperform the individual diagnostic yield, highlighting the utility of the FCBF algorithm and MLP neural networks to assess complementarity between features and to diagnose pediatric OSA, respectively. When comparing classic against OSA-specific region models, the latter generally obtained a higher performance. In the 1 e/h severity cutoff, despite the unbalanced Se/Sp pair, MLP1As far as we know, this is the first study where HRV bispectral analysis of pediatric OSA was conducted. In this sense, a direct comparison of classification results with other research studies using an HRV bispectral approach is not possible. However, HRV analysis to diagnose pediatric OSA has been previously performed. A research group carried out three classification studies ,55,56, wBW1 individually) and 0.592 AUC (LDA band of interest model). With 5 e/h as the severity threshold, the highest results were 76.6% Acc (RPBW2) and 0.688 AUC (LDA band of interest model). Finally, in the 10 e/h severity cutoff, the highest results reached were 82.8% Acc and 0.796 AUC (both using the LDA band of interest model). It can be observed from Classic and LDASpecific models also outperform the diagnostic performance obtained in the previous work, being only surpassed in terms of Acc in the 1 e/h threshold. Thus, the diagnostic utility of the features extracted from the bispectral HRV region analysis is clearly demonstrated, with the MLP models reaching the highest diagnostic performance in the literature when using HRV features exclusively to generate an automated classification of pediatric subjects into the presence or absence of OSA and to estimate the severity grouping. Moreover, the higher diagnostic performance reached by the bispectral analysis highlights the usefulness of this analysis in the pediatric OSA context, which seems to be more accurate than traditional frequency analysis to evaluate HRV alterations. The presence of HRV nonlinear dynamics demonstrated in Thus, our previous study  is the oSpecific obtained similar Se results. It is worth noting that, among the studies included in the meta-analysis, none of them considered HRV signals. Furthermore, the sample size from seventeen out of nineteen studies included in the systematic review was markedly smaller than the databases analyzed here, only being surpassed by the cohort included in the studies of Hornero et al. [Finally, although the results of the diagnostic performance from studies using physiological data from different sources must be carefully compared, it is interesting to contrast our results with the meta-analysis of a recently published systematic review . In thiso et al.  and Vaquo et al. . TherefoDespite the high diagnostic performance obtained with the methodology followed in this study, some limitations deserve mention. First, although the sample size included is markedly large, accounting for 1738 overnight HRV recordings, some imbalance between severity groups is apparent. In this sense, an increase in the population included in future studies, trying to balance the severity groups’ distribution, would be desirable to raise the robustness and generalizability of our results.Additionally, none of the features extracted from the region bound by BW1 frequencies (0.001–0.005 Hz) were selected. This frequency range has been linked to sleep fragmentation due to OSA, and its usefulness in spectral analysis has been demonstrated . Thus, aA previous study performing bispectral HRV analysis in the adult OSA context only analyzed the non-redundant bispectral region . In thisAdditionally, the highly satisfactory results achieved illustrate the utility of bispectral HRV analysis to characterize and diagnose pediatric OSA, outperforming the previous spectral analysis diagnosis yield . TherefoFinally, despite the high diagnostic performance achieved here through bispectral analysis and the MLP models constructed, the promising results obtained by deep learning techniques in healthcare issues in recent years highlight the potential utility of these methods to automate the diagnosis of pediatric OSA . AccordiRP_Diag, showed its utility, generally achieving the highest individual diagnostic performance as well as the highest correlations with polysomnographic indices. Furthermore, the MLP models outperformed the previous results of diagnostic performance based on spectral analysis, with the MLP1Specific, MLP5Specific and MLP10Classic models achieving the highest diagnostic yield from the study for each severity cutoff. These results highlight the usefulness of bispectral HRV analysis in the pediatric OSA context, especially when analyzing bispectral regions bounded by OSA-specific frequency ranges. Thus, we conclude that information extracted from HRV bispectra allows for the characterization and diagnosis of pediatric OSA, leading us to propose this approach as a potential alternative to PSG.To the best of our awareness, this is the first work where bispectral HRV analysis has been conducted to characterize and diagnose pediatric OSA. Our methodology allowed us to obtain two feature subsets, one containing information regarding bispectral regions based on classic HRV frequency ranges, and the other one with OSA-specific bispectral regions. Those subsets were formed by features containing complementary information about alterations in the non-Gaussianity, nonlinearity and irregularity behavior of the HRV due to OSA. Among the features selected, a novel bispectral measure presented here,


The extracellular matrix (ECM) is involved in many physiological processes that occur in the ovary and affect reproduction in animals and humans. The ECM has been shown to significantly affect folliculogenesis, ovulation, and corpus luteum formation. This is mainly due to the involvement of ECM in intercellular signaling. In the present study, we report the gene expression profile of porcine granulosa cells during their primary in vitro culture. The genes presented are related to ECM formation but also to cadherins and integrins that influence intercellular dialogue. During the study, it was shown that most of the genes were upregulated. A detailed understanding of the expression of genes such as POSTN, CHI3L1, CAV-1, IRS1, DCN in in vitro culture of granulosa cells may provide a basis for further studies on the molecular mechanisms occurring within the ovary. Knowledge of ECM-related gene expression within granulosa cells can also be used to study the recently discovered stemness of these cells. Moreover, the presented data may serve for the development of assisted reproduction techniques, which, especially in vitro, are becoming increasingly common.The extracellular matrix (ECM) in granulosa cells is functionally very important, and it is involved in many processes related to ovarian follicle growth and ovulation. The aim of this study was to describe the expression profile of genes within granulosa cells that are associated with extracellular matrix formation, intercellular signaling, and cell–cell fusion. The material for this study was ovaries of sexually mature pigs obtained from a commercial slaughterhouse. Laboratory-derived granulosa cells (GCs) from ovarian follicles were cultured in a primary in vitro culture model. The extracted genetic material  were subjected to microarray expression analysis. Among 81 genes, 66 showed increased expression and only 15 showed decreased expression were assigned to 7 gene ontology groups “extracellular matrix binding”, “extracellular matrix structural constituent”, “binding, bridging”, “cadherin binding”, “cell adhesion molecule binding”, “collagen binding” and “cadherin binding involved in cell-cell adhesion”. The 10 genes with the highest expression  and 10 of the most down-regulated  were selected for further analysis. The results were validated by RT-qPCR. The current results may serve as preliminary data for further analyses using in vitro granulosa cell cultures in assisted reproduction technologies, studies of pathological processes in the ovary as well as in the use of the stemness potential of GCs. The extracellular matrix (ECM) is an extremely important structure present in all tissues of the animal organism. It exhibits various functions and is composed of many substances of different origins ,2. The mIt is important to consider the role of cell signaling