Fish skin microbiomes are rarely studied in inland water systems, in spite of their importance for fish health and ecology. This is mainly because fish species distribution often covaries with other biotic and abiotic factors, complicating the study design. We tackled this issue in the northern part of the Jordan River system, in which a few fish species geographically overlap, across steep gradients of water temperature and salinity.Using 16S rRNA metabarcoding, we studied the water properties that shape the skin bacterial communities, and their interaction with fish taxonomy. To better characterise the indigenous skin community, we excluded bacteria that were equally abundant in the skin samples and in the water samples, from our analysis of the skin samples. With this in mind, we found alpha diversity of the skin communities to be stable across sites, but higher in benthic loaches, compared to other fish. Beta diversity was found to be different among sites and to weakly covary with the dissolved oxygen, when treated skin communities were considered. In contrast, water temperature and conductivity were strong factors explaining beta diversity in the untreated skin communities. Beta diversity differences between co-occurring fish species emerged only for the treated skin communities. Metagenomics predictions highlighted the microbiome functional implications of excluding the water community contamination from the fish skin communities. Finally, we found that human-induced eutrophication promotes dysbiosis of the fish skin community, with signatures relating to fish health.Consideration of the background water microbiome when studying fish skin microbiomes, across varying fish species and water properties, exposes patterns otherwise undetected and highlight among-fish-species differences. We suggest that sporadic nutrient pollution events, otherwise undetected, drive fish skin communities to dysbiosis. This finding is in line with a recent study, showing that biofilms capture sporadic pollution events, undetectable by interspersed water monitoring.Video abstract. The importance of the cutaneous mucus in fish is well established. The teleost epidermal mucus provides mechanical protection against physical and biological harm; thanks to its viscosity and high turnover , 2, and Vibrio spp. [Vibrio spp. appear to constitute small fractions of normal microbiomes and culture-dependent techniques grossly over-represent them [Although the skin microbiome in fish has not been the focus of microbiome research, some important progress has been made by a few research groups. The skin microbiome is known to be affected by both environmental and fish species dependent factors , 15, witrio spp. . Conversent them . Additioent them  and probent them , both wient them , 23, 24.ent them .While most of the current research is targeted at fish species with commercial relevance , 26–29 oIn this work, we have sampled the upper reaches of the Jordan River system and Springs Valley streams, north and south of the Sea of Galilee, respectively. This range includes heterogeneous sites, differing in fish community composition and water properties. We sampled mostly in nature reserves, although three of the sites suffer human-induced eutrophication, one of which is a settling pool and two others receiving fish farm and fishpond outlets. The geographic range of a few fish species in this part of the system partially overlap, thus allowing us to study host and site-dependent effects on fish skin microbiomes. Due to the sensitivity of the sampled ecosystem, we employed a non-destructive sampling procedure, swabbing the captured fish on site and immediately releasing them. Our results reveal effects of both fish species and sampling site on the skin microbiome, highlight the importance of considering the background microbial contamination of the swab samples by the water and show that eutrophication may drive the skin microbiome to dysbiosis.Capoeta damascina (Cyprinidae) were collected from all sites in the Hermon (H) and Snir (S) streams, and from two sites in the Jordan River (J). The species most co-occurring with C. damascina was Oxynoemacheilus insignis (Nemacheilidae), which was found in three H sites, one S site and one J site. Unlike C. damascina, O. insignis was also captured in Tel-Saharonim Stream . Another relatively widely dispersed group included the Tilapiine (Cichlidae) species Coptodon zillii (formerly Tilapia), Sarotherodon galilaeus and hybrids of Oreochromis aureus, which were found in three J sites in the northern basin, co-occurring with C. damascina in one site, and in the two southern basin streams, co-occurring with O. insignis in one site. The remaining species, belonging to Cyprinidae, Haplochrominae (Cichlidae), Poeciliidae and Mugilidae, had a narrow geographic rage and a small geographic overlap with other species  and two streams to its south (one and two sites per stream). We will hereafter denote the two regions the “northern” and “southern” basins , although the 95% CI overlapped for Bacilli and Fusobacteriia. Prominent genera , Anaerobacillus sp.  and Skermanella sp. .The bacterial classes recovered from raw swab communities, having the highest median relative abundances  index  and an up value = 1.8 × 10− 11) and fish family or tribe  were significant factors, with many significant pairwise differences among streams .Alpha diversity results are summarised in Fig. q value = 0.015 and Additional file q value = 0.015 and Additional file μ values were lower than for the raw swab samples for all sites . The Faith PD values of the water communities covaried with those of the raw swab communities, with the exception of sites with low water temperatures . Kernel PCA, while agnostic to the ASV phylogeny, allows to compare a linear kernel with a radial basis function (RBF) kernel, which does not assume linearity . We thenp value = 0.001), as well as pairwise stream comparisons . However, significant differences between pairs of fish taxa that co-occur geographically were found only for the corrected skin communities. These pairs included Nemacheilidae and its co-occurring fish taxa . Differences between co-occurring fish taxa were not recovered for raw swab communities.Both stream-based , a salinity bioindicator [Anaerobacillus (ASV e942) for the corrected communities , but this effect was mostly lost for the corrected skin community values  and a weak correlation with the dissolved oxygen was observed instead . The second PCoA axis had weaker correlations with any of the water measurements than the first axis. For the second axis, the raw swab community values correlated with pH and dissolved oxygen measurements  and the corrected skin community values correlated with the temperature measurements (R2 = 0.14).The explanatory ASVs changed between the raw and corrected communities, with ndicator , having ndicator  Phycisphus ASV e92 for theus ASV e92 for theus ASV e92 for theus ASV e92 for theTo summarise, beta diversity in the raw swab communities is best explained by the water salinity or temperature. The corrected skin communities, however, are less affected by water characteristics, of which dissolved oxygen level is the strongest. Accordingly, in the raw swab communities, a salinity bioindicator bacterium varies the most, whereas for the corrected skin communities we detect large variations in anaerobic bacteria. It is important to note that dissolved oxygen measurements were not taken in the H stream, and thus, the strength of this finding is tentative.R2 depended on the kernel but was similar between the raw swab and corrected skin datasets. For PC2, the dissolved oxygen was not dominant either, independently from assumptions of linearity  for both the raw and corrected communities.To further investigate the relationship between the fish taxonomy and the skin microbiome, we carried out another PCoA, separating the northern and southern basins to increase the geographic range overlap of the included fish taxa in each analysis Fig. . This anThe ratio between Proteobacteria and Bacteroidetes is associated with fish health, with compromised individuals having increased Bacteroidetes relative abundances . We compTo understand the effect of water background contamination on the inference of fish skin microbiomes and the reconstruction of metabolic models that would be recovered from their metagenomes, we employed PICRUSt . PICRUStMugil cephalus in marine environments and found it to have an uncharacteristically high relative abundances of Alphaproteobacteria, compared to the strictly marine fish they have sampled. A similar excess of Alphaproteobacteria was found in wild Salmo salar [Salvelinus fontinalis [S. salar fry [Silurus glanis, a catfish caught in the wild [Few studies have investigated skin microbiomes in freshwater fish, and it is not clear if it is fundamentally different than those of marine fish. Larsen et al.  have sammo salar  and Salvntinalis , anadromntinalis . In staralar fry , and alsthe wild . Of the In this study, we have investigated freshwater fish and identified Alphaproteobacteria as having the highest median relative abundance, highlighting their dominance as a possible feature of some freshwater fish skin microbiomes, compared to marine fish , 48. SucR2 = 0.75 and 0.66 for the first PCoA axis). However, when the water background contamination is addressed, these effects are lost completely for alpha diversity, and become much weaker, in the case of beta diversity, where the percent dissolved oxygen and temperature emerge as two weak factors .Based on alpha and beta diversity analyses, the raw swab communities of some of the sites are clearly different from the water communities in the same location, particularly when water temperature is low. This may form the impression that the raw swab communities properly represent the skin microbiome in fish. However, our results show that this may be misleading and the water background contamination should be formally addressed. Water measurements, especially the temperature, may seem to govern alpha and beta diversity of fish skin communities. The temperature and conductivity may be perceived as overwhelmingly strong effects on beta diversity in particular , a site in the Snir Stream downstream a fish farm outlet (S.3) and a fishpond outlet on the Jordan River (J.1). The relative abundances of the two phyla, Proteobacteria and Bacteroidetes, strongly covary with these levels of interrupted and uninterrupted sites, where Bacteroidetes relative abundances increase at the expense of Proteobacteria, at the interrupted sites. The relationship between these two phyla is a hallmark of dysbiosis and reduced fish health, in the skin microbiome , althoug, a site To predict the implications of background noise treatment for functional inference, we compared