We concluded that the new Prototic Coproparasitological Test for Dogs (PC-Test Dog) allowed a better visualization of the parasitic structures and showed a favorable result for the diagnosis of intestinal parasites in dogs.Because canine intestinal parasites are considered cosmopolitan, they carry significant zoonotic potential to public health. These etiological agents are routinely diagnosed using microscopic examination commonly used because of its low cost, simple execution, and direct evidence. However, there are reports in the literature on the poor performance of this test due to low to moderate sensitivity resulting from frequent errors, procedures and interpretation. Therefore, to improve the diagnostic efficiency of microscopic examination in veterinary medicine, we developed and evaluated a unique new protocol. This system was tested in a study involving four genera of highly prevalent canine intestinal parasites in an endemic region in São Paulo state, Brazil. Fecal samples from 104 animals were collected for this research. The new protocol had a significantly higher ( Toxocara spp., Ancylostoma spp., Trichuris spp., and Giardia spp. [Because canine intestinal parasites are considered cosmopolitan, they present a significant zoonotic threat to public health ,2. In redia spp. ,4.Toxocara canis species is the most prevalent in pet dogs, presenting as the most frequent clinical symptoms diarrhea, flatulence, abdominal distension, dehydration, and developmental delay [Among the three genera of multicellular parasites mentioned above, the al delay ,6. This Toxocara spp. in developing countries, infection with the parasite Ancylostoma spp. can cause serious organic problems in dogs and humans [Ancylostoma spp. Although less prevalent than d humans . In dogsd humans ,8. In tuTrichuris vulpis can cause considerable abdominal pain, protein loss, inflammation, intestinal bleeding, and diarrhea in infected dogs. Depending on the intensity of infection and animal immunological capacity, this parasite can cause severe anemia resulting from iron deficiency [The multicellular species ficiency ,9,10.Giardia spp., the eukaryote species Giardia duodenalis is considered of great anthropozoonotic importance by the World Health Organization (WHO) and is effectively subdivided into different genotypic groups. Infection with this enteroparasite has diarrhea as a common symptom in humans and in more than twenty breeds of vertebrate animals, including dogs [Regarding the genus of protozoan ing dogs ,12. The ing dogs ,14,15.Parasites of the intestinal tract of dogs are routinely diagnosed by microscopic examination of the fecal samples, mainly because of its the low cost, simple execution, and direct evidence ,18,19,20Despite the benefits previously mentioned for routine laboratory microscopy examination, there are reports in the literature that this test has limited effectiveness, resulting from low to moderate diagnostic sensitivity. According to several authors ,14,21,23For about fifteen years a parasitological technique known as the Three Fecal Test (TF-Test) has been used with significant results in several academic studies in Brazil ,21,23,25Currently, a broad scientific and technological study for automated diagnosis is being developed in the Health Medicine and Computer Science areas at the University of Campinas (UNICAMP) in Brazil, seeking to reduce the two types of errors previously described, and consists of a parasitological protocol, computer and customized equipment containing a high-resolution digital camera, appropriate optical tube, and motorized stage ,27,28,29We aimed in this effective study to develop and evaluate new protocol to detect four genera of highly prevalent canine intestinal parasites in an endemic region of São Paulo state, Brazil, and to compare it with visual diagnosis and automatic recognition after a coproscopy by the flotation method using zinc sulfate using the TF-GII/Dog and Prototic Coproparasitological (PC)-TEST DOG technique.The 104 dogs used in this study were of no defined breed, gender, and age (puppies to adults), and they came from shelters located in Araçatuba, SP, an endemic region for canine parasitic diseases in São Paulo State, Brazil. The study was approved by the Animal Experimentation and Ethics Committee (CEEA) of the School of Dentistry (FO) of UNESP, Araçatuba, SP, protocol 00968-2017.To investigate the parasite infections, fecal samples were collected in two steps, in triplicate, from each animal using appropriate TF-Test tubes, containing 5 mL of 7.5% formalin neutral solution each , which wIn this study we used the technique known as TF-GII/Dog  and the The samples collected were analyzed as follows: Step 1—Laboratory evaluation of the Parasitological Protocol (PC)-TEST DOG for the DAPI system and creation of an image database composed of parasites and fecal impurities; Step 2—The TF-GII/Dog technique  and the http://www.bio-brasil.com/produto/tf-test) and the PC-TEST DOG to determine the positive samples that were used to form the image database.The first sampling step consisted of obtaining fecal samples