on reproductive efficiency, including via the ECM, cadherins, and integrins ,24,25,26A total of 40 crossbred Landrace gilts with a median age of 170 days and weight of 98 kg were used in this study. All animals were housed under identical conditions. The animals in the study reached sexual maturity as pigs become sexually mature at 4–6 months of age. n = 80) were recovered at slaughter and transported to the laboratory at 38 °C in 0.9% NaCl within 30 min. In the laboratory, the ovaries of each animal were placed in PBS supplemented with fetal bovine serum . Thereafter, single preovulatory large follicles, with a diameter estimated greater than 5 mm (n = 300), were opened into a sterile Petri dish by puncturing using a 5 ml syringe and 20 G needle, and the cumulus-oocyte complexes (COCs) and follicular fluid (FF) were recovered. The transcriptomic profile of mural GCs, which constitute a significant majority among the GCs population was analyzed. The follicular fluid was used to isolate GCs, whereas the COCs were discarded. The extracted follicular fluid after discarding COCs was filtered through sterile nylon cell strainers with a mesh diameter of 40 µm  to eliminate tissue debris and larger cell aggregates (including blood cells) or epithelium. The resulting suspension was centrifuged at room temperature for 10 min, 200 rpm, to obtain individual cell fractions. The GCs pellet was then resuspended in collagenase type I solution  1 mg/1 mL DMEM and incubated 10 min in a 37 °C water bath and centrifuged (under the same conditions). The obtained cell pellet was resuspended in culture medium to establish in vitro culture under the conditions described below. Granulosa cells collected from ovarian follicles were pooled to homogenize the sample.Ovaries . The number of cells and their viability were assessed using the ADAM Automatic Cell Counter . From the cell suspension, a 20 µL sample for number and viability analysis was stained with propium iodide and examined in a fluorescence analyzer on disposable microchips. By staining the cell nuclei, the counter is able to distinguish single cells in aggregates. Only those samples with viability above 85% were used for further studies. Cells in culture were kept until culture termination when the material was collected at 0 h, 48 h, 96 h, 144 h. The culture medium was changed every 72 h. A primary in vitro culture model was used in this study with four time intervals for each biological repeat. For microarray expressions, cultures were maintained in two biological replicates for each time interval. For validation by RT-qPCR, cultures were maintained in a triplicate biological sample model for each time interval. Primary cultures were established from GCs in four bottles with 3 × 102). Once the adherent cells were more than 80% confluent, they were detached with 0.05% trypsin-EDTA  for 3 min. and then passaged. The morphology of the GCs was evaluated using an inverted phase-contrast microscope, and the results of these observations are presented in Culture medium consisted of Dulbecco’s Modified Eagle’s Medium , 2% fetal calf serum (FCS) , 10 mg/mL ascorbic acid , 0.05 μM dexamethasone , 200 mM L-glutamine , 10 mg/mL gentamycin , 10,000 units/mL penicillin and 10,000 μg/mL streptomycin . Cells were cultivated at 38.5 °C under aerobic conditions . Obtained cDNA was biotin labeled and fragmented by Affymetrix GeneChip® WT Terminal Labeling and Hybridization . Biotin-labeled fragments of cDNA (5.5 μg) were hybridized to Affymetrix® Porcine Gene 1.1 ST Array Strip  (48 °C/20 h). Then, microarrays were washed and stained according to the technical protocol using Affymetrix GeneAtlas Fluidics Station . Subsequently, the array strips were scanned by the Imaging Station of the GeneAtlas System . The preliminary analysis of the scanned chips was performed using Affymetrix GeneAtlasTM Operating Software. The quality of gene expression data was checked according to quality control criteria provided by the software. Obtained CEL files were imported into the downstream data analysis software. All of the presented analyses and graphs were performed by Bioconductor and R programming language. Each CEL file was merged with a description file. In order to correct background, normalize and summarize results, we used Robust Multiarray Averaging (RMA) algorithm. The Affymetrix procedure was previously described by Trejter et al.  and usedp-value was corrected for multiple comparisons using Benjamini and Hochberg’s false discovery rate. The selection of significantly changed gene expression was based on a p-value under 0.05 and expression fold higher than 2. Differentially expressed genes were subjected to the selection of genes involved in cadherin and collagen binding and structuralization of extracellular matrix components. Differentially expressed gene lists were uploaded to DAVID software , where “extracellular matrix binding”, “extracellular matrix structural constituent”, “binding, bridging”, “cadherin binding”, “cell adhesion molecule binding”, “collagen binding” and “cadherin binding involved in cell-cell adhesion” GO MF terms were obtained. Expression data of these genes were subjected to a hierarchical clusterization procedure and presented as a heatmap graph. Detailed analysis of genes belonging to selected GO MF terms was presented as plots using “GOplot” library [Statistical significance of analyzed genes was performed by moderated t-statistics from the empirical Bayes method. Obtained  library  and “Clu library . In the Using STRING10  software, interactions between genes and the proteins they encode have been investigated. The STRING database includes information on protein/gene interactions, including experimental data, computational prediction methods, and public text collections. The STRING database engine supplied the molecular network of interactions formed between the chosen genes. Search criteria are provided based on gene/protein co-occurrence in scientific texts (textmining), coexpression, and experimentally observed interactions. The functional interaction between genes that belong to the chosen GO BP terms was investigated by the REACTOME FIViz application to the Cytoscape 3.8.2 software . The Reactome FIViz app is designed to find pathways and network patterns related to cancer and other types of diseases. This app accesses the pathways stored in the Reactome database, allowing to do pathway enrichment analysis for a set of genes, visualize hit pathways using manually laid-out pathway diagrams directly in Cytoscape, and investigate functional relationships among genes in hit pathways. The app can also access the Reactome Functional Interaction (FI) network, a highly reliable, manually curated pathway-based protein functional interaction network covering over 60% of human proteins. ® Green I  as a detection dye, and target cDNA was quantified using the relative quantification method. The relative abundance of analyzed transcripts in each sample was standardized to the internal standard glyceraldehyde-3-phosphate dehydrogenase (GAPDH). For amplification, 2 µL of cDNA solution was added to 18 µL of QuantiTect® SYBR® Green PCR  and primers . The RNA samples were resuspended in 20 µl of RNase-free water and stored in liquid nitrogen. RNA samples were treated with DNase I and reverse-transcribed (RT) into cDNA. RT-qPCR was conducted in a LightCycler real-time PCR detection system  using SYBR primers . One RNAp-value was corrected for multiple comparisons using Benjamini and Hochberg’s false discovery rate.To quantify the specific genes expressed in the GCs, the expression levels of specific mRNAs in each sample were calculated relative to PBGD and ACTB. To ensure the integrity of these results, the additional housekeeping gene 18S was used as an internal standard to demonstrate that PBGD and ACTB mRNAs were not differentially regulated in GC groups. The gene for 18S rRNA expression has been identified as an appropriate housekeeping gene for use in quantitative PCR studies. Expression of PBGD, ACTB, and 18S mRNA was measured in cDNA samples from isolated GCs. The statistical significance of the analyzed genes was performed using moderated t-statistics from the empirical Bayes method. The obtained ® Porcine Gene 1.1 ST Array Strip  for the microarray gene expression analysis of porcine