the KEGG pathway composition between the raw and corrected predicted metagenomes. We have found that the removal of variants that equally occur in the skin and water microbiomes fundamentally changes the variety of potential pathways in the metagenome. The largest change between the raw and corrected microbial skin communities was in KEGG pathways related to the biosynthesis of secondary metabolites, microbial metabolism in diverse environments and the biosynthesis of antibiotics. These categories are fundamental to the way bacteria interact with their environment and a metagenomic analysis of this sort would be more accurate, taking background noise effects on the microbiome composition into consideration.In this study, we highlight the importance of a formal consideration of water background noise in fish skin microbiomes, when studying heterogenous inland systems, in which fish species and environmental conditions covary. In the northern Jordan River system, north and south of the Sea of Galilee, we identify a consistent alpha diversity among sites, indicating that the limiting factors of alpha diversity in the skin microbiome are set by the mucus itself, and not by water properties. We further identify the dissolved oxygen to play a role in governing the community composition on the skin, in accordance with a previous research. Finally, we find the ratio of Proteobacteria and Bacteroidetes in the skin microbiome, a useful and informative biomarker for freshwater habitat monitoring.Oxynoemacheilus insignis  is the only loach in the system. Astatotilapia flaviijosephi  is the only haplochromine in the system, and it is therefore the only cichlid with egg-shaped marks on its anal fin. Coptodon zillii  is a cichlid with a dotted tail fin and 8–9 protrusions per gill raker. Sarotherodon galilaeus  is a cichlid with a clear convex tail fin and over 13 protrusions per gill raker and a black mark on the operculum. Oreochromis hybrids are cichlids with striped tail fins and over 17 protrusions per gill raker. Gambusia affinis  is the only killifish in the study area and identifiable by its size (< 5 cm) and superior mouth. Mugilidae individuals escaped from fish farms in the regions (The Jordan River system has no marine outlet.) and were identified by their general mugilid form. Within Cyprinidae, Carasobarbus canis  and Barbus longiceps  each have two pairs of barbels. B. longiceps with an elongated head and over 50 scales along its lateral line. C. canis is distinguishable by its short head and very large scales, less than 40 along the lateral line. Capoeta damascina  has one pair of barbels and very small scales, over 70 along its lateral line. Garra nana  and Garra jordanica Geiger and Freyhof, 2014 have small, barely visible barbels. G. jordanica has a suction cup and G. nana a fold, under the lower lip. Acanthobrama lissneri Tortonese, 1952, has elongated and compressed body, with deeply forked tail and up to 12 cm adult total length, and Pseudophoxinus kervillei  is of similar size, has circular body section and a forked tail with a black stain at the base.Samples were collected between August and October 2017 from 17 sites in the Northern Jordan River water system, nine of the sites representing the upper reaches tributaries Hermon and Snir, five sites representing the northern Jordan River itself and three additional sites from the Springs Valley Jordan River tributaries  sites and pH at two Jordan River (J) sites.https://bit.ly/2YWRTvC).DNA was extracted from the swabs and filters using the DNeasy PowerSoil and PowerWater DNA extraction kits (Qiagen), respectively, following the manufacturer’s instructions. Metabarcoding libraries were prepared using a two-step PCR protocol, in which the first PCR reaction is designed to amplify the genetic marker along with artificial overhang sequences and the second PCR reaction is designed to attach sample specific barcode sequences and Illumina flow cell adapters. The forward and reverse PCR primers in the first reaction were ′5-tcgtcggcagcgtcagatgtgtataagagacagCCTACGGGNGGCWGCAG-′3 and ′5-gtctcgtgggctcggagatgtgtataagagacagGACTACHVGGGTATCTAATCC-′3, respectively, including the target-specific primers for the V3–V4 region  with ovehttps://git.io/fjFZo (DOI: 10.5281/zenodo.3583001) as a Jupyter Notebook (https://bit.ly/2z7teFm), coupled with raw data and intermediate and output files. Sequence data trimming, amplicon sequence variant (ASV) prediction and taxonomic identification were carried out in Trimmomatic 0.39 [https://bit.ly/2Hcv6AZ) and DADA2 1.12 [https://bit.ly/2OZXrkl). The resulting ASV biom table was filtered with QIIME2 2019.4 [https://bit.ly/30euZwh). Following alpha rarefaction analysis (https://bit.ly/2NeetsA), the ASV biom table was further filtered to exclude samples with less than 1000 sequences. A subset of the ASV biom table was created to represent the skin microbiome without ASVs that are likely to belong strictly to the water (https://bit.ly/2Z4vXhp). In this subset, we included ASVs that were unique to the swab samples, or that had a significantly higher relative abundance in the swab samples than in water samples, based on Benjamini–Hochberg corrected [U test [https://bit.ly/2HbEyEP). To carry out this test, we used SciPy 1.2 [p value” and “q value”, respectively. This process is regarded as “background noise treatment”, and the subset as the “corrected skin community” throughout the text. To study the taxonomic composition of the samples (https://bit.ly/2TMkEFl) and the relationship between Proteobacteria and Bacteroidetes in the different sampling sites (https://bit.ly/33GUgkL), we collapsed the ASV biom table to taxonomic tables (https://bit.ly/2z9Qlic) using QIIME2 2019.4 [The bioinformatics analysis is provided on GitHub, at tic 0.39  (https:/DA2 1.12  (https:/2 2019.4  to excluorrected  Mann–Whi [U test  (https:/ciPy 1.2  and StatciPy 1.2 . The ori2 2019.4 .https://bit.ly/2OZPfR2). Faith PD depends on the number of ASVs in the sample, their pairwise phylogenetic distances and their relative abundances. We further tested the correlation of Faith PD values with the water measurements using SciPy 1.2 [https://bit.ly/2z6v217).To study the factors shaping alpha diversity, we computed Faith phylogenetic diversity (Faith PD) indices  for eachciPy 1.2 . This wahttps://bit.ly/2OZPfR2). The factors considered were the stream of origin and the family or tribe of the host fish. For the latter, we carried the analyses per basin to increase the geographic overlap of the fish species. This procedure was carried out for both the raw swab communities and corrected skin communities. We further tested the correlation of the water measurements with the values along the first and second PCoA axes, in order to explain these axes, using SciPy 1.2 [https://bit.ly/2KV9Vod). Finally, we executed ANCOM tests [https://bit.ly/2KH1M7O).To study the factors shaping beta diversity, we produced unweighted UNIFRAC matrices  which weciPy 1.2  (https:/OM tests  to identhttps://bit.ly/2rf396V).The UniFrac-based PCoA analysis and subsequent Pearson’s correlation assume linear relationships in various steps. To gage the importance of such assumptions in biasing the results, we utilised the kernel principal component analysis (PCA) and support vector regression (SVR), implemented in Scikit-learn . Kernel μ) of the different site and fish family levels, as well as the μ relative abundance of bacterial taxa in the raw swab and corrected skin samples. The analysis was carried out in PyMC3 [https://bit.ly/2M5P5nm), using normal priors with the sample mean. We additionally computed posterior distributions for the difference between each pair of compared μ values, expressed as the subtraction of μ1 and μ2, divided by the pooled standard deviation of both parameters. The pooled standard deviation was expressed as the sum of standard deviations, divided by 2. Posteriors were sampled at least 11,200 times per parameter, in four chains, until convergence.We have taken a Bayesian approach to evaluate the posterior Faith PD mean (in PyMC3  (https:/https://bit.ly/2ZcYSf4). ENZYME term abundances were converted to relative abundances using pandas 0.42 [p value” and “q value”, respectively. The KEGG PATHWAY categories of each significantly different entry were retrieved with Biopython’s REST KEGG API [To predict the differences in relative abundances of metabolic pathways between the raw and treated swab communities, we predicted their metagenomes and abundances of KEGG ENZYME terms , using Pdas 0.42  and the das 0.42  Wilcoxondas 0.42  in SciPydas 0.42  and Statdas 0.42 . The oriKEGG API .Additional file 1: Figure S1. Alpha rarefaction curves for each fish family or tribe, denoted by the color legend. The x axis is the size of sequence reads subsample and the y axis is the Shannon diversity in the subsample.Additional file 2: Figure S2. Posterior distributions of μ pairwise differences (A and B) and of μ (C and D) in streams and fish families. Each confidence interval represent one of four posterior sampling chains that were carried out for each comparison or for each level. Grey intervals represent the raw swab samples and the orange interval represent the skin corrected samples.Additional file 3: Figure S3. A comparison of R2 values between approaches with and without assumptions of linear relationships. To evaluate the effect that assumptions of linear relationships have on the proportion of principal component values that is explained by environmental factors, we compared the R2 values that were obtained with a linear kernel with those obtained with an RBF kernel. This was carried out with kernel PCA followed by SVR between PC1 or PC2, and one of the water physicochemical measurements.Additional file 4: Table S1. The number of fish swabs collected from each fish species in each site. Sites are sorted according to stream and basin. Fish species are sorted by family or tribe. Site codes correspond with Fig. N northern basin, north of the Sea of Galilee; S southern basin, south of the Sea of Galilee; IH exotic haplochromine; OH Oreochromis hybrid.Additional file 5: Table S2. Pairwise Kruskal–Wallis tests for alpha diversity differences among streams, for raw swab bacterial communities and corrected skin bacterial communities.Additional file 6: Table S3. Pairwise Kruskal–Wallis tests for alpha diversity differences among fish families or tribes, for raw swab bacterial communities and corrected skin bacterial communities.Additional file 7: Table S4. Pairwise PERMANOVA tests for beta diversity differences among streams, for raw swab bacterial communities and corrected skin bacterial communities.Additional file 8: Table S5. Pairwise PERMANOVA tests for beta diversity differences among fish families or tribes, for raw swab bacterial communities and corrected skin bacterial communities.