from each one of the 104 dogs, on alternating days, using three TF-Test collection tubes , with 1.18 g/mL specific density, was added. This tube was further homogenized using a pipette, and it was supplemented with ZnSO4 until a meniscus formed on the top. A microscope slide was placed over the formed meniscus and left to stand for 15 min. After this, the slide was removed with a sudden inversion movement, stained, and taken for reading under conventional light microscopy.To this suspension, 3 mL of zinc sulfate (ZnSO3COOC2H5) pro analysis were added. Afterward, the three tubes were homogenized again in identical conditions to obtain a uniform suspension of fecal particles.The three collection tubes containing fecal samples from each animal were homogenized using a vortex mixer at 3000 rpm (g) for 30 s. To each tube, 25 µL of 10% sodium dodecyl sulfate (SDS) anionic surfactant and 3 mL of ethyl acetate  In the image segmentation step, an image may contain several parasite structures such as eggs and cysts, and many impurities  with size, shape, color, and texture similar to those of the parasites. An enhanced image is computed to increase the contrast and make parasites and some similar impurities brighter and the rest of the image darker. The enhancement explores differences between the color channels since red and green components tend to be higher than blue for the relevant objects. This image is thresholded, and the objects with area outside the range of parasites are eliminated, and the remaining ones are submitted to feature extraction.(b) An object representation is based on color, shape, and texture. In this work, we used the pixel connectivity, object area, perimeter, symmetry, major and minor axes of the ellipse that best fit the object; and for classification they were used the difference between the ellipse and the object, energy, entropy, variance, and homogeneity of the co-occurrence matrix as measures to describe the objects.(c) The object recognition step uses a Support Vector Machines (SVM) classifier trained to separate impurities and each of the 4 species of canine intestinal parasites. Ancylostoma spp.—155 eggs, Trichuris spp.—136 eggs, Toxocara spp.—277 eggs, and Giardia spp.—2564 cysts) and 7567 fecal impurities consisting primarily of cells, organic and plant structures of undigested digestive waste, according to To train the SVM classifier, we used a database consisting of 10,699 components, composed of 3132 parasite images , slight (0–2.0), fair (0.21–0.40), moderate (0.41–0.60), substantial (0.61–0.80), and almost perfect (0.8–1.0) [In order to compare the proportion of positive parasite detection results between the mean obtained by the new PC-TEST DOG protocol and the TF-GII/Dog laboratory protocol, this study used parametric descriptive analyses using the McNemar test and Kappa coefficient of agreement (k), and the results were considered significant with 0.8–1.0) .p < 0.0001) had 80.88% sensibility, with a substantial agreement (Kappa coefficient = 0.7636).The TF-GII/Dog technique showed a high number of impurities , as demoOn the other hand, In p < 0.0156) and an almost perfect Kappa agreement (k = 0.8660).The statistical analysis shows that the PC-TEST DOG readings were 6.73% higher with 52.88% (55/104) average positive compared to 46.15% (48/104) of the DAPI system, indicating a significant difference   for the The samples collected from the 104 dogs were satisfactory for the study using the new PC-TEST DOG protocol, and a parasite infection occurrence of 52.88% was detected for the animals housed in shelters located in Araçatuba, SP. Giardia spp. and Ancylostoma spp. eggs. (p < 0.0001). The findings of our study indicate that this expressive difference results from the fact that flotation principle techniques use high-saturation chemical reagents, such as 1.18 g/mL ZnSO4 for 10 to 15 min in TF-GII/Dog, which is able to decompose morphologically the parasites that have thin external lipoprotein membranes, like the two parasite genera mentioned above [4) was used in this study without changing the egg morphology of the two parasites Trichuris spp. and Toxocara spp. that have thick membranes containing a bimolecular lipid stratum and a single protein  in a study involving four genera of canine parasites. Samples of 104 dogs were collected for this work using a new parasitological protocol, named PC-TEST DOG, which was evaluated and validated against a parasitological technique, TF-GII/Dog. A database composed of 10,699 components was created for researching the DAPI system, which was examined against the PC-TEST DOG results obtained by two parasitology specialists. Although this automated diagnostic system advanced the parasitological diagnosis, our findings indicate the need for further research to create a more robust database with better images showing parasites free of impurities that should be used in the image analysis techniques based on segmentation and pattern classification.