granulosa cells allows the study of the gene expression of 27,558 transcripts at 0, 48, 96, and 144 h of in vitro granulosa cell culture. Genes with more than 2-fold changes and corrected p-values less than 0.05 were selected for downstream analysis. A total of 3380 differentially expressed genes (DEGs) were identified according to the above criteria. The list of DEGs was uploaded to the GEO database (ID:GSE134361). Microarray gene expression analysis with subjecting the list of DEGs to DAVID software, which showed that the genes can be assigned to 1901 GO BP , 162 GO MF (Gene Ontology Molecular Function), and 182 GO CC (Gene Ontology Cellular Component) terms. Gene Ontology is the knowledge database of gene function and provides the basis for computational analyses used in molecular biology and genetics. This paper focused on the genes involved in cadherin and collagen binding and structuralization of extracellular matrix components. The DAVID software indicated the following GO MF terms, which cover the above processes: “extracellular matrix binding”, “extracellular matrix structural constituent”, “binding, bridging”, “cadherin binding”, “cell adhesion molecule binding”, “collagen binding” and “cadherin binding involved in cell-cell adhesion”. The 81 genes involved in those processes were clustered using hierarchical clustering and presented as heatmaps  or increased . The z-scores were presented as segments of inner circles in Subsequently, the interaction between the seven selected ontological groups of the 81 genes was evaluated. One of the most visually appealing ways of presenting such interaction is by using a dendrogram . ClusterIn the gene ontology database, single genes may belong to many ontological terms. In our study, among 15 down-regulated genes, 5 of them belonged to two or more ontology groups. Of the 66 up-regulated genes, 33 showed belonging to more than one GO. Therefore, plots with visualization of fold change values and the relationship between genes and selected GO MF terms were employed to depict the data A,B. The A STRING interaction network was used to demonstrate the interaction between the 10 most up-regulated and the 10 most down-regulated genes analyzed, which can predict the relationship between the protein products of the genes described . In the Results from microarray expression were confirmed by quantitative RT-qPCR on porcine granulosa cells. These data sets were collected, compared, and presented as bar graphs . All 20 The contact of the cell with the external environment, including the ECM, is crucial for the continuity of the communication, the reception of stimuli, and the occurrence of basic processes such as migration, proliferation, differentiation, morphogenesis, as well as providing tissue homeostasis . The higIt has been shown that ECM components have a direct effect on biological processes within the ovary such as folliculogenesis, ovulation and steroidogenesis. Glycoproteins, which play an important role in ovulation are involved in signaling with other ECM components including proteins, integrins and growth factors, deserve special attention. These include, among others: DCN (decorin), FN1 (fibronectin) and POSTN (periostin). These genes showed a significant increase in expression during in vitro culture of GCs compared to the reference value. The description of increased expression of DCN in mural granulosa cells (MGCs) than cumulus granulosa cells (CGCs) may indicate involvement in ovulation. The effect of LH/hCG on the upregulation of this gene expression was also shown to be mediated through PKA, PKC, ERK/MEK, and PI3K pathways . The rolIn turn, integrins are bound by various ECM components, including fibronectin . The typIt is worth considering that the expression of this gene in the follicular fluid of lambs is lower than in sheep  indicatiAnother glycoprotein showing roles in ECM remodeling is CHI3L1 (chitinase-3-like protein 1 CHI3L1) gene, which also showed up-regulation during conducted culture. Its high expression level is an indicator of poor prognosis for ovarian cancer patients  and can Not only the ECM glycoproteins mentioned above are involved in these processes within the ovary. CAV1 (caveolin-1) protein is strictly responsible for the composition of the extracellular matrix. It is involved in the formation of exosomes and sorting of protein components of the extracellular matrix as well as their deposition . Thus, iA group of widely distributed and important proteins are integrins, adhesion molecules, as transmembrane proteins are composed of α and β components . ITGA isOne of the main components of lamina basalis is laminin, which binds to collagen. Increased expression of the laminin subunit beta 1 (LAMB1) gene, which belonged to the ontology group  is illustrated in COL1A2, COL3A1, COL5A2, COL12A1, COL15A1, COL6A3) in the theca interna of the bovine ovarian follicle, leading to their consideration as cellular markers of this structure [Another important component of the lamina basalis is collagen, which during the development of the ovarian follicle may change its conformation, amount, and proportion of isoforms . In the tructure .IGF1 (insulin-like growth factor 1) is one of the described regulators of folliculogenesis and oogenesis and is also involved in cell proliferation. Expression of this gene was the most strongly reduced during in vitro culture of porcine GCs relative to the reference value. The number of its receptors in the ovary changes depending on the presence of the corpus luteum and is therefore dependent on steroid hormones . In turnThe down-regulated PAICS gene (phosphoribosylaminoimidazole carboxylase and phosphoribosylaminoimidazolesuccinocarboxamide synthase) encodes an enzyme that catalyzes steps 6 and 7 of the de novo purine biosynthesis pathway  and was Both physical and functional interactions between particular genes are confirmed by STRING interaction . InteracThe incompletely understood molecular basis of processes within the ovarian follicle leading to ovulation and luteinization requires precise determination of the gene expression profile within, among others, granulosa cells. Therefore, the results can be broadly applied not only to understand the physiology of the ovary but also to study pathological processes and assisted reproduction techniques. In addition, compared to in vivo studies, in vitro methods are less expensive, easier to perform, and do not require the approval of ethical committees if the material used in the study comes from commercially slaughtered animals. The expression profile of genes in porcine granulosa cells cultured in vitro, associated with cadherin and collagen binding and structuralization of extracellular matrix (ECM) components were carefully evaluated, and the culture of GCs in vitro showed increased expression of genes highly responsible for extracellular matrix (ECM) formation. ECM is involved in the regulation of many processes within the ovarian follicle, including its development, maturation, ovulation, and corpus luteum formation. The pig has been used as a model in human medicine, so the results of this study may be valuable in determining pathological mechanisms within the ovary, including finding targets for clinical therapy in humans. Of special attention is the description of the expression levels of genes such as POSTN, CHI3L1, CAV-1, IRS1, DCN, which according to literature data are associated with diseases such as POI, PCOS, and ovarian cancer. The current and previously demonstrated increased expression of POSTN, FN1, ITGB3, ITGA2, LAMB1, DCN genes in granulosa cells, associated with the processes of growth, proliferation, maturation and differentiation, may become potential new molecular markers of proliferation of these cells in vitro what can be valuable in assisted animal reproduction techniques. Additionally, the described expression profile of genes related to extracellular matrix structuralization, which plays an important role in stem cell differentiation and regeneration processes, may provide important knowledge in this aspect. Signaling pathways and interactions associated with the described genes demonstrate the multidirectional role of the ECM in both spatial structure formation, intercellular signaling describing even more fully the role of selected genes.