Linearbandkeramik , the first full farming culture in this area. Skeletal remains of several dozen victims of this prehistoric warfare are known from different sites in Germany and Austria. Here we show that the mass grave of Halberstadt, Germany, a new mass fatality site from the same period, reveals further and so far unknown facets of Early Neolithic collective lethal violence. A highly selected, almost exclusively adult male and non-local population sample was killed by targeted blows to the back of the head, indicating a practice of systematic execution under largely controlled conditions followed by careless disposal of the bodies. This discovery significantly increases current knowledge about warfare-related violent behaviour in Early Neolithic Central Europe.The later phase of the Central European Early Neolithic witnessed a rise in collective lethal violence to a level undocumented up to this date. This is evidenced by repeated massacres of settled communities of the  Linearbandkeramik (LBK) communities are evidenced by mass graves from the Early Neolithic of Central Europe. Here, Meyer et al. describe a newly discovered mass grave from Germany revealing the execution of a predominantly adult male group of non-local individuals.Prehistoric warfare and massacres of  Linearbandkeramik , which widely replaced the Mesolithic foraging way of life. While the LBK has long been considered well-researched3, recent studies have revealed substantial differences between the genetic compositions of Mesolithic and Early Neolithic populations. These major discontinuities were caused by an influx of Neolithic farmers originating from southeastern Europe, where the LBK itself developed7. Spreading from there, LBK farmers introduced domesticated animals and crops to Central Europe as the economic basis of a new way of life in this region. With time these new settlers colonised large parts of Central Europe, mainly targeting the fertile loess soils2. By firmly establishing the Early Neolithic agricultural system these people significantly changed their natural environment by clearing forests and establishing permanent settlements and farming plots, in which considerable amounts of time and effort were invested8. It is assumed that kinship and membership in probably pioneering lineages played an important part in the organisation of LBK communities10. Specific patterns known so far from biomolecular studies include differentiation in the funeral rite connected to the place of origin and very likely virilocal residence patterns in addition to the large-scale genetic changes during the Mesolithic–Neolithic transition9.In Central Europe, the first full Neolithic farming culture is the 11. Detailed analyses of how and why the deceased became part of their respective burial features allow important insights into the complex interplay of actors, factors, and events that shaped the contemporary LBK experience of communities and individuals12. Usually, LBK burials are found as individual inhumations or cremations in dedicated cemeteries13. Overall, there is a large variety but left-crouched inhumation burials are consistently encountered, often equipped with grave goods. The most common grave orientation is roughly East–West, but extended, right-crouched, unfurnished, or differently orientated burials do occur as well14. A substantial part of the LBK skeletal record stems from settlement burials, which do not differ greatly from the cemetery burials15. Multiple burials of carefully arranged bodies are rare16.The skeletal remains of the overall LBK population form the biogenic basis of these summary studies, but the specific contexts in which they are discovered also hold key information for characterising their actual life and death experiences as a bioarchaeological record17. The highly intense manipulation of corpses and possible ritualistic cannibalism practised at Herxheim represent behaviours that are still difficult to unravel, but knowledge about them has affected the interpretation of other sites that have long been considered as evidence for LBK warfare and, more specifically, massacres19. These sites are the mass grave of Talheim, Germany and the enclosure at Asparn/Schletz, Austria23. Both yielded human skeletal remains bearing clear signs of perimortem lethal violence such as blunt force cranial injuries and possible arrow wounds. While the bodies at Talheim had been quickly collected and deposited in a disorganised mass grave, those at Asparn show signs of carnivore gnawing and partial disarticulation that occurred before natural sedimentation in the ditches23. Recently, a third massacre site has been analysed: the mass grave of Schöneck-Kilianstädten, Germany24. It is assumed that at these three sites LBK communities were wilfully destroyed by unknown attackers, most likely members of other groups labelled today as LBK, possibly even close neighbours. The recent interpretation of the Kilianstädten mass grave as a further violence-related mass fatality site that shows similar patterns to Talheim and Asparn/Schletz again strengthens the interpretation of these massacres as warfare-related24.Also found in various LBK contexts are isolated human body parts and partial skeletons. These occur scattered within settlement pits, in natural caves, or in enclosure pits and ditches and might be the remains of disturbed older burials or have had ritualistic significance. This last point is exemplified by the large enclosure site of Herxheim, Germany, where human bodies were subjected to complex postmortem manipulation and disarticulation before being deposited in mixed assemblages with selected animal bones and various artefacts25, the victimised population samples seem to be representative of settled LBK communities, including balanced numbers of children and adults of both sexes. The high percentage of subadults among the massacre victims and their burial within settlement areas are both strongly indicative of surprise attacks on villages, a practice encountered among many different societies27. One further LBK mass grave at Wiederstedt, Germany is even dominated by subadult individuals , but in the absence of recognisable perimortem injuries another cause of death seems more likely in this case. However, like the other mass graves, it was located in a settlement context28, thereby demonstrating again the close association of LBK children with village sites. This is also evident from the settlement burials where they generally dominate29.Signs of careful funeral ritual, clearly evident in the regular graves of the LBK, are absent for the massacre victims. While some younger women might have been captured rather than killed, which is suggested for all three massacre sites currently known30. Once the victims expired, their physical remains likely held no more meaning for the aggressors and apparently were disposed of without further ritual treatment12. This postmortem neglect again suggests that the massacre victims were casualties of intergroup warfare rather than part of complex ritual actions charged with greater meaning31.Lacking from the massacre or mass grave sites is a clearly focussed and patterned postmortem treatment like that found for example at Herxheim. Although targeted destruction of the lower leg bones was observed at Kilianstädten, this apparently occurred within the context of the massacre itself and thereby likely ante-mortem. It does not fit within a framework of complex long-term cyclic actions like at Herxheim. Mutilation and torture are well known to occur as part of massacres, which are clearly defined as being limited in timeInto this tableau of LBK collective lethal violence and mass burial we add a new site: the mass grave of Halberstadt, Germany. It contained the skeletal remains of irregularly deposited and severely traumatised individuals. Careful excavation of the block-lifted feature and subsequent laboratory analyses revealed some similarities to the other LBK mass fatality sites, but, more importantly, also significant and currently unique deviations from them. By applying osteological, palaeopathological, taphonomic, and isotope analyses , this study adds important and previously unavailable information to the discussion of the scope and frequency of Early Neolithic violence and its contextual interpretation.32. In the same area, at least 38 regular LBK inhumation burials have been excavated so far, which relate spatially mainly to the houses and therefore are examples of settlement burials. Overall, these graves represent a carefully deployed LBK burial site including subadults and adults of both sexes33.The mass grave was discovered in 2013 during archaeological excavations prior to housing development in the southern outskirts of Halberstadt, a town located just north of the Harz mountains in the German state of Saxony-Anhalt historic activities. This made it difficult to precisely define the edges of the actual feature Fig. , which l34. The remaining seven individuals all fall between 25 and 40 years of age. This highly peculiar, almost exclusively adult male demography (89%) deviates significantly from that of the nearby settlement burial site33, which shows the standard