Desalinization was faster than salinization in the transplanted sediments. The salinity of the transplanted sediment increased and decreased in the salinization and desalinization simulation experiments, respectively. The TOC contents of the transplanted sediments were lower than that of their undisturbed counterparts in the salinization experiments, whereas they had a strong negative linear relationship with salinity in the desalinization experiments. Microbial diversity decreased in response to salinization and desalinization, and microbial community dissimilarity significantly (P < 0.01) increased with salinity differences between the transplanted sediments and their undisturbed counterparts. Microbial groups belonging to Gammaproteobacteria and Actinobacteria became abundant in salinization whereas Bacteroidetes and Chloroflexi became dominant in desalinization. Among the predicted microbial functions, hydrogenotrophic methanogenesis, methanogenesis through CO2 reduction with H2, nitrate/nitrogen respiration, and nitrification increased in salinization; in desalinization, enhancement was observed for respiration of sulfur compounds, sulfate respiration, sulfur respiration, thiosulfate respiration, hydrocarbon degradation, chemoheterotrophy, and fermentation, whereas depressing was found for aerobic ammonia oxidation, nitrate/nitrogen respiration, nitrification, nitrite respiration, manganese oxidation, aerobic chemoheterotrophy, and phototrophy. Such microbial variations could be explained by changes of transplantation, salinity, and covarying variables. In summary, salinization and desalinization had profound influence on the geochemistry, microbial community, and function in lakes.Uncovering microbial response to salinization or desalinization is of great importance to understanding of the influence of global climate change on lacustrine microbial ecology. In this study, to simulate salinization and desalinization, sediments from Erhai Lake  and Chaka Lake  on the Qinghai–Tibetan Plateau were transplanted into different lakes with a range of salinity of 0.3–299.3 g/L, followed by  Lakes are distributed globally and respond sensitively to regional and/or global environmental changes . Lake saPrevious investigations about salinity effect on microbial community compositions have largely been limited to estuaries with a continuous salinity gradient  and to sTransplanting experiments provides a valid field experiment strategy for studying microbial response to salinity variation . Transpl2) (The Qinghai–Tibetan Plateau (QTP) hosts thousands of lakes (>1 km2) . These l2) . In addi2) . Hence, 2 and maximum water depth of 2.0 m (2 and maximum water depth of 23.6 m. Gahai Lake2 (GHL2)  has a surface area of 35 km2 and maximum water depth of 15.0 m. Xiaochaidan Lake (XCDL)  covers an area of 71.5 km2 with the maximum water depth of 1.0 m. Chaka Lake (CKL) is a shallow lake , with a surface area of 105 km2 and an average water depth of 20–30 cm  with a full range of salinity  on the northern Qinghai–Tibetan Plateau  were selected in this study . The stuof 2.0 m . Tuosu L20–30 cm . The spaIn September 2013, surface sediment (with a mean depth of 1–5 cm) samples were collected in triplicate (the distance is about 200–300 m among the triplicate sampling sites) from the five investigated lakes by using a grab-bucket collection sampler . The triplicate sediments from each lake were mixed into a unique representative analytical sample . Then the sediments from EHL (representing the freshwater lake end) and CKL  were split into two technical replicates  and were separately collected into dialysis bags (∼20 cm long and 10 cm in diameter)  to establish transplanting cages as described previously . “ExperiAfter 50-day incubation, all the 20 sediment cages were recovered from each lake in November 2013. Upon retrieval, approximately 5 g of sediments in the center of each dialysis bag was aseptically collected into Eppendorf tubes. For comparison, the surface sediment sample of the five incubating lakes were also collected in an identical way in September (beginning of the incubation) and November (end of the incubation). These lakes were suffixed with “Sep” and “Nov,” indicating the sampling time. Note that one transplanted sediment sample replicate (XCDL_E1_1) was lost. A total of 29 samples  were obtained. All the collected samples for geochemical and microbial analyses were stored in dry ice during transportation and in a −80°C freezer in laboratory until further analysis.g for 30 min. The sediment pore water was used for pH determination by a digital soil pH meter . Major cation and anion concentrations  of sediment porewater were measured by using ion chromatography . Salinity was calculated as the sum of the concentrations of the measured ions  in the unit of grams per liter. The total organic carbon (TOC) content in the sediment was measured using a multi N/C 2100S analyzer .In laboratory, sediment porewater was obtained by centrifugation at 7200 × Total DNA was extracted from all the collected sediments by using the FastDNA Spin Kit for Soil  according to the manufacturer’s instructions. The universal V4 hypervariable region of the 16S rRNA genes was PCR amplified with the use of a set of bar-coded