This observational study aimed to investigate the possible influence of sexual behavior and demographic characteristics in the expression of HPV-related biomarkers in a colposcopy population. Lifestyle factors that impacted HPV DNA positivity in a statistically significant manner were younger age at first sexual intercourse, a history of more than four sexual partners as well as a partner change during the last year before HPV DNA assessment. Although ambitious, the development and validation of lifestyle scoring systems that combine molecular and epidemiological patient data to effectively predict underlying cervical pathology will represent a milestone in the quest of cervical cancer prevention.p < 0.0001). Similarly, a reported partner change during the last year before HPV DNA assessment contributed to 2.5 times higher odds for DNA positivity (p = 0.0006). From this perspective, the further development and validation of scoring systems quantifying lifestyle factors that could reflect cervical precancer risk seems paramount.Despite the significant scientific evolution in primary and secondary cervical cancer prevention in the battle started by George Papanicolaou in the previous century, global cervical cancer mortality rates remain disappointing. The widespread implementation of HPV-related molecular markers has paved the way to tremendous developments in cervical cancer screening, with the transition from cytological approach to the more accurate and cost-effective HPV testing modalities. However, the academic audience and different health systems have not yet adopted a universal approach in screening strategies, and even artificial intelligence modalities have been utilized from the multidisciplinary scientific armamentarium. Combination algorithms, scoring systems as well as artificial intelligent models have been so far proposed for cervical screening and management. The impact of sexual lifestyle inherently possesses a key role in the prevalence of HPV-related biomarkers. This study aimed to investigate any possible influence of sexual behavior and demographic characteristics in the expression of HPV-related biomarkers in a colposcopy population from October 2016 to June 2017, and corroborated the determining role of age at fist intercourse; the older the age, the lower the probability for DNA positivity. Multivariate analysis illustrated additionally that a number of sexual partners exceeding 4.2 was crucial, with women with ≤5 partners being approximately four times less likely to harbor a positive HPV DNA test ( Cervical cancer remains the fourth most frequently diagnosed cancer and the fourth leading cause among cancer deaths, with an estimated 604,000 new cases and 342,000 deaths worldwide in 2020 . In spitThe deployment of cytology-based cervical cancer screening strategies with early detection and treatment of precancerous lesions has significantly reduced both the incidence and mortality from cervical cancer in the developed world ,6. HowevFurthermore, in Greece, despite several initiatives, no national cervical screening program is currently in place, and most women are being opportunistically screened, predominantly by cytology and lately by HPV DNA or mRNA co-testing . AccordiWe conducted a study in a colposcopy clinic population of women of reproductive age, studying the expression of HPV-related biomarkers (HPV DNA and mRNA E6 and E7) in relation to dependent variables such as cytology and colposcopy, using as gold standard cervical histology . We also sought to determine possible co-variations with demographic characteristics and lifestyle sexual factors .http://HPVGuard.org, accessed on 25 June 2021, Project Number: 11ΣΥΝ_10_250, Cooperation framework, Protocol Number: ΕΥΔΕ—ΕΤAΚ 1788/1-10-2012). The study included women of reproductive age who were referred in the Larisa Primary Health Centre Colposcopy clinic for evaluation of abnormal cytology. This clinic is affiliated with the University Hospital of Larisa, covering a large proportion of referrals from Central Greece. Experienced gynecologists and board-accredited colposcopists staff the aforementioned department.We conducted a prospective pragmatic observational study within the framework of a multidisciplinary research protocol in cervical pathology (Ministry of Education and Religious Affairs), under the frame of the HPVGuard research project ) or either positive or negative mRNA E6 and E7 test as tested by APTIMA® HPV Assay  at the initial visit. There were no prerequisites regarding the anti-HPV vaccination status in terms of the eligibility to participate in the study.We included all women of reproductive age who signed the informed consent form harboring any grade of cytological abnormalities and/or abnormal colposcopy (LGSIL/HGSIL). We also included individuals who tested positive for HPV DNA genotyping   were also recorded. ® )HPV DNA genotyping ).Detection of E6/E7 mRNA from the 14 high-risk HPV types  sample was obtained using a Rovers™ Cervex-brush just prior to the colposcopic evaluation. This was transferred in PreservCyt solution and subsequently underwent cytological and bio-molecular analysis for established HPV-related biomarkers, in particular:® HPV2 allows the simultaneous detection of 35 different HPV genotypes  by PCR amplification of a fragment within the highly conserved L1 region of the virus [® HPV Assay is a commercially distributed kit allowing the identification of E6 and E7 mRNA of the 14 high-risk HPV types  utilizing a transcription mediated amplification of viral mRNA, while testing and analysis were basically performed on an automated system  by using 1 mL from the ThinPrep sample transferred into an APTIMA Specimen Transfer tube. All specimens were analyzed for cytological and biomolecular evaluation, centrally at the University General Hospital “Attikon” of Athens.The cytological examination was expressed according to the Bethesda classification (TBS 2001 system) ,12. The he virus . The APTWomen with referral abnormal cytology of any grade as well as those who tested HPV DNA and/or mRNA E6 and E7 positive underwent colposcopic evaluation. Women with negative colposcopy had no biopsies taken and were referred for a repeat cytological and colposcopic assessment six months later. Women with low-grade colposcopy underwent colposcopically directed single or multiple punch biopsies. All women with high-grade colposcopy had either biopsies taken or loop excision of the transformation zone (LLETZ) of the cervix. LLETZ conization is considered the gold standard for histological diagnosis since it includes the entirety of the cervical transformation zone and was preferred over multiple punch biopsies in women who had completed childbearing . The subAs stated, based on the study design all women underwent a colposcopic evaluation in order to document any cytologic and/or biomolecular discrepancies. All colposcopic examinations were performed by expert board-accredited colposcopists. In all HPV unvaccinated individuals, a strong recommendation for HPV vaccination was given irrespectively to the study’s cytological and or colposcopical findings, since this represents universal standard clinical policy of the department.All women were informed about the scope of the study and were asked to undersign a consent form before entering the study. The study’s protocol has been approved by the Greek Central Government and subsequently received additional approval from the coordinating authority “Attikon” University Hospital Ethics Committee (Code: ΕΒΔ 623/14-5-13) .p < 0.05 and all tests were two sided.Statistical analysis was performed via the SAS for Windows 9.4 software platform . Descriptive values were expressed as mean ± standard deviation (SD) also reporting minimum and maximum values, as well as median and first and third quartiles  and for the categorical data using frequencies and percentages. Comparisons between groups for the qualitative parameters were made using the chi-square test and if required the Fisher exact test. We examined the relation between the number of sexual partners and the coitarche using the Spearman correlation coefficient since normality could not be ensured for these variables. We evaluated if these two characteristics can be used as single predictors of HPV DNA and mRNA positivity using the receiver operating characteristics (ROC) area under curve (AUC). The significance level for all statistical tests was set to The performances of cytology and colposcopy, as well as HPV DNA and mRNA detection in respect to the histological results, were set as primary outcomes of the study, and were assessed by counting the true positive and negative cases as well as the false positive and negative cases for various cytological and colposcopical cut-offs as well as for various histological thresholds. Subsequently, we calculated the sensitivity, specificity, positive and negative predictive value , and false positive and false negative rates . Moreover, as single number indicators of the overall performance we calculated the overall accuracy and the diagnostic odds ratios (DOR).In total 336 women participated in the study; both their demographic and medical baseline characteristics are depicted in At enrollment, about one in three (36.9%) were current