demography of almost equal numbers of male and female and adult and subadult individuals . The absence of children from the Halberstadt mass grave is especially noteworthy, as these usually are numerous in the regular individual and irregular mass burial sites, as well as in other non-standard deposition types35. For example, both massacre mass graves of Talheim and Kilianstädten have balanced ratios of subadults to adults from which the Halberstadt mass grave significantly differs . The difference to the child-dominated mass grave of Wiederstedt is even more obvious . In contrast, the encountered sex ratio with a pronounced lack of females is reminiscent of the massacre sites of Asparn/Schletz and Kilianstädten24. But overall, the mass grave of Halberstadt clearly represents a population sample different from all other attritional or catastrophic LBK burial assemblages known so far. The clear dominance of younger adult males and the absence of children have to be especially stressed, as these characteristics, in combination, indicate important contextual differences from the other sites of LBK mass burial and/or collective lethal violence.The pit contained nine individuals, seven of them diagnosed as male (78%). The remaining two are a probable male 11%) and a probable female (11%). The youngest male was ca. 16–20 years old, the probable female ca. 21–26 years  on tooth enamel and carbon and nitrogen isotope analyses (δ13C and δ15N) on bone collagen disclosed non-local origins and distinct dietary habits of the individuals interred in the mass grave in comparison to the regular settlement burials at the same site , while the δ15N exhibited values between 8.6 and 10.2‰ (vs. AIR). They form a narrow data cluster of distinctly lower δ13C and higher δ15N values than the settlement burials41 , Germany and focused on osteologically defined individuals until all skeletal remains and other finds had been removed.65. Age estimation was based on a summary assessment of skeletal and dental development, maturation, wear and degeneration of all available skeletal elements. Sex estimation primarily made use of dimorphic pelvic features, followed by cranial features and comparative osteometric assessment of the postcranial skeleton. Body height was calculated using the Pearson formulae also assembled for easy use in the standard manuals65. Pathological and taphonomical alterations of the bones were assessed in reference to the respective standards of the field66. The bone fragments collected prior to the block-lifting process were reattributed to the respective individuals during the osteological analysis as far as possible. Some bones could not be securely reattributed, but these did not raise the number of individuals represented and did not carry significant further information relevant for this study. For statistical testing Fisher’s Exact Test (one-tailed) has been used67.After the successive removal of the skeletal remains from the block-lifted feature, the adhering loose soil was removed from the bones with cold water and soft toothbrushes. After this step, most bone surfaces were still covered by extensive and irregular layers of compact calcareous encrustations. In part, these were carefully removed mechanically during the drying process at room temperature. In cases where the encrustations could not thus be removed without damaging the bone substance, the bones were soaked in 10% acetic acid until most of the adhering concretions were dissolved, then rinsed again repeatedly with water and dried at room temperature. Overall, encrustations were removed as far as possible from the diagnostically relevant bones to enable a full osteological analysis. Morphognostic methods were used in age and sex estimation of the individuals, as summarised in detail in the standard manuals68. Enamel samples were cut from tooth crowns and all surfaces and dentine were removed using diamond-coated dental cutting and drilling equipment. The samples were then powdered in an agate mortar, pre-treated with buffered acetic acid and ashed. Sr was separated under clean-room conditions using Eichrom Sr-Spec resin. Sr concentrations were determined by Quadrupole-Inductively Coupled Plasma-Mass Spectrometry (Q-ICP-MS) and 87Sr/86Sr ratios by High-Resolution Multi Collector-ICP-MS (Neptune) at the Curt-Engelhorn-Centre for Archaeometry in Mannheim, Germany. Raw data were corrected according to the exponential mass fractionation law to 88Sr/86Sr = 8.375209. Blank values were lower than 10 pg Sr during the whole clean lab procedure. The NBS 987 and Eimer & Amend (E & A) standards run along with the human samples yielded 87Sr/86Sr ratios of 0.71024 ± 0.00001, 2σ; n = 5 and 0.70802 ± 0.00001, 2σ; n = 4, respectively.Strontium isotope analysis followed previously established protocols69 with omission of the ultrafiltration step. Compact bone samples were cut, the surfaces removed, and demineralised in 10 ml of 0.5 N HCl at initially 4 °C and later at room temperature for 14 days, rinsed to neutrality and reacted with 10 ml of 0.1 M NaOH for 24 h at 4 °C, rinsed again to neutrality and gelatinised in 4 ml of acidified H2O (pH 2–3) for 48 h at 75 °C. Insoluble particles were separated using EZEE filter separators, and the collagen frozen and lyophilised. C and N contents and the stable isotopic compositions were determined in triplicates using a Thermo Flash 2000 Organic Elemental Analyzer coupled to a Thermo Finnigan Mat 253 mass spectrometer at the Department of Applied and Analytical Palaeontology, Institute of Geosciences at the University Mainz. The raw data were calibrated against the international Standards USGS 40 and USGS 41. Interspersed samples of IAEA CH6 gave a mean δ13C value of −10.35 ± 0.01‰ and IAEA N2 gave a mean δ15N value of 20.61 ± 0.06‰.Sample preparation for carbon and nitrogen isotope analysis followed previously published protocolsσ date ranges are used.Two independent and securely attributed bone samples were collected from inds 2, 3 and 7 each during excavation and were submitted for AMS radiocarbon dating at the Klaus-Tschira-Laboratory for Radiometric Dating Methods, Mannheim, Germany. Results were calibrated and further analysed using the online version of OxCal v4.3/IntCal13  are housed at the State Office for Heritage Management and Archaeology of Saxony-Anhalt in Halle , Germany under catalogue numbers 6500:100202:1-154.Supplementary Information


Species loss from upper trophic levels can result in some major changes in community structure and ecosystem functions. Here, we experimentally excluded macroconsumers  in a Brazilian karst tropical stream during the dry season to investigate if their loss affected the accrual of calcium, dry mass (DM) and ash-free dry mass (AFDM) of sediment, benthic invertebrates, and chlorophyll-a. We found that the exclusion of macroconsumers decreased accrual of calcium. The absence of fish and shrimp may have promoted increased grazing by mayflies and snails in the electrified treatment as expressed by the depressed calcium accrual and shift in periphyton community composition. However, the exclusion of macroconsumers had no effect on DM and AFDM, chlorophyll-a, or total abundance of invertebrates. Our findings shed new light on the impact of macroconsumer loss and consequences for calcium accrual in karstic streams. Human activities can disproportionally affect macroconsumers in the upper trophic levels of food webs . Loss of macroconsumers can result in rapid changes in community structure and ecosystem function –3. In stIn karstic streams, benthic algae and insect larvae such as Hydropsychidae and Chironomidae are known to affect the deposition of calcium carbonate and the formation of travertine or tufa sedimentary structures –19. HoweAncistrus formoso and Trichomycterus dali) [Streams in karstic watersheds of the Neotropics are particularly threatened by human activities  where thus dali) .These streams have complex hydrological connectivity with groundwater inflows due to the surrounding limestone-dominated watershed. Consequentially, the streams have high levels dissolved calcium and bicarbonate . These iThe purpose of this study was to evaluate whether the presence of macroconsumers  affect the accumulation of calcium and organic sediments, as well as benthic invertebrate abundance, and biofilm production in the benthos of a representative karstic stream in the Bodoquena Plateau. We hypothesized that experimental exclusion of fish and shrimp would decrease bioturbation of sediments and increase accumulation of calcium-rich sediments. Additionally, we expected that the absence of predation would increase the abundance of grazing aquatic insects and gastropods. This increased grazing would decrease biofilm biomass . Our hypotheses were based on related research that concluded the absence of macroconsumers in stream ecosystems can increase the accumulation of inorganic sediment and organic material ,15. BiofPermits of animal handling for this study were approved by the Ministério do Meio Ambiente (MMA), Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio), Sistema de Autorização e Informação em Biodiversidade (SISBIO) under protocol number (7028–1) and authentication code (91743927). Permission to work at the National Park  was granted by the ICMBio  that approved the protocols for this project. This study did not involve endangered or protected species.The study was conducted in a headwaters stream, Córrego Taquaral, located in the Bodoquena Plateau, Mato Grosso do Sul, Brazil  during 12–26 June 2016. The Bodoquena Plateau is characterized by karstic watersheds and is one of the most extensive continuous karst aquatic systems in Brazil . The CórThe Córrego Taquaral flows from an elevation of 400 to 358 m above sea level, ranges from 8 to 30 m wide, and was 50–70 cm deep during the study. Flow is groundwater fed and relatively uniform during the dry season when this experiment was conducted. Substrate consists primarily of sand as well as large boulders and cobble-sized inorganic sediments with travertine deposition. High calcium carbonate concentrations and elevated pH during photosynthesis result in the formation of numerous calcareous tufa formations, travertine waterfalls, and calcium carbonate deposits along pool and riffles downstream. The steep waterfalls are natural barriers to movements of some species of predatory fishes .Stigeoclonium sp.), diatoms, and cyanobacteria [Xenurobrycon macropus), Crenuchidae (Characidium zebra), and Heptapteridae (Rhamdia quelen). In addition, there are algivous fish: Loricariidae (Ancistrus sp.) and (Hypostomus sp.) that feed on periphyton [Macrobrachium brasiliense) and a crab  that consume algae and insects [Due to the broad channel width (>20 m), the Córrego Taquaral possesses an open canopy and benthic algae (periphyton) provide the primary resource base for the food web. The periphyton assemblage is dominated by Chlorophyceae (bacteria . The benbacteria . Macrocobacteria –47: Char insects .-1); salinity (0.2 ± 0.005 ppt); pH (7.7± 0.27); and dissolved oxygen (4.0± 0.96 mg l-1) were measured once daily with a multi-parameter meter . Total nitrogen  and total phosphorus  were also recorded during the experiment. TN was measured using persulfate methods and cadmium reduction, while TP was measured using persulfate and with stannous chloride. Mean values were determined using spectrophotometry [Multiple physical and chemical parameters were recorded during the experiment to characterize the study site. Water quality parameters, including temperature 20.4°C ; conductivity (407 ± 6.90 μ.Sotometry .2), round limestone rocks to promote colonization of invertebrates  were deployed for each section of the pool . Macroconsumer exclosures consisted of 20 × 20 × 10 cm plastic baskets with 50 mm mesh opening to maintain some limited flow through the rock substrates and with a 50 mm mesh in the bottom of the baskets  . The mestebrates . These rWe standardized measurements of periphyton colonization and inorganic matter accumulation inside each basket by installing a 23 × 6 cm thick acrylic panel with four circular holes of 4.2 mm diameter . Each hoThe experiment was conducted during 15 days. The duration of the experiment was based on our review of the literature and on our previous studies as well as being constrained by the variable duration of the dry season in this region. We expected that: (i) colonization time of benthic macroinvertebrates would be rapid in tropical streams ,5,55,  then ashed for 2 h at 550°C and reweighed to estimate the ash-free dry mass . Total sediments deposited on filters were then calculated as DM and AFDM. We sampled the baskets for benthic invertebrates by disrupting the sediments by hand in front of a 0.5 mm mesh kick net positioned immediately downstream to catch any organisms that had colonized the stones. Benthic invertebrates were also sorted from the substrate in each basket and were preserved in 80% ethanol for subsequent identification in the laboratory. All insects were identified, usually to family, using taxonomic keys and original descriptions ,57.All stones in each basket were scraped and washed into a bucket to create a 200 ml slurry for analysis of primary producer mass in the form of chlorophyll-a. We used a syringe to extract a 50 ml subsample from the slurry for periphyton analysis. Each periphyton sample was obtained using pre-ashed glass fiber filters . Chlorophyll-a was extracted from each filter in 10 ml 80% ethanol for 24 h in a freezer in the absence of light  and concWe selected 18 replicates, nine from each treatment, with four filters each to analyze calcium from the concentration of dry mass. Filters were macerated using acid digestion and peroxide of hydrogen (method 3050B)  and lateWe applied repeated measures linear mixed or generalized linear mixed models to test for the effects of the exclosure treatment on all dependent variables of interest. For each statistical model, the stream section  was treated as a random block factor. Continuous dependent variables, including DM, AFDM, calcium, and chlorophyll-a were modeled using linear mixed models. Data were log-transformed  to fit the assumption of normality when appropriate. For discrete dependent variables, such as the abundance and richness of benthic invertebrates, generalized linear models assuming Poisson-distributed data were derived also using the stream section as a random block factor. All models included day of trial and experimental status as independent variables. Models for dependent variables representing accrual on tiles included the abundance of scrapers and collector-gatherers as independent variables as well. Degrees of freedom for mixed models were estimated using the Satterthwaite approximation .We also tested for differences in benthic invertebrate communities among treatments using permutational multivariate analysis of variance (PERMANOVA) on Bray-Curtis dissimilarities derived from log-transformed abundance data. To determine if these parameters changed over the course of the study, we assessed linear regression models of readings with time as an independent variable.All analyses were performed in open-source software R . Mixed mMost physical and chemical measures, including pH and dissolved oxygen remained uniform throughout the study period (p>0.05), while temperature, conductivity, and salinity did increase linearly (p<0.05). These increases were likely due to the advancement of the dry season during the study period. The total phosphorus was within the range designated for unpolluted streams in the region .2) and electrified (0.00024–0.00006 mg/m2). However, the exclusion of macroconsumers had no effect on DM (sediment) or AFDM  (2) and electrified treatments (0.104 mg/cm2).We found a significant difference in patterns of calcium accrual between treatments, with an increase in the controls compared with the electrified treatments . The calaterial) . The meaThere were no differences in chlorophyll-a concentration between treatments , suggestWe observed no significant differences between treatments in the total richness and abundance of invertebrates colonizing the stony substrata . We collCommunity composition, as assessed by a PERMANOVA model, was unrelated to treatment  or an interaction term between treatment and time  .Our findings suggest that excluding macroconsumers did not directly affect the abundance of benthic invertebrates, chlorophyll-a concentration or dry mass, and AFDM. Previous studies in the Bodoquena Plateau during the dry season and in tropical streams of Hong Kong have also reported that the absence of macroconsumers did not increase accumulation of sediments, periphyton accrual, or benthic invertebrate abundance ,34,66.The most interesting finding of our study was the increased accumulation of calcium in the presence of macroconsumers. The changes in calcium accumulation could be related to both direct and indirect effects of macroconsumers on trophic interactions. The indirect trophic interactions were likely associated with changing the behavior of some benthic invertebrates, particularly the insect larvae associated with travertine formation –19. The Simothraulopsis [Benthic stream invertebrates have behavioral and morphological adaptations to avoid predators , especiaaulopsis  occurredaulopsis ,73 can aKarstic streams are rich in dissolved calcium carbonate, carbon dioxide, and bicarbonate. When the dissolved carbon dioxide is used in photosynthesis, pH increases and deposition of calcium carbonate increases among algae and macrophytes in these streams ,17. This2 and increase pH that further enhances the accrual of calcium carbonate [Aquatic insect larvae such as the Hydropsychidae and Chironomidae build retreats (cases) using sediment particles or construct silken nets for filtering suspended organic particles ,74. Duriarbonate . Althougarbonate . These wIn our study, we found a low abundance of benthic invertebrates in both treatment and control baskets when compared to other experimental studies in tropical streams . Calcium, calcium carbonate-depositing portions of some karst streams are known to provide microhabitats and high biodiversity for algae and insect larvae [Calcium carbonate deposition is potentially related to many ecological attributes in karst stream ecosystems. For instancet larvae , and otht larvae . This stS1 Table(PDF)Click here for additional data file.