primers of 515F (5′-GTGYCAGCMGCCGCGGTAA-3′)/806R (5′-CCGGACTACHVGGGTWTCTAAT-3′). PCR conditions followed a previous study . Briefly1. Briefly, raw sequences were quality trimmed and assigned to samples based on unique sample tags. Sequences containing ambiguous nucleotide bases and mismatches during filtering were discarded. Chimera checking was performed using UCHIME  in the “vegan” package based on Bray–Curtis dissimilarity. One-way and two-way PERMANOVA  were performed, using the “adonis” function in “vegan” package  under the BioProject ID PRJNA248705.The raw reads were deposited into the NCBI Sequence Read Archive database  with the TOC contents of the transplanted sediments in experiment 2 , whereas no significant correlation  was observed between salinity and the TOC contents of the transplanted sediments in experiment 1  (Geochemistry of the caged sediments changed after transplantation . In expeization) .P < 0.001) correlation was observed between salinity and the microbial diversity  with transplantation and salinity , and with salinity, TOC contents and major ions including K+, Na+, Mg2+, Ca2+, Li+, Cl–, SO42–, and NO2– in experiment 2   with the salinity difference between the transplanted sediments and their undisturbed counterparts  . Meanwhiterparts .Proteobacteria, Bacteroidetes, Chloroflexi, Firmicutes, and Actinobacteria in a decreasing order. In the CKL sediments, the dominant phyla were Proteobacteria, Euryarchaeota, Bacteroidetes, Actinobacteria, and Firmicutes in September, and were Proteobacteria, Bacteroidetes, and Actinobacteria in November. In the transplanted sediments, microbial communities were dominated by Proteobacteria, Firmicutes, Bacteroidetes, and Chloroflexi in experiment 1 , and by Proteobacteria, Firmicutes, Bacteroidetes, and Halanaerobiaeota in experiment 2  collected in September and November, the microbial communities were predominately composed of Chloroflexi, Alphaproteobacteria, and Actinobacteria decreased (decreasing variation > 1%) whereas the Firmicutes increased in the transplanted sediments along the increasing salinity of the incubating lakes. The Gammaproteobacteria increased only in the transplanted sediments incubated in the (hyper)saline lakes  whereas it decreased in those incubated in low salinity lakes (EHL and TSL). In contrast, the relative abundance of the Betaproteobacteria exerted an opposite variation along the increasing salinity of the incubating lakes  and their undisturbed counterparts . Specifing lakes . In expe and TSL .The suppressive effect of salinization and desalinization was obvious at the ASVs level. Venn diagrams demonstrated the microbial similarities (shared ASVs) and differences (unique ASVs) between the transplanted sediments (incubated in different lakes) and their undisturbed counterparts. Specifically, in experiment 1 , a total of 812, 905, 813, and 973 ASVs were only identified (disappearing) in the lake sediments EHL_Sep compared with TSL, GHL2, XCDL, and CKL, respectively. In contrast, a total of 453, 284, 571, and 410 ASVs were only identified (newly appearing) in the transplanted sediments incubated in TSL, GHL2, XCDL, and CKL, respectively . LikewisMethanobacteriales were higher in the sediments incubated in high-salinity lakes  than that in EHL    were analyzed in greater detail . In expeization) , in contization) .2 reduction with H2, nitrate/nitrogen respiration, and nitrification showed significant (P < 0.05) positive correlation with the salinity in the transplanted sediments (P < 0.01), negatively correlated with the salinity in the transplanted sediments, whereas the relative abundance of aerobic ammonia oxidation, nitrate/nitrogen respiration, nitrification, nitrite respiration, manganese oxidation, aerobic chemoheterotrophy, and phototrophy displayed a significantly (P < 0.01) positive correlation with the salinity in the transplanted sediments  were predicted from the 16S rRNA gene sequences in all the collected sediments. In the sediments of the incubating lakes, the predicted microbial functions were similar between September and November, and mainly consisted of sulfate respiration, respiration of sulfur compounds, fermentation, and (aerobic) chemoheterotrophy. In contrast, the predicted microbial functions in the transplanted sediments at the incubation end (S_Nov) were distinct from their beginning counterpart (S_Sep) and from that in the sediment of the incubating lakes . Among tediments . Similarediments .The desalinization process in the transplanted sediments was faster than salinization. After 50-day incubation, the salinity of the transplanted sediments in the desalinization simulation experiment was in equilibrium with the incubating lakes; however, such an equilibrium was not observed for the salinity in the salinization simulation experiment. This finding could be ascribed to the following fact: in the salinization simulation experiments, osmotic stress resulted in salt-in and water-out, and thus dry microenvironment was formed in the caged sediments, which did not avail to further exchange water and salt between inside and outside of the