smokers and similarly 34.5% were vaccinated, the vast majority (81%) with quadrivalent Gardasil. On average, the number of lifetime sexual partners was 6; 33.9% of the women had ≤3 lifetime partners and a similar percentage of 36.3% had more than 5. For the whole population, the average percentage of condom use during intercourse was 29.8%; 150 women were never users (44.6%) and 14 (4.2%) were consistent users (100% rate of condom utilization).In terms of medical conditions, this special colposcopy clinic population illustrated 53.6% aberrant cytology rates, while an even higher percentage (62.5%) harbored abnormal colposcopic findings. HPV DNA was found positive in 152 (45.24%) of the participating women, and of these 60 (39.5% of the HPV positive population) had co-infection with multiple HPV genotypes . In addition, 92 women (27.4%) were found to be HPV mRNA positive while no further mRNA typing was performed. Finally, history of warts (active or treated) was reported in 18 women (5.4%).Regarding the expression of HPV’s genotypes in our population, the results of the HPV DNA assay detected a distribution of particular HPV subtypes that concurs with reported geographical data ,17. Almop < 0.0001) related with negative cytology, . The results are presented in ogy, See  whereas p = 0.0108). Actually, if we use the number of lifetime sex partners as a predictor for HPV DNA positivity then the AUC of the ROC curve was 65.39% (95% CI: 59.39–71.39%) indicative that is a rather good predictor. Moreover, for the age of coitarche yielded an AUC of 44.88% (95% CI: 38.80–50.96%).In our study, the number of sexual partners gained importance for HPV DNA positivity when it exceeded the threshold of three partners (per lifetime)—actually for this cut-off the OR was 1.9 (95% CI: 1.2–3.0); in other words, women with more than three partners per lifetime had two times higher odds for a positive HPV DNA test. Notably, the odds were increasing with increasing numbers of sexual partners, for instance for five partners OR reached 3.8 and for 10 OR reached 4.2. In terms of sexual behavior, a recent (within the last 12 months) change of partner was also predictive of a positive HPV DNA outcome . Interestingly, vaccination status, history of warts and condom use did not yield statistical significance in our study (p = 0.0014), as 55% of HPV DNA negative women were smokers, in contrast to the higher percentage (72%) of HPV DNA positive women who were non-smokers, and this could be perhaps attributed to the relatively small study sample [Oddly in our patients smoking appeared to a play a “protective” role , indicative that the higher this age the lower the probability for DNA positivity. Interestingly the age of first sexual intercourse was found to be statistically important . Furthermore, a partner change during the last year before HPV DNA assessment conferred 2.5 times higher odds for DNA positivity . Finally, both smoking  and HPV vaccination  had a positive impact for a negative HPV DNA outcome. Among the various cut-offs for the number of sex partners, the multivariate analysis demonstrated that a cut-off of five sexual partners was crucial; specifically, women with ≤5 partners were about four times less likely to harbor a positive DNA test , indicative that these two characteristics were not related.Characteristic plots depicting graphically the role of age at first sexual intercourse and number of sexual partners vs. smoking and vaccination are depicted in p < 0.0001) compared to 5.4 for HPV DNA, a finding indicative that HPV mRNA might represent a better predictor of underlying cervical pathology. All mRNA positive cases also harbored colposcopical abnormalities . Moreover, despite the finding that positive HPV DNA and mRNA results were strongly interrelated, the study data analysis did not detect a role of multiple infections in HPV mRNA expression rates.In a similar manner, as for HPV DNA, we examined the role of all the studied variables in relation to HPV mRNA positivity (see results in p = 0.0019) and similarly there is a trend that the odds are increasing with rising number of sexual partners. The number of lifetime sex partners was also tested as a predictor for HPV mRNA positivity; the AUC of the ROC curve was 62.88% (95% CI: 56.11%–69.66%), indicative that it is a rather good predictor. Moreover, the age of coitarche yielded an AUC of 46.08% (95% CI: 39.20%–52.97%). A reverse role of smoking was also documented regarding mRNA positivity rates .In terms of the role of the number of previous sexual partners, data analysis illustrated that a history of more than one partner might affect mRNA expression . Finally, vaccine type did not affect HPV mRNA expression rates.p-value < 0.1 in the univariate analysis (see p < 0.0001), while as observed for DNA positivity, non-vaccinated women and oddly non-smokers illustrated higher rates of mRNA positivity .Similar to DNA positivity, we applied multivariate analysis for addressing any possible confounding factors influencing mRNA positivity. The factors entered in the multivariate models were those with a ysis see . As was Additionally, we calculated various performance indicators for the role of cytology, colposcopy, HPV DNA and HPV mRNA as tests which affect the corresponding cervical status —note that per protocol all women which simultaneously presented with negative cytology, HPV DNA and colposcopy were considered as being histologically negative. These results are summarized in Clearly, regarding cytological and histological cut-offs of ≥LSIL, sensitivity of cytology reached 72% while specificity was above 87% see .Similar to cytology, a colposcopical and histological threshold of ≥LSIL provided rather balanced results  [The low overall prevalence of HG disease in this cohort predictably had an impact on our findings; the main study’s results are in line with the existing epidemiological literature ,45. In a 0.0001) .p = 0.0006), a finding also consistent with the literature [In our study a partner change during the last year before HPV DNA assessment contributed to 2.5 times higher odds for DNA positivity (terature . In the terature .Particularly in recent times, the clinician should carefully balance between a too intrusive or disengaged approach during history taking in the colposcopy clinic; the presence of a midwife is helpful in this direction. After reassuring the patient on privacy concerns and that all data will be anonymized, women should be encouraged to provide a scheduled history covering basic aspects of reproductive and sexual demographic data. Epidemiological co-factors which augment hrHPVs in the development of severe precancerous lesions possess an established all-important role in cervical carcinogenesis which should not be overlooked or even dismissed whatsoever, in light of the elucidated role of hrHPV-mediated molecular mechanisms . AnotherFrom this perspective, the further development and validation of scoring systems quantifying lifestyle factors that could impact cervical precancer risk seems paramount . The recResearch in emerging HPV-related biomarkers should be coupled with the development, integration and validation of lifestyle scoring systems. These systems quantify lifestyle factors that could impact cervical precancer risk and together with artificial intelligence tools accurately predict underlying cervical pathology.


Percutaneous endoscopic lumbar discectomy (PELD) is a relatively safe and effective minimally invasive surgery in the treatment of calcified lumbar disc herniation (CLDH). However, studies on percutaneous endoscopic interlaminar discectomy (PEID) and percutaneous endoscopic transforaminal discectomy (PETD) for CLDH have rarely been reported. This research aimed to compare the clinical efficacy of PEID and PETD for L5-S1 CLDH.n = 28) and PETD (n = 26) group according to the surgical methods. The demographic characteristics and surgical results of the two groups were compared. Clinical outcomes were estimated by the visual analog scale (VAS) for leg pain, Oswestry disability index (ODI) and modified MacNab criteria.We retrospectively analyzed 54 consecutive patients with L5-S1 CLDH treated with PELD at our institution from August 2016 to August 2020. Patients were divided into PEID group (P = 1.000), whereas the PEID group exhibited superior results for operative time (min)  and fluoroscopy times (n)  compared with the PETD group.All patients were successfully operated on by PEID or PETD. No significant differences in the demographic characteristics, intraoperative blood loss, postoperative hospital stay and complication rate were noted between the PEID and PETD groups. The excellent and good rates in the PEID group were similar to those in the PETD group (89.29% vs 88.46%, PEID has achieved good clinical efficacy as PETD for L5-S1 CLDH. Compared with PETD, PEID has the advantages of shorter operative time and a reduced number of fluoroscopy times in the treatment of CLDH. Calcified lumbar disc herniation (CLDH), with a low incidence rate, is a special type of lumbar disc herniation (LDH). Calcified disc herniation, with hard structure, usually adheres extensively to surrounding tissues such as nerve roots and the dural sac. Most patients with CLDH have severe low back and leg pain