In this study, we evaluated the association between thyroid echogenicity on ultrasonography (US) and thyroid function in pediatric and adolescent Hashimoto's thyroiditis (HT) patients.P < .001). In contrast, free thyroxine levels were significantly decreased with increasing US grade (P < .001).In 86 pediatric and adolescent HT patients, the association between echogenicity and thyroid function and microsomal autoantibody status was evaluated. Among patients with overt hypothyroidism, 89.2% (33/37) showed a US grade of 3 or 4. All of the patients at grade 4 presented with overt hypothyroidism. In contrast, 97.8% (44/49) of the patients with subclinical hypothyroidism or euthyroidism showed grades 1 or 2. Patients with increased thyroid-stimulating hormone titer also tended to have increased US grades (P < .001).In conclusion, patients with higher US grades had decreased thyroid function ( It is a chronic disorder with a prevalence of 1.3% to 9.6% in children and adolescents, and is characterized by the loss of tolerance to self-thyroid antigens. Changes in echogenicity have been demonstrated in subclinical hypothyroidism.–10 Decreased echogenicity or irregularity in the echo pattern during a thyroid US in patients with elevated thyroid-stimulating hormone (TSH) could possibly be taken as early signs of thyroid failure. Thyroid US in subclinical hypothyroidism has a significant predictive value for treatment outcome that is comparable to other well-known prognostic parameters, such as antithyroid peroxidase antibody (TPOAb) or high TSH levels; this is because subclinical thyroid dysfunction tends to develop into overt thyroid dysfunction. Progression to overt hypothyroidism, with the associated adverse effects on lipid metabolism and the cardiovascular system, are major concerns for patients diagnosed with subclinical hypothyroidism. The risk of progression to overt hypothyroidism in subclinical hypothyroid patients is known to be higher in those with underlying thyroid disease. However, it is difficult to predict the risk of natural disease progression to a more severe form of thyroid dysfunction. There is no consensus on the clinical parameters associated with prognosis for this mild form of thyroid dysfunction, although elevation of serum TPOAb and decreased or heterogeneous echogenicity on US are commonly observed. Thyroid US in conjunction with TPOAb assay for the initial assessment of a patient with subclinical hypothyroidism appear to be more helpful than TPOAb alone for predicting the clinical course and establishing a treatment plan, especially in cases with mild elevation of serum TSH.Previous studies have reported ultrasonography (US) findings of thyroiditis and the association between echogenicity of the thyroid gland and thyroid function. Decreased echogenicity of the thyroid gland is associated with overt hypothyroidism.To the best of our knowledge, no studies have evaluated the relationship between thyroid US echogenicity and thyroid function in pediatric and adolescent patients. Therefore, this study retrospectively evaluated echogenicity in US scans and thyroid function in pediatric and adolescent patients with Hashimoto's thyroiditis (HT).22.1This retrospective study was approved by our Institutional Review Board and the requirement for informed patient consent was waived. The study was performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments.At our institution, 117 patients were diagnosed with pediatric or adolescent HT between February 2006 and July 2016. Of these, 88 had undergone thyroid US. Two patients were excluded because the US scans showed diffusely scattered microcalcifications, making a true evaluation of thyroid echogenicity difficult. Ultimately, 86 patients were enrolled in this study. Laboratory data obtained from the medical records were evaluated retrospectively, including the serum levels of free triiodothyronine (T3), free thyroxine (FT4), TSH, antimicrosomal antibody, and antithyroglobulin autoantibodies (TGAbs). Radioimmunoassays were used for the laboratory examinations  and were performed on the day closest to the US examination date. Diagnosis of HT was based on the presence of autoantibodies (TPOAbs and TGAbs). Some patients also underwent fine needle aspiration biopsy (n = 1) or core biopsy (n = 2) for lymphocytic thyroiditis, or surgery due to associated nodules .Thyroid function was subdivided into overt hypothyroidism (Ia), subclinical hypothyroidism (Ib), euthyroid (II), and hyperthyroidism (III). Overt hypothyroidism was defined by decreased concentrations of T4 and elevated serum concentrations of TSH (>20 mU/L). Ib was defined as an increased serum TSH level and normal free thyroid hormone levels. Patients with normal levels of TSH and free thyroid hormones were deemed as II. The normal ranges of TSH, FT4, and FT3 were 0.27 to 4.2, 0.93 to 1.71, and 0.85 to 2.02 mU/L, respectively. Antimicrosomal antibody titers were analyzed in 84 patients.2.2US and color Doppler examinations were performed using a 5 to 12 MHz linear array transducer . One of 3 radiologists with 5, 8, and 25 years of experience in thyroid imaging performed the US examination. The US findings were retrospectively reviewed by radiologists with 5 and 25 years of experience in thyroid imaging and consensus was achieved; the reviewers were blinded to the hormonal status. The US patterns were classified into 4 grades to compare the thyroid echogenicity and anterior strap muscle echogenicity, which was used as a reference: Grade 1 (G1): diffusely enlarged gland with a normoechoic US pattern   or Fisher exact test were used for the categorical variables. For the continuous variables, Student t test was used to compare 2 groups and the Kruskal–Wallis test was used to compare the 4 levels of US grade after testing for normality and equivalent variance. To determine interobserver reproducibility, both observers performed their evaluations of the ultrasonographic findings independently. Interobserver and intraobserver agreement were calculated using the Cohen kappa test. A 2-tailed P-value of <.05 was considered statistically significant. All statistical analyses were performed using R software .Data are reported as means ± standard deviation for continuous variables and frequency (percentage) for categorical variables. To compare clinical characteristics between groups, the chi-square (33.1P = .887) and sex (P = 1).The mean age of the patients was 11.1 ± 3.7 years (range 4–18 years). There were 12 (14.0%) males and 74 (86.0%) females. Levels of microsomal antibodies were evaluated in 84 patients. Comparisons of the total patients and patients with microsomal Ab were tabulated, and no significant difference was found for age (3.2P < .001). Thyroid function differed significantly among the 4 US groups (P < .001) and patients with higher grades had reduced thyroid function. The US grade increased with the TSH titer (P < .001), while the FT4 level significantly decreased with increasing US grade (P < .001). The microsomal antibody titer was not significantly related to US echogenicity. Interobserver agreement was 0.74 and intraobserver agreement was 0.85.Table 4–3 HT is the most common cause of hypothyroidism in iodine-sufficient countries, although the incidence varies with geographic location. Overt hypothyroidism is a common but potentially fatal medical condition evident in approximately 10% of HT patients; onset is insidious and the condition may not become clinically apparent until the full range of symptoms develop. While subclinical and overt hypothyroidism share similar etiologies, the symptoms of the former are nonspecific and signs of hypothyroidism are typically absent. Consequently, a diagnosis of subclinical hypothyroidism depends solely on thyroid function tests. Although subclinical hypothyroidism is a somewhat benign abnormality compared to other thyroid diseases, its absence of specific, related symptoms, and the difficulty in determining whether treatment is required, remain as challenges to both physicians and patients. Problems of diagnosing and treating subclinical hypothyroidism are becoming progressively more important as human life expectancy increases, because thyroid dysfunction, including subclinical hypothyroidism, is found more often in the elderly.,13–15 Subclinical hypothyroid patients with underlying thyroid disease have a greater risk of progression to overt hypothyroidism, which is associated with adverse effects on lipid metabolism and cardiovascular function. However, predicting disease progression and assessing the risk of progression to a more severe state of thyroid dysfunction are challenging. There is no consensus regarding the clinical parameters associated with the prognosis for this mild thyroid dysfunction, although elevated serum TPOAb and decreased or heterogeneous echogenicity (diffuse thyroid disease) on US are often observed.HT, also known as lymphocytic thyroiditis, is a goitrous form of AIT, and the most common thyroid disorder in children and adolescents. HT is characterized by diffuse lymphocyte and plasma cell infiltration, fibrous replacement, and eventual atrophy of the parenchyma.,10 A change in echogenicity is also seen in subclinical hypothyroidism.,5 However, previous studies included only adults or adults and adolescents; the present study evaluated this association in a pediatric population.Many studies have examined US in thyroiditis and its association with echogenicity, mainly hypoechogenicity, of the thyroid gland and thyroid function. The association between decreased echogenicity of the thyroid gland when examined with US and overt hypothyroidism is well known in HT. younger patients show a stronger association between decreased echogenicity and a higher TSH, and the relationship is stronger still when the changes are recent. Decreased echogenicity in thyroid US scans in patients with elevated TSH might be an early sign of thyroid failure.,9 Thyroid US in subclinical hypothyroidism has significant predictive value in terms of treatment outcome, comparable to other well-known prognostic parameters such as microsomal antibodies and high TSH levels. In our study, there was no significant association between echogenicity and microsomal antibody level. Of the few studies that investigated the relationship between thyroglobulin antibody and hypoechogenicity, none have found any such association.,17 Thyroid US in conjunction with a microsomal antibody assay during initial assessment of a patient with subclinical hypothyroidism appear to be more helpful than microsomal antibody alone in predicting the clinical course and establishing a treatment plan, particularly in cases with mildly elevated TSH levels. Thyroid echogenicity can be used to predict thyroid failure.We found that pediatric and adolescent HT patients also show a significant correlation between thyroid US echogenicity and thyroid function, not only in overt hypothyroidism but also in subclinical and euthyroidism. There was also a strong association between decreased echogenicity and TSH level in pediatric and adolescent patients. According to Vejbjerg et al, Another study demonstrated that all patients who developed hypothyroidism during follow-up had decreased echogenicity at baseline. This suggests that hypoechogenicity is an early marker of hypothyroidism. Further studies that follow up on changes in thyroid echogenicity and function are needed. Another limitation was that US is a subjective measure based on the experience and skills of the sonographer. Although we achieved consensus among 3 experienced radiologists, the reliability of the results is open to dispute. There has been some effort to quantify sonographic echogenicity,,20 but it is not used widely. Therefore, subjective sonographic findings are still important.There were some limitations to this study; it used a retrospective design and included a relatively small number of participants who underwent US examinations at a single institution. Therefore, a larger series should be conducted to validate our results. Furthermore, we did not conduct a follow-up US of consecutive patients. A previous study examined apparently healthy subjects with a 3-year follow-up and found that none of those who developed thyroid dysfunction had normal echogenicity in thyroid US scans at the time of inclusion.5In our pediatric and adolescent HT patients, there was an association between decreased echogenicity and thyroid dysfunction and high TSH levels, even in subjects with normal thyroid function, or subclinical or overt hypothyroidism. This suggests that US can be useful for supplementing biochemistry assays in the evaluation of thyroid status. Further investigations with follow-up studies are needed to evaluate the usefulness of US to predict the progression of abnormal thyroid function.We are grateful to Bora Lee  for the statistical analysis.Conceptualization: Hyun Sook Hong.Data curation: Hyun Sook Hong, Ji Ye Lee.Formal analysis: Hyun Sook Hong, Sun Hye Jeong, Ji Ye Lee.Investigation: Hyun Sook Hong, Sun Hye Jeong, Ji Ye Lee.Methodology: Hyun Sook Hong, Sun Hye Jeong, Ji Ye Lee.Project administration: Hyun Sook Hong.Resources: Hyun Sook Hong, Sun Hye Jeong, Ji Ye Lee.Validation: Hyun Sook Hong, Ji Ye Lee.Visualization: Hyun Sook Hong.Writing - Original Draft: Hyun Sook Hong, Sun Hye Jeong.Writing - Review and Editing: Hyun Sook Hong, Ji Ye Lee.Hyun Sook Hong orcid: 0000-0003-3210-9740.