cages; whereas an opposite water–salt exchange trend  took place in the desalinization simulation experiments, and water–salt exchange became easier as desalinization proceeded . So it iP < 0.01) in experiment 2  in the salinization simulation experiments, whereas the salinity in the transplanted sediments decreased from 299.3 to 3.2 g/L (minimum) in the desalinization simulation experiments . Such sariment 2 . Such a riment 2 . This coriment 2 . A largeriment 2 . The chariment 2 , which triment 2 . This poIt is notable that microbial diversity in the transplanted sediments was lower than that in the sediments of the incubating lakes in both experiments. Microbial diversity decreased in response to salinization, which was understandable in that microbial diversity decreased with increasing salinity . In contFirmicutes and Gammaproteobacteria increased whereas Betaproteobacteria and Chloroflexi decreased in the transplanted sediments incubated in the lakes with high salinity  existing in the beginning lake sediments evaded detection  but may Halomonos, Pseudoaltermonas, and Vibrio within the Gammaproteobacteria in low-salinity lakes showed stronger carbon utilization ability than their counterparts from high-salinity lakes  microbial communities could change with time, which could be evidenced by the microbial difference between the incubating lake sediments sampled in September and November; and (2) anthropogenic perturbations  would change the redox conditions of microenvironments in the caged sediments, leading to a shift of the microbial community compositions. So it is not surprising to observe that the microbial communities in the transplanted sediments retrieved from both EHL_E1 and CKL_E2 were different from that in their undisturbed counterparts. Given that incubation time may affect the microbial community structure , it is iOn the other hand, salinity accounted for the observed difference among the samples, and the greater the salinity difference, the greater the community difference. Specifically, in experiment 1 , the salinity in the caged sediments were not fully equilibrated with  their new incubating lakes after 50-day incubation . The salIn conclusion, salinization and desalinization had strong effects on sedimentary geochemistry, microbial community compositions, and functions in the transplanted sediments, and the desalinization process was faster than salinization. In response to salinization and desalinization, microbial diversity decreased and microbial taxonomic and functional compositions succeeded with the emergence of a transitional microbial community. The variations in microbial community compositions and predicted functions are likely dependent on integration of transplantation, salinity, and covarying variables. Microbial communities might adapt to salinization and desalinization by regulating the relative abundance and functions of certain taxa. Taken together, this study expands our current knowledge about microbial response to salinization or desalinization in lakes with salinity from freshwater to salt saturation.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ HJ conceived the study. JY, HJ, and GW collected the samples and performed on-site simulation experiments. JHu, WL, BW, HX, and JHa analyzed geochemistry and microbiology of the samples. JHu analyzed the sequencing data. HJ and JHu drafted the article. All authors reviewed results and commented on the article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To assess the prevalence and missed reporting rate of potential clinically-significant incidental findings (IFs) in the neck CTA scans.All consecutive patients undergoing neck CTA imaging, from January 1, 2017 to December 31, 2018, were retrospectively evaluated by a radiologist for the presence of incidental findings in the upper chest, lower head and neck regions. These incidental findings were subsequently classified into 3 categories in terms of clinical significance: Type I, highly significant, Type II, moderately significant; and Type III, mildly or not significant. Type I and Type II IFs were determined as potential clinically significant ones and were retrospectively analyzed by another 2 radiologists in consensus. The undiagnosed findings were designated as those that were not reported by the initial radiologists. The differences in the rate of unreported potential clinically significant IFs were compared between the chest group and head or neck group.2 = 32.151, χ2 = 31.211, χ2 = 65.286, respectively; P < .001 for all).A total of 376 potential clinically significant IFs were detected in 1,698 (91.19%) patients, of which 175 IFs were classified as highly significant findings (Type I), and 201 (53.46%) as moderately significant findings (Type II). The most common potential clinically significant findings included thyroid nodules , pulmonary nodules , sinus disease , intracranial or cervical artery aneurysms , enlarged lymph nodes , and pulmonary embolism . In addition, 184 (48.94%) of them were not mentioned in the initial report. The highest incidence of missed potential clinical findings were pulmonary