symptoms and even severe neurological symptoms in the acute stage . HoweverPatients with CLDH are typically treated by traditional open surgery. Traditional open surgery completely resects the calcified intervertebral disc with good clinical outcomes. However, traditional open surgery, with a long incision, extensive stripping of the paravertebral muscles and laminectomy, has some deficiencies, such as significant tissue damage, considerable intraoperative blood loss, muscle denervation and atrophy, and even spinal instability , 3.Percutaneous endoscopic lumbar discectomy (PELD), including percutaneous endoscopic interlaminar discectomy (PEID) and percutaneous endoscopic transforaminal discectomy (PETD), is a minimally invasive operation for the treatment of LDH. Some studies demonstrated that PELD had similar clinical results as traditional open discectomy , 5. PELDn = 28) and PETD (n = 26) groups according to the surgical methods. The inclusion criteria were as follows: 1) symptoms of pain and numbness of lower limbs; 2) L5-S1 CLDH confirmed by preoperative lumbar X-ray, computed tomography (CT) and magnetic resonance imaging (MRI); 3) failure of conservative treatment or no significant improvement in symptoms for greater than 3 months; and 4) the follow-up time was greater than 12 months. The exclusion criteria were as follows: 1) multisegmental lesions; 2) noncalcified lumbar disc herniation; 3) lumbar spinal stenosis, lumbar instability, lumbar tuberculosis, lumbar infection or spinal tumor. A percutaneous transforaminal endoscopic spine system , tip-flexible bipolar radiofrequency system , ultrasonic osteotome  and endoscopic instruments  were used.This research obtained the support of the Ethics Committee of our institution and informed consent of all patients. From August 2016 to August 2020, we collected 54 consecutive patients with L5-S1 CLDH treated with PEID and PETD in our hospital. These patients were divided into PEID , duration of symptoms, type of calcification, location of calcification and follow-up time, were compared between the PEID and PETD groups. Surgical outcomes, such as operative time, intraoperative blood loss, intraoperative fluoroscopy times, postoperative hospital stay and complications, were compared between the two groups. Clinical outcomes indicators, including the preoperative and postoperative visual analog scale (VAS)  for leg The SPSS 25.0 program  was used for statistical analysis of all data. Comparisons between the two groups were analyzed by independent-sample t tests or Wilcoxon test, whereas paired-samples t tests were used for intragroup comparison. Sex, type of calcification, location of calcification and complications were compared by chi-square tests. The modified MacNab criteria were compared by the Mann–Whitney U test. P < 0.05 indicates a statistically significant difference.All patients were successfully operated on by a trained and experienced spine surgeon in our hospital. We followed up all 54 patients (28 in the PEID group and 26 in the PETD group). The demographic characteristics  of the two groups were significantly similar as shown in Table P = 1.000) as shown in Table VAS for leg pain and ODI scores were used to evaluate the clinical results before and 1 day, 1 month, 3 months, 6 months and 12 months after the operation. The postoperative VAS and ODI scores of the two groups were obviously lower than those before surgery as shown in Table In the PEID group, 1 patient had a dural sac tear but no cerebrospinal fluid leakage; 1 patient had postoperative dysesthesia. In the PETD group, 1 patient had postoperative dysesthesia; 1 patient had residue of herniation. The symptoms of patient with residue were relieved after the second operation while the rest of patients were cured after conservative treatments. During the follow-up period, there were no reports of epidural hematoma, infection or lower extremity deep vein thrombosis.Although both PEID and PETD could achieve good clinical efficacy in the treatment of L5-S1 CLDH, our study showed that PEID group had shorter operative time and fewer intraoperative fluoroscopy times compared with the PETD group.Compared with children, CLDH is more common in adults. Children usually are treated by conservative treatments, while adults fail to. Calcified disc herniation adheres extensively to nerve roots and the dural sac, which not only increases the difficulty of removal by PELD, but also enlarges the risk of nerve root injury and dural sac tear. Some previous studies showed that PELD was applied to treat CLDH –14. DaboIn our study, postoperative outcomes demonstrated that the symptoms of all patients were significantly relieved by PEID or PETD. For the treatment of CLDH, we used an ultrasonic osteotome to remove the calcified disc. Ultrasonic osteotome had selective osteotomy properties and retains the adjacent soft tissue , 18. ComCompared with PEID, PETD has some deficiencies in the treatment of L5-S1 CLDH. PETD is a relatively complex procedure with a long learning curve. Multiple punctures and adequate foraminoplasty are needed, especially in the L5-S1 level with foraminal stenosis and a high iliac crest, which not only increases the operative time and fluoroscopy times, but also enlarges the risk of the exiting nerve root injury. Moreover, surgeons and patients are also exposed to much radiation . The worIn our research, no significant difference was observed in the complication rate between the PEID and PETD groups, which may be due to the small number of cases or short follow-up time. In the PEID group, 1 patient had a dural sac tear but no cerebrospinal fluid leak, and 1 patient had postoperative dysesthesia. In addition, 1 patient had postoperative dysesthesia, and 1 patient had residue of herniation in the PETD group. According to our experience, repeated punctures and foraminoplasty may cause the nerve root injury, which may lead to postoperative dysesthesia. The tight adhesion between calcified intervertebral disc and surrounding nerve tissues may result in nerve root injury, dural sac tear and even cerebrospinal fluid leakage. Residue of herniation may be caused by the poor position of the working channel due to the obstruction of the high iliac crest, resulting in insufficient decompression.There were some limitations in our research. First, this was a retrospective study. Moreover, the sample size is small and the follow-up period is short. Further studies with multicenter, large sample and long-term follow-up will be conducted in the future.Both PEID and PETD has achieved good clinical outcomes for L5-S1 CLDH. The clinical efficacy of PEID and PETD in the treatment of CLDH at L5-S1 level are comparable. Compared with PETD, PEID has the advantages of shorter operative time and a reduced number of fluoroscopy times in the treatment of L5-S1CLDH.


Inia geoffrensis, n = 25) and Bolivian  river dolphins of the Amazon basin and in selected tissue samples of franciscanas  stranded or bycaught in southeastern Brazil. Additionally, available franciscana tissue samples were examined by histopathology. Herpesvirus DNA was amplified in 13 Bolivian river dolphins  and 14 franciscanas . All Amazon river dolphins were herpesvirus-negative. Two different herpesviruses were found in Bolivian river dolphins: a previously known gammaherpesvirus detected in blood and/or skin samples of all positive individuals and a novel alphaherpesvirus in the skin of one animal. A new gammaherpesvirus was found in several franciscana samples—the first herpesvirus recorded in Pontoporiidae. Intranuclear inclusion bodies consistent with herpesvirus were observed in the lymph node of one franciscana. The high divergence among the obtained herpesviruses and those previously described can be explained by viral-host coevolution, and by the fact that these populations are fairly isolated.River dolphins are a highly threatened polyphyletic group comprised of four odontocete families: Iniidae, Pontoporiidae, Lipotidae, and Platanistidae, the first two endemic to South America. To address the knowledge gap regarding infectious agents in this cetacean group, we surveyed the presence of herpesviruses by PCR in skin and/or blood samples of live-captured Amazon ( They are likely relict representatives of originally diverse marine taxa that precede the radiation of the Delphinidae and remained in riverine ecosystems or coastal waters 1. These families are characterized by certain morphological similarities, such as narrow and elongated rostrum, elongated and fused mandibular symphysis, small eyes, flexible neck and broad pectoral fins3. River dolphins are currently the most threatened group of cetaceans4, including the last functionally extinct cetacean species—the baiji (Lipotes vexillifer)5.The polyphyletic group “river dolphins” comprises four different odontocete families: Iniidae, Pontoporiidae, Lipotidae and Platanistidae6 and Hrbek et al.7, the Iniidae comprises three species: the Araguaian boto (Inia araguaiaensis), the Bolivian river dolphin (I. boliviensis), and the Amazon river dolphin or boto (Inia geoffrensis). The Iniidae are widely distributed throughout rivers and lakes of the Amazon, Orinoco and Araguaia-Tocantins basins8, and are strongly dependent on river margins and flooded forests10. The