Background: Since most patients with relapsing-remitting multiple sclerosis (RRMS) are women, the present study aimed to determine whether treatment of patients with MS by cytotoxic agents is associated with an increased risk of cervical dysplasia. Cancer screening is often neglected in the chronic diseases such as MS, so more attention in this field was needed. Decreasing morbidity and mortality due to cervical cancer is the most important goal of screening in female MS patients especially in child bearing age. Thus, it can be said that this is the first study which investigated this important issue.Methods: A total of 129 individuals participated in this cohort study. They were assigned into 3 groups including 43 patients with MS who were treated with cytotoxic drugs, 43 patients with MS on immunomodulators, and 43 normal healthy controls. Pap smears were performed following standard methods and the results obtained from the three groups were compared by statistical analysis. Demographic data, Expanded Disability Status Scale (EDSS), and Pap smear changes were analyzed by SPSS software.Results: The most commonly detected abnormality in all examined patients and healthy controls was inflammation. Five patients with MS who were treated with cytotoxic agents revealed benign cellular changes (BCC) in their Pap smear that were statistically significant in comparison with other groups (P = 0.03). Patients who took Mitoxantrone presented BCC more than other groups . There was no significant difference between mean duration of MS diagnosis (P = 0.12), mean duration of previous MS treatments (P = 0.25), and mean duration of current MS treatments (P = 0.21) in patients with BCC compared to normal healthy controls or inflammatory change.Conclusion: According to the results of present study, BCC is more frequently observed in patients with MS who were treated with cytotoxic agents with immunosuppressive effect. Since BCC is a ‘premalignant condition’, the authors suggest that mandatory annual Pap smear should be performed for patients with MS who are treated with cytotoxic agents irrespective of their age in order to detect early signs of malignancy. Acute myeloid leukemia,6 bladder cancer,7 and solid tumors8 are life-threatening malignancies which have been reported to be the result of prolonged utilization of these cytotoxic agents. However, as far as the authors of the present study are concerned, the risk of other types of malignancies or pre-cancerous conditions in this group of patients has not been fully assessed and determined.Multiple sclerosis (MS) is a chronic inflammatory demyelinating and neurodegenerative disease of human central nervous system (CNS), which often results in the development of various neurological manifestations. Cytotoxic agents such as Mitoxantrone, Mycophenolate mofetil, Cyclophosphamide, and Azathioprine are prescribed to prevent severe relapses by inducing immunosuppression in certain patients with secondary progressive MS (SPMS) or progressive relapsing MS (PRMS).Most patients with relapsing forms of MS are women of child bearing age, and the incidence of certain premalignant conditions such as cervical dysplasia may be higher in these patients and easily ignored by the clinicians. Cancer screening is often neglected in the chronic diseases such as MS, so more attention in this field was needed. Decreasing morbidity and mortality due to cervical cancer is the most important goal of screening in female MS patients especially in child bearing age. Thus, it can be said that this is the first study which investigated this important issue.In this regard, this study aimed to explain cervical dysplasia or other Pap smear (Pap) abnormalities in female MS patients receiving immunomodulators (Interferon-β 1a and b) or immunosuppressive medications.This cohort study was approved by Ethics Committee of Tehran University of Medical Sciences , and was performed in Sina hospital, Multiple sclerosis Research Center, Neuroscience Institute, Tehran University of Medical Sciences, Tehran, Iran.During three years, since Jan 2011 to Jan 2013, 129 individuals including three groups, 43 patients with relapsing-remitting (RRMS), 43 patients with SPMS, and 43 healthy individuals, were recruited in this study. All patients received their treatments without any intervention during this time.Multiparty women aged between 18 to 70 years old, diagnosed with RRMS or SPMS based on McDonald’s criteria (2010), within expanded disability status scale (EDSS) between 0-5, and having only one sex partner participated in this study.Patients who were identified as current smokers, had history of using oral contraceptive pills (OCPs) or intrauterine device, diagnosed with active infection of sexually transmitted diseases (STDs) including human papillomavirus (HPV), herpes simplex virus (HSV), chlamydia trachomatis, trichomonas vaginalis, etc., were pregnant, and had history of exposure to diethylstilbestrol were excluded from the study.All eligible patients had to sign informed consent for enrollment. In this study, a neurologist performed neurologic exams including EDSS evaluation. Three general practitioners (GPs) evaluated patients’ baseline characteristic information  and performed Pap smear on patients. Simple Pap smear examinations were performed under standard conditions.Three groups were enrolled in this study: the first group consisted of 43 patients with RRMS who were under treatment by immunomodulators (Interferon-β 1a and b) within 3 years before conducting this study. The second group consisted of 43 patients with SPMS who were under treatment by immunosuppressive medications  within 3 years before conducting this study. The first and the second groups were examined by three GPs for performing Pap smear. The third group consisted of 43 individuals  whose Pap smears results were sent to the same laboratory during these three years. The results obtained from this group were evaluated for determining Pap smears differences between normal population and the two MS patients group.9 Statistical analysis was performed using SPSS software . Demographic data, EDSS, and Pap smear changes were analyzed. The study reported mean values of baseline characteristic information to describe numeric variables.To avoid any bias, the researchers labeled all samples with participants' number, initials, and age. All samples were reviewed by a professional pathologist in Danesh laboratory who gave her opinion about the grade of Pap smears according to the revised Bethesda system.A total of 129 individuals participated in this study. The baseline characteristic information of participants is demonstrated in The mean ± standard deviation (SD) age of participants in this study in the immunomodulator group, the immunosuppressive group, and the control group were 38.84 ± 8.29, 39.21 ± 8.55, and 40.23 ± 9.03 years, respectively. There was no statistically significant difference between the mean ± SD age of participants in these three groups according to one-way ANOVA (P = 0.74), as shown in The mean ± SD age of all participants was 39.43 ± 8.58 and the minimum and the maximum ages were 24 and 66 years, respectively.The main current MS treatments received by the patients before enrollment in the study included Avonex  in the first group, and Mitoxantrone  in the second group. Moreover, the main previous MS treatments received by the patients before enrollment included nothing  in the first group and Betaferon  in the second group. The detailed information of each group is presented in The mean ± SD time of MS diagnosis were 35.28 ± 22.73 and 48.81 ± 22.17 months in the first and the second groups, respectively. The mean ± SD EDSS at time of performing samples were 1.13 ± 0.90 and 3.89 ± 0.44 in the first and the second groups, respectively . The maxMean ± SD duration of current MS treatment in the immunomodulator group was 23.26 ± 19.72 months for Avonex, 33.50 ± 16.72 months for Betaferon, and 27.50 ± 10.99 months for Rebif. Mean ± SD duration of current MS treatment in the immunosuppressive group was 24.67 ± 11.01 months for Azathioprine, 36.00 ± 0 months for Methotrexate, 22.89 ± 20.18 months for Cellcept, and 13.40 ± 7.59 months for Mitoxantrone.As showed in After categorizing the Pap smear results into two groups , in binary logistic regression, the relationship between using immunosuppressive drugs (immunomodulatory and immunosuppressive) and Pap smear results  tended to be significant. Moreover, there was a significant relationship between immunosuppressive drugs and Pap smear results (P = 0.01) after adjusting for potential confounders including age and EDSS. Patients who took Mitoxantrone presented BCC more than other groups  .The mean ± SD of EDSS in patients with and without BCC were 4.50 ± 0.00 and 2.38 ± 1.52, respectively; the results of t-test showed that the difference was