embolism and pathologic fractures and erosions (100% for both). The unreported rate of the chest group was significantly higher than that of the head or neck one, regardless of Type I, Type II or all potential clinically significant IFs (χImportant clinically significant incidental findings are commonly found in a proportion of patients undergoing neck CTA, in which nearly half of these patients have had potential clinically significant IFs not diagnosed in the initial report. Therefore, radiologists should beware of the importance of and the necessity to identify incidental findings in neck CTA scans. The latter has long been considered as standard diagnostic procedure, an imaging modality for assessing the vessels of lower extremity, cardiac, pulmonary artery.With the rapid technological advance in image acquisition and spatial resolution, computed tomographic angiography (CTA) serves increasingly as a quick, accurate, and attractive noninvasive clinical procedure upper extremity and pulmonary arterial system.,9 Although some incidental findings, such as the degeneration of skeleton system, may have little clinical significance, the detection of others, such as early detection of curable tumors, may mean a huge difference to the patients.In addition to diagnosing the target vessel, CTA can detect a trail of incidental findings (IFs) within the anatomic coverage. Incidental finding refers to an observed abnormality on the images that is not related to the initial indication for the examination. Previous studies have assessed the incidence and significance of incidental findings detected on CTA of the thorax aorta, evaluating vascular and other incidental findings on head and neck CTA, recorded clinically significant findings in 21.5% of cases. But the enrolled case size was not large enough and the issue of underreported IFs was spared.Indications for neck CTA include acute ischemic stroke, vascular stenosis, etc., and the scanning coverage extends from the aortic arch to the frontal sinus, so the incidental findings of the cerebral parenchyma, soft tissues and skeleton of the head and neck, and upper thorax could be obtained. However, indications for neck CTA may vary from person to person in the patient population, which may influence the missed-report rate and incidence of incidental findings. This reality may account for the scarcity of related research in the literature. To our best knowledge, little evidence is available to probe into the missed reporting rate of incidental findings on neck CTA. A retrospective study by Kanesa-Thasan R et alThe current study attempted to assess the prevalence and the missed reporting rate of potential clinically significant IFs during neck CTA examination and to evaluate the reasons that may influence their omission by radiologists. The study will help radiologists to avoid misinterpretations and clinicians in their clinical decision-making process.2This study was approved by the Ethics Committee of Fujian Medical University Union Hospital, and the stipulation for informed consent was waived.2.1One radiologist with 9 years of experience, blinded to the initial CT reports, retrospectively assessed all neck CTA studies, between January 1, 2017 and December 31, 2018, only for detecting IFs. All cases were distinguished by searching our PACS  between the above-mentioned dates using the keyword “neck CTA.” Participants were excluded in our study if there was substantial artifact or the images were not retrievable. For patients receiving repeated neck CTA during the study period, only the first examination was included in the study. In the remaining cases, if one or more incidental findings were found in a case, it was recorded as a “potential IF” case.2.2All examinations were performed on one of two 64-multidetector CT (MDCT) scanners  at our imaging center, utilizing test bolus software to acquire the optimal trigger time of CTA. FOV spanned from the aortic arch to the frontal sinus. On the basis of the patient's weight, a dose of 40 to 80 ml of nonionic iodinated contrast media  and 20 to 30 mL saline chaser were injected via an antecubital vein through an 18-gauge peripheral intravenous line at a flow rate of 4 to 5 ml/s with a power injector. Scan parameters included 120 kVp, auto mAs, and slice thickness of 0.6 to 1 mm.2.3Axial images were first analyzed and coronal and sagittal multi-planar reformation (MPR) were also reviewed if necessary. Images were assessed on a PACS  work-station. The images were evaluated with windows and levels suitable for the structures under consideration. For this reason, lung scans were evaluated using a window both for the mediastinum  and for the lung parenchyma , whereas for scans of head and neck, a window and level suitable for evaluating both parenchymatous organs  and bone structures  was employed. A window fit for vessels  was utilized to evaluate vessel IFs. all studies were classified into three categories in terms of their clinical significance: type I, type II, and type III. Type I refers to highly clinically-significant findings that required further imaging follow-up