genus Inia is considered Endangered by the IUCN Red List and the Brazilian Ministry of Environment12 especially due to illegal hunting13, but also to several other factors such as habitat fragmentation, mining-related pollution (e.g. mercury), deforestation and burning of primary forest, oil exploitation16, fishing interaction and bycatch17, and increased ship traffic18.Two families of river dolphins are endemic in South America: Iniidae and Pontoporiidae. According to da SilvaPontoporia blainvillei), also known as toninha or La Plata dolphin. The franciscana is a small cetacean endemic of the southwestern Atlantic Ocean, that inhabits coastal and occasionally estuarine waters from the state of Espírito Santo, southeastern Brazil (18º25′S–30º42′W) to the Chubut province, in the northern Argentinean Patagonia (42º35′S–64º48′W), with some gaps in their distribution19. This species is classified as Vulnerable by the IUCN Red List20 and as Critically Endangered by the Brazilian Ministry of Environment12. The main threats to franciscanas are bycatch in gillnet fisheries20, followed by overfishing21, chemical pollution23, and ingestion of plastic debris24.The Pontoporiidae family comprises only one species—the franciscana 51, which might be able to impact reproduction in affected populations52.Herpesviruses (HV) are enveloped double-stranded DNA viruses within the family In light of the scarce literature regarding herpesvirus infections in riverine dolphins, our goals were to (1) establish and compare the presence of herpesviruses in threatened wild Amazon river dolphins, Bolivian river dolphins and franciscanas in Brazil and (2) elucidate the phylogeny of the detected herpesviruses.32. Regarding the gB gene, it was amplified in 9.1%  of the cutaneous samples—collected from different animals than those with skin samples positive to DPOL gene, and also in 4.5%  of blood samples. Detailed descriptions of DPOL and gB positive and negative samples are provided in Supplementary Table Herpesvirus DNA was amplified solely in the Bolivian river dolphins of Guaporé River. Overall, herpesvirus DNA was detected in 59.1%  of these individuals by DPOL and/or gB PCR, including cutaneous samples from four animals and blood samples from eleven animals. Two dolphins had concomitant herpesvirus infection in skin and blood (Boto 6-GR and Boto 20-GR). The DPOL gene was amplified in 9.1%  of skin samples and 55.6%  of blood samples of these dolphins. Of note, the DPOL sequence amplified from a proliferative skin lesion of Boto 20-GR was previously published by our groupDelphinus delphis) from Portugal  and one from the Canary Islands, Spain (MN179655). It also presented the highest amino acid identity (61.7%) to alphaherpesviruses from a Cuvier's beaked whale  of the Mediterranean coast of Spain and a harbor porpoise  from Portugal. Regarding the gB gene, the same sequence was found in the skin samples of two individuals  and in blood sample of another specimen (Boto 20-GR). This gB sequence was most similar  to a gammaherpesvirus sequence detected in a squirrel monkey . Overall, gammaherpesvirus sequences were detected in 13.6%  of the skin samples and 61.1%  of the blood samples of Bolivian river dolphins, and an alphaherpesvirus was found in 4.5% of the tested skin samples .Two different DPOL sequences were identified by sequencing; one was present in the proliferative skin lesion of Boto 20-GR  and in blood samples of ten additional dolphins, corresponding to a gammaherpesvirus. The other DPOL sequence was found in a single apparently healthy skin sample of Boto 6-GR, which presented the highest nucleotide identity (62.2%) to the alphaherpesvirus sequences described in two common dolphins  and Tapajós  rivers were negative for herpesvirus on both DPOL and gB PCRs.Balaenoptera acutorostrata) from Spain. Regarding gB, the same sequence was found in 24 samples , prescapular lymph node [n = 3], mesenteric lymph node [n = 3], spleen [n = 2], mediastinal lymph node [n = 1], thymus [n = 1], unidentified lymph node [n = 1], heart [n = 1], liver [n = 1], skin [n = 1], spinal cord [n = 1], skeletal muscle [n = 1], and testicle [n = 1]) of 12 franciscanas, and had the highest nucleotide (68.9%) and amino acid identities (72.7%) with gammaherpesvirus sequences found in a common vampire bat  and squirrel monkey , respectively. Detailed descriptions of DPOL and gB positive and negative samples are provided in Supplementary Table Herpesviral DNA was amplified in 51.9%  of the tested franciscanas; 28.6%  of calves, 75%  of juveniles and 80%  adults. The same DPOL sequence was identified in 14 tissue samples  to the gammaherpesvirus sequence KP995688, found in a common minke whale , MZ223443  and MZ223444 . Representative gB sequences were submitted under accession numbers MZ209258 (gammaherpesvirus obtained from the skin of the Bolivian river dolphin Boto 14-GR) and MZ209259 . Unfortunately, it was not possible to obtain the complete HV genome sequence in any of the HV-positive samples tested by metagenomics .Inia. Regarding the HV-positive franciscanas, one of them (MM568) presented intranuclear inclusion bodies consistent with typical herpesviral inclusions in lymph nodes , a Bolivian river dolphin , a dwarf sperm whale (Kogia sima), and an Atlantic spotted dolphin 53. To the authors’ knowledge, this is the first report of an alphaherpesvirus in a Bolivian river dolphin worldwide, and also of herpesvirus in a franciscana. One Bolivian river dolphin (Boto 6-GR) had a gammaherpesvirus and an alphaherpesvirus. The presence of different herpesviral sequences in the same individual has been previously described in other cetacean species, as in Atlantic bottlenose dolphin (Tursiops truncatus)42, striped dolphin 55, dwarf sperm whale32 and common dolphin (Delphinus delphis)56.This study reports the presence of herpesviruses in Bolivian river dolphins and franciscanas in Brazil. All Amazon river dolphins were negative. Gammaherpesvirus sequences were detected in 64.7% of the blood and 13.6% of the skin samples of Bolivian river dolphins, and an alphaherpesvirus was found solely in the skin sample of one individual. Additionally, a hereinto unknown gammaherpesvirus sequence was found in 51.9% of the tested franciscanas. In South American cetaceans, the presence of herpesviruses had been reported in two Guiana dolphin , reinforcing our hypothesis. The three herpesviruses found in river dolphins (Bolivian river dolphins and franciscanas) were highly divergent from the closest sequences available in GenBank. The gammaherpesvirus found in Bolivian river dolphins was previously tentatively denominated Iniid herpesvirus 132. According to the current HV nomenclature used by the ICTV39, we propose renaming the virus as Iniid gammaherpesvirus 1. The novel unique sequences found in this study likely correspond to novel herpesvirus species, herein proposed as Iniid alphaherpesvirus 1 for the novel alphaherpesvirus found in a Bolivian river dolphin skin sample, and Pontoporiid gammaherpesvirus 1 for the gammaherpesvirus discovered in franciscanas. The large differences between the herpesviruses found in river dolphins from South America and those previously described in marine cetaceans can be explained by the co-evolution of the viruses with their hosts, as described for other members of the family Herpesviridae59. According to McGowen et al.60, the families Iniidae and Pontoporiidae, as well as Lipotidae, separated from the other odontocete cetaceans around the Late Oligocene, between 23.03 and 24.92 million years ago. We hypothesize the herpesviruses detected in the South American river dolphin co-evolved with their host during millions of years, which likely explain the large differences observed in well-preserved herpesviral genes (DNA polymerase and glycoprotein B) when compared to those detected in other cetacean families. Another possibility would be the cross-species transmission from unidentified host species. Further studies are necessary to explore these hypotheses.High gammaherpesvirus prevalence was observed in the studied Bolivian river dolphins and franciscanas, indicating that these species are likely the natural host of the detected viruses61. Specifically, the Tapajós River Basin hosts the largest small-scale gold mining (“garimpo” in Portuguese) district in the world62, an activity known for using large amounts of mercury, water siltation, and habitat fragmentation and degradation65. In mammals, mercury intoxication causes neurological and reproductive disorders, and immunosuppression68. High total mercury loads (from 0.61 to 2.3 mg/kg wet weight) were recently described in muscle samples of eight of the Amazon river dolphins from the Tapajós River69, also analyzed in the present study. Five Amazon river dolphins from the Tapajós River were in poor nutritional condition, with three individuals presenting loss of nucal fat and one of them also presenting marked scapula and rib cage. By contrast, all the Amazon river dolphins from Negro River and Bolivian river dolphins from Guaporé River apparently were in good nutritional condition. It is well known that immunosuppression, as a result of concurrent disease, stress and reproductive effort, may reactivate latent herpesvirus infections70, leading to