statistically significant (P < 0.01). In addition, the mean ± SD of age in patients with and without BCC were 36.20 ± 6.05 and 39.55 ± 8.66 years, respectively; the difference was not statistically significant (P = 0.39). Furthermore, there was no significant difference between mean of duration of MS diagnosis (P = 0.12), duration of previous MS treatments (P = 0.26) and duration of current MS treatments (P = 0.21) in patients with BCC compared to normal healthy controls or inflammatory change .In this study, it was shown that patients with MS who took immunosuppressive drugs as current treatment had more chance to present BCC in their Pap smear results. Mitoxantrone had more relation on presenting BCC among immunosuppressive drugs. Furthermore, previous treatments in these patients were immunosuppressive drugs as well. MS is a relatively common disease in young women, and the mean age and duration of MS diagnosis in this study revealed importance of Pap examinations in them. It should be noted that in the chronic diseases such as MS, other aspects of the patient’s life is under the influence of the main disease and cancer screening is neglected usually in them. 9 It has been shown that some patients, for whom BCC was reported in Pap examinations, had cervical intraepithelial neoplasia (CIN) in their biopsies.10,11 As BCC was detected only in patients who were immunocompromised due to cytotoxic medications, and not in the other two groups, this should be considered as a complication of cytotoxic agents. Higher rates of cervical dysplasia in patients with other diseases requiring immunosuppressive medications have been reported previously.12-14 Bernatsky, et al., for instance, studied 1015 patients with lupus and evaluated the incidence of abnormal Pap smears and some other risk factors such as smoking status, history of STDs, and usage of OCP.13 They found that 13.3% of their patients had abnormal Pap smears. Although several different factors could increase the risk of cervical dysplasia in these patients, the authors concluded that immunosuppressive agents might be a trigger for facilitating HPV to induce abnormal cell differentiation and predispose those patients to premalignant conditions.13The rate of cervical dysplasia or other Pap abnormalities has not been studied in patients with MS previously, as far as the authors of the present study are concerned. Thus, it can be said that this is the first study which investigated this important issue. The findings of the present study revealed an increase in BCC rate in patients with MS who were treated with immunosuppressive drugs. Although BCC was previously mentioned as a benign process caused by infection, inflammation, or reactive cellular changes; nowadays, there is an intention to separate BCC from those Pap changes recognized as “Within Normal Limits” (WNL).15Although World Health Organization (WHO) suggested guideline for cervical cancer screening , American Society for Colposcopy and Cervical Pathology (ASCCP) recommends Pap test every 3 years for initializing, and co-testing (including HPV testing and Pap test) every 5 years for women with negative results of both initial tests.12In another study, Bernatsky, et al., reviewing the evidence obtained from systemic lupus erythematous (SLE) patients’ susceptibility to different malignancies, concluded that the impairment of HPV clearance and the effect of immunosuppressive medications may be two of the most crucial causes of cervical dysplasia.14 Furthermore, HPV may be a predisposing factor for non-melanoma skin cancer,10 the incidence of which was reported in previous studies to be higher among immunocompromised patients.14Dreyer, et al. revealed that HPV-dependent malignancies including cervical dysplasia, and anogenital and oropharyngeal cancers have higher rates among SLE patients.16-20 Although recent study revealed special guideline for screening and follow-up of cervical cancer in immunosuppressant women such as women with SLE, irritable bowel syndrome (IBS) and other immunocompromised women, perhaps MS may be different from other diseases.21Recent studies in immunocompromised women suffering from SLE revealed that these patients have increased chance of presenting more premalignant lesions (with or without HPV infection) than the general population.Despite the lack of co-testing for evaluation of our patients, our study provided special data on MS immunocompromised women which may have different condition for presenting abnormal cervical cytology. In the present study, patients with MS with different levels of immunosuppression revealed precancerous cervical lesions which may be different from findings of patients with SLE in other studies, so it would be of interest for a future study to include different type of diseases treated with immunosuppressive drugs for evaluating role of disease on presenting abnormal cervical cytology.According to the mentioned evidences, HPV may play an important role in developing BCC and other probable cervical dysplasia in patients with MS who receive immunosuppressive medications; it should be noted that clearance defect of HPV is the most significant cause of cervical dysplasia and other Pap smear abnormalities in these patients. Cytotoxic agents change the nature of immunity and immunological defense against different infections; thus, HPV can induce abnormal cell differentiation and lead to premalignant and malignant conditions.22 Moreover, similar to normal individuals, patients with MS can be affected by these predisposing factors. In addition, according to the results obtained from the present study, immunosuppressive agents should be considered as additional risk factors for cervical dysplasia in patients with MS. Thereafter, the authors highly recommend that annual Pap smear should be done for patients with MS receiving cytotoxic medications. This strategy may protect them against cervical malignancy through early diagnosis.CIN is a significant predisposing factor for cervical cancer. There have been several factors which make women more likely to suffer from these threatening conditions including infections, smoking, status, and history of infertility.Moreover, duration of MS diagnosis and duration of (previous or current) treatments may have a role in BCC change in Pap examination, which necessitates having more narrow observation for performing annual Pap smears.One of the limitations of this study is low use of Novantrone in the presence of other drugs which have a high potential and are increasingly used for the treatment of MS. Among these drugs are Fingolimod, Tysabri, Rituximab, Alemtuzumab, and Eculizumab. These drugs question the use of drugs that have various side effects such as Novantrone. Although this limitation is one of the drawbacks of this study, it should be noted that the potential side effects of the drugs such as Rituximab, alemtuzumab, or Eculizumab are still unknown. These drugs have significant effects on the patient's immune system which may cause side effects such as those mentioned in the present study. Moreover, patients with MS are mostly young individuals among them women have considerably the most ratio. This increases the risk of getting such diseases in young women during treatment that regarding their low age is long. Thereafter, in addition to emphasizing on paying attention to women’s disorders, the present study proposed a framework for further studies.23 Ronco, et al. suggested that HPV screening leads to over-diagnosis of regressive low grade lesions in Pap tests of young women.24Moreover, Szarewski and Sasieni estimated that 92.5% of low-grade abnormalities regressed within 24 months and 6.5% progressed in young women.Another aspect that must be taken into account is that the abnormal lesions detected in the most patients may regress in follow-up observations; so, HPV testing in combination with Pap test (co-testing) and having numerous follow-up samples of our patients is another limitation of our study.The present study had some other limitations including small number of participants. Besides, the study could not detect which immunosuppressive drug is more associated with cervical dysplasia. Moreover, the study could not judge about the risk of cytotoxic monotherapy versus poly therapy, and find a cut-off cumulative dose of treatment for increased risk of cervical dysplasia. These issues should be addressed in other large multi-center cohort studies in future.According to the results of the present study, BCC is more frequently observed in patients with MS who were treated with cytotoxic agents with immunosuppressive effect. Since BCC is a ‘premalignant condition’, the authors suggest that mandatory annual Pap smear should be performed for patients with MS who are treated with cytotoxic agents irrespective of their age in order to detect early signs of malignancy.