or immediate therapeutic intervention, including malignancy or curable tumor, pulmonary embolism, and intracranial aneurysms. Type II was defined as moderately clinically-significant findings that required no immediate action but necessary follow-up, including enlarged lymph nodes, thyroid lesions, sinusitis, and ground-glass opacities. Type III was designated as minor or no significant findings that required no follow-up, such as degeneration of spine, arachnoid cyst, and atelectasis, and were excluded from this study.Similar to that conducted by Lumbreras and his colleagues,Type I and type II IFs were regarded as potential clinically significant findings and assessed in our study. Another two radiologists with 10 and 26 years of experience retrospectively reviewed each “potential clinically significant IF” case seen on the neck CTA images, respectively. If both radiologists agreed with the initial radiologist, it was classified as potential clinically significant IF . Any discrepancies in extracted data were discussed, and disagreements were resolved by consensus with a fourth radiologist with 21 years of experience resulting in a diagnostic agreement of IFs. All IFs were recorded and compared with the original report.2.4P value of less than .05 was considered statistically significant.All data were analyzed using the Statistical Package for the Social Sciences . Discrete and continuous data were reported as counts and percentages, and as mean ± SD, respectively. Comparisons of categorical variables were performed and Chi-square test was used when appropriate. A 3A total of 1862 consecutively-registered cases underwent neck CTA during the 2-year study period, of which 135 (7.3%) cases were excluded due to artifacts, 2 (0.1%) due to irretrievable images, and 27 (1.5%) for repeated CTAs. The remaining 1698 cases consisted of 922 males and 766 females with an average age of 54.7 ± 10.6 years (age range: 34–86 years).P < .001, Chi-square test].A total of 376 potential clinically significant IFs were identified in 355 (20.91%) patients. The rest 1,343 (79.09%) patients had type III or no IFs and were thus not included in this study. Of the 376 IFs, 159 (42.29%) were found in the chest and 217 (57.71%) in the head or neck; 175 (46.54%) were regarded as type I and 201 (53.46%) as type II. The most common identified potential significant IFs were thyroid nodules , pulmonary nodules , sinus disease , intracranial or cervical artery aneurysms , enlarged lymph nodes , and pulmonary embolism . Other IFs were rare. Among these 376 IFs, 184 (48.94%) were not mentioned in initial CTA reports. The unreported proportion of chest IFs was 73.58% (117/159), significantly higher than that of head or neck IFs [30.88% (67/217); χ2 = 65.286, 2 = 32.151, P < .001, Chi-square test). Examples of Type I IFs are provided in Figures The highly clinically significant findings were summarized in Table 2 = 31.211, P < .001, Chi-square test). Examples of type II IFs are shown in Figures For the moderately clinically-significant group, the detailed distribution of IFs were outlined in Table 4Computed tomographic angiography is not uncommon for noninvasive evaluation of patients with neck or head diseases. As the field of view in CTA examinations includes some additional organs, such as the pulmonary parenchyma or soft tissue structures, it is vital to inspect all those structures for potential diseases. The present study investigated the frequency, distribution and unreported rate of potential clinically significant IFs on neck CTA images. To our best knowledge, this is the first study focusing on evaluating IFs on neck CTA scans from a large non-selected cohort. This might be expected, as we found some IFs, that is, pulmonary embolism, which were not reported before, even though our data of neck CTA cases have smaller coverage and fewer images.A total of 376 potential clinically significant IFs were identified in 20.91% of the patients, of which 46.54% of IFs were considered as highly clinically significant incidental findings and 53.46% as moderately clinically significant. The rate of clinically significant IFs in our study was not different from that of a previous similar study, which identified actionable, incidental findings in 21.5% of patients receiving acute stroke intervention combined with CTA of the head and neck.,13 but is comparable to that in patients undergoing CT cervico-cerebral angiography. The low incidence of thyroid nodule may result from the inclusion criterion set in the current study, in which we only included thyroid nodule (>1 cm) into our research, for the previous study shows that failure to diagnose thyroid cancers (≤1 cm) is unlikely to influence morbidity and mortality.Thyroid nodule is one of the most familiar IFs on imaging studies that involve the neck. In our study, the prevalence of thyroid nodule (>1 cm) was 5.18%, representing the most common of potential clinically significant IFs. This is lower than the reported average incidence from CT scans targeting the region of neck from the literature,,14 However, pulmonary embolism has been identified in 