periodic or continuous shedding of infectious virions and destruction of the infected cell71, consequently increasing the likelihood of their detection. The absence of herpesvirus detection in the Amazon river dolphins may be explained by (1) the low number of tested samples from Amazon river dolphins captured in Tapajós River (2) apparent good health status of Amazon river dolphins from Negro River, (3) a lack of sensitivity of the employed methods to detect another divergent herpesvirus infecting Amazon river dolphins or (4) differences in the evolutionary history of Amazon and Bolivian river dolphins and their herpesviruses, once herpesviruses and their natural hosts are intimately related, usually sharing a common evolutionary history and mutual adaptation59. Future studies with a larger sample size are necessary to clarify the difference in herpesvirus prevalence between Amazon and Bolivian river dolphins.The absence of herpesvirus detection in the Amazon river dolphins was unexpected, especially from those of Tapajós River, which inhabit an area intensely affected by anthropogenic activities32. Additionally, an alphaherpesvirus was amplified from a cutaneous sample of another Bolivian river dolphin (Boto 6-GR). Unfortunately, only the proliferative cutaneous lesion of Boto 20-GR was histologically evaluated, due to the lack of available fixed samples from the remaining HV-positive individuals. No gross cutaneous lesions were observed in other HV-positive Bolivian river dolphins aside from Boto 20-GR. In franciscanas, no evidences of herpesvirus infection were found aside from amphophilic intranuclear inclusion bodies in one individual. The lack of associated lesions was also observed in 12 herpesvirus-positive striped dolphins  from Italy72. It is important to remark that many herpesviruses only cause mild disease in their natural host59, and the absence of HV-associated lesions in the examined franciscanas with gammaherpesvirus and in most of the alpha- and gammaherpesvirus-positive Bolivian river dolphins may be examples of the low pathogenicity of some HVs. Nevertheless, the Iniid alphaherpesvirus 1 and Pontoporiid gammaherpesvirus 1 pathogenicity should be further investigated.Regarding the potential pathogenicity of the riverine dolphin herpesviruses, the gammaherpesvirus detected in several Bolivian river dolphins was previously identified in a proliferative skin lesion of one individual from the same population (Boto 20-GR), and might be associated with its etiology75. Cardiomyocyte and myocyte hypereosinophilia have been also described in human drowning cases76. The pathological findings are in agreement with the franciscanas’ records since several of these animals were directly recovered entangled in fishing nets and handed by the fisherman to the researchers. Bycatch is considered the main threat affecting franciscana’s conservation, especially entanglement in gillnets20. The population recovery from the current high levels of bycatch across this species range is limited due to franciscana’s short life span and low reproductive potential78. Although several studies tested alternative approaches and techniques with artisanal fisheries, no solutions have significantly reduced the mortality rates associated with this impact80. The high occurrence of fisheries interaction observed in our study reinforces the need to develop management strategies to reduce the impact of incidental captures of this species in the studied region.The main pathological findings observed in the herpesvirus-positive franciscanas were linear cutaneous lacerations and imprints, multi-organ congestion, pulmonary edema and myocyte and cardiomyocyte degeneration and necrosis. In cetaceans, although non-specific, these lesions are usually related to peracute underwater entrapment syndrome associated with bycatchHerein we described the presence of two herpesviruses in Bolivian river dolphins: a previously known gammaherpesvirus detected in blood and skin samples, and a novel alphaherpesvirus species present in a skin sample. A new gammaherpesvirus species was also found in several tissue samples from franciscanas, the first herpesvirus ever recorded in the family Pontoporiidae. Based on the high gammaherpesvirus prevalence observed in Bolivian river dolphin  and franciscana  and the absence of associated lesions in most of the animals (with the exception of the skin lesion observed in Boto 20-GR), these species are likely their natural hosts. Of note, the obtained sequences were highly divergent when compared to other herpesviruses available on public databases, which could be explained by viral-host coevolution—once these riverine species diverged from the other odontocete cetaceans millions of years ago, and by the fact that these populations are fairly isolated to this date. Future studies are warranted to elucidate the pathological potential and impact of these novel viruses in highly threatened riverine cetaceans.Inia  were collected in expeditions conducted in the Brazilian Amazon basin in 2015 and 2017 , Bolivian river dolphins from Guaporé River (22 skin samples and 18 blood samples) and Amazon river dolphins from Tapajós River (9 blood samples). Blood samples were collected from the ventral caudal peduncle and placed in Vacutainer tubes with EDTA. Biopsied skin samples were collected from the caudal and dorsal fins (n = 36) or cutaneous lesions (n = 1). Blood and skin samples were kept at − 80 °C until analysis.82. The age class was established according to total body length, as described by Rosas and Monteiro-Filho83, using the values of the Franciscana management area II . In three franciscanas the exact age was determined through the histology of the teeth85. Selected tissue samples were fixed in 10% formalin at room temperature. An additional set of samples was frozen at − 20 °C or − 80 °C until analysis. Additional data regarding the franciscanas are displayed in Table Overall, 91 tissue samples  of 27 franciscanas stranded or bycaught in the coast of southeastern Brazil . The PCR protocol described by Ehlers et al.87 using the primer set GH1 to amplify the glycoprotein B (gB) gene of gammaherpesviruses was also performed in all available samples.Total DNA was extracted from frozen samples using the kit DNA Blood and Tissue . The panPCR nested protocol described by VanDevanter et al.32. The skin sample of Boto 20-GR was also tested for HV gB32. A Magellanic penguin alphaherpesvirus and a fur seal gammaherpesvirus were selected as positive controls89. DPEC water was used as no template control. Positive amplicons were purified using ExoSap-IT  and directly sequenced by Sanger using the ABI 3730 DNA Analyser. The obtained sequences were aligned in MEGA Software 7.0 by Muscle to obtain the consensus sequences90. After editing out the primers, these consensus sequences were compared to those available at GenBank by Blast search, and p-distance was calculated to obtain the percentage of identity ([1 – p-distance) * 100]). Finally, DPOL and gB Maximum likelihood phylograms based on the deduced amino acid sequences of the herpesviruses found in this study, representative herpesvirus species recognized by the International Committee on Viral Taxonomy (ICTV) and herpesvirus previously identified in cetaceans were constructed using MEGA 7.0  and one blood sample (Boto 20-GR) of Bolivian river dolphins captured at Guaporé River, as well as a skin sample from one Amazon river dolphin of Negro river (Boto 12-NR), were previously tested by DPOL PCRTissue samples from selected HV-positive animal were submitted to complete sequencing at the University of Florida.Available formalin-fixed samples from HV-positive cases were embedded in paraffin wax, processed as routine, sectioned at 5 μm and stained with hematoxylin and eosin for light microscopic examination.The Amazon river dolphins from the Tapajós river were sampled as part of the South American River Dolphin (SARDI) integrated strategy for the conservation of South American river dolphins, funded by WWF. The Amazon river dolphins from Negro river were sampled as part of Projeto Mamíferos Aquáticos da Amazonia and sponsored by Ampa/Petrobras Socioambiental. All study samples were collected in full compliance with specific federal permits issued by the Brazilian Ministry of Environment (MMA) and the Chico Mendes Institute for Biodiversity Conservation (ICMBio), and approved by the Biodiversity Information and Authorization System , ABIO Nº 1169/2019 and ICMBio/MMA (13157). Three fransciscanas were collected as part of the Santos Basin Beach Monitoring Project (Projeto de Monitoramento de Praias da Bacia de Santos—PMP-BS), licensed by the Brazilian Institute of the Environment and Renewable Natural Resources (IBAMA) of the Brazilian Ministry of Environment (ABIO Nº 1169/2019). All procedures were performed in accordance with the Ethical Committee in Animal Research of the School of Veterinary Medicine and Animal Sciences, University of São Paulo or the Ethical Committee of Animal Use of the National Institute of the Amazon Research, the latter responsible for the approval of the protocol for handling and removing of small samples of epithelial tissue and blood from the botos . ARRIVE guidelines: not applicable.Supplementary Information.