0.8% to 7.6% studies by other different research methods.,17 Therefore, the incidence of pulmonary embolism in patients undergoing neck CTA examination is probably underestimated, as neck CTA only involves a limited part of the upper portion of pulmonary artery but the lower lung artery is the predilection site of pulmonary embolism. The other possible cause is that not all the arteries in neck CTA images meet the appraisal criteria of pulmonary embolism.Of note, in the current study, the prevalence of pulmonary embolism in our study was 1.12%. Pulmonary embolism has not been described as IF in CT imaging of the head and neck CTA examinations specialized in IFs. The observed high unreported rate may be attributed to the following factors: firstly, a phenomenon known as “inattentional blindness” may contribute to under-reporting. CT is usually performed to find the actual cause of a known disease or symptom. Therefore, radiologists often focus more on the clinical interest than on the potentially more clinically significant IFs. Another phenomenon called “satisfaction of search” may also be attributable when a lesion to answer a clinical question was found, leading to the termination of evaluation of other abnormalities, because the radiologist is contented with the detected findings. A third reason may be due to too much work and not enough manpower in our hospital.The unreported rate of potential clinically significant IFs in the primary report was 48.92% , which is lower than that of a study of chest imaging. The latter found that potential significant cardiovascular IFs were unreported in 63.2% of patients undergoing non-ECG-synchronized chest CT examinations.As seen, the reported rate of IFs in the head or neck group was higher than that of the chest group. Several factors may account for the relatively higher rate of under-reporting of chest findings. The most important one is that the suitable CT window and level for evaluating lung was not provided in previous images. The other possible reason is a lack of sufficient training and low confidence on the part of the initial reporting radiologists in identifying chest IFs on standard neck CTA. On the contrary, all incidental pulmonary embolisms were missed in the initial report, though they were identified in 19 (1.12%) patients. One possible cause is that radiologists are inclined to concentrate on carotid arteries rather than on pulmonary vessels. Meanwhile, the diagnosis of PE on neck CTA scans seems more difficult than that on standard pulmonary CTA images, for the conspicuity of pulmonary vessels is not comparable to that from pulmonary CTA.Thyroid nodule accounted for a large fraction of potential clinically significant IFs (23.40%), but most of these (68/88) were reported by initial radiologists. Thyroid nodules are almost of low density and can easily be detected in neck CT, because normal thyroid gland contains iodine and presents a high attenuation even on non-contrast CT.Several possible limitations are present in this study. Firstly, it was a single-center study, and the regional patient population may have pathogenic factors and diseases different from those of other regions. Thus, the results may not be readily replicated to all hospitals, and their clinical significance may be limited. Secondly, as a retrospective study, the follow-up data of all patients with IFs were not available, so all IFs were not subjected to further investigations and follow-up. The discovery of some IFs  are largely dependent on the radiologist's professional experience. However, the IFs diagnostic criteria used in the current study are relatively strict. Thirdly, the use of arterial phase imaging in the examination of the neck, head and upper thorax may limit the evaluation of hypo-vascular lesions and pathology, which may be more reliably detected in a delayed phase. Moreover, we did not consider whether the incidental findings were already known to the clinicians. However, we assume that this did not substantially influence the reporting rate of the IFs, for clinical information on these diseases is seldom provided to radiologists before reporting.5The current paper reveals the high prevalence of potentially life-threatening incidental findings in unselected patient population undergoing neck CTA and exposes that nearly half of these IFs, especially those in the chest region, are not mentioned in the original report. The results signify that due care should be given to incidental findings during neck CTA. To reduce the missed rate of incidental findings, a definite categorization of incidental findings in neck CTA is necessary for interpreting significance of such findings. Moreover, the clinicians should provide sufficient clinical information of the patients and radiologists should endeavor to access as detailed data of the patients as possible before the imaging examination.Conceptualization: Guang-Liang Chen, Yun-jing Xue, Qing Duan.Data curation: Guang-Liang Chen, Yun-jing Xue, Jin Wei.Formal analysis: Guang-Liang Chen, Yun-jing Xue.Investigation: Guang-Liang Chen.Methodology: Guang-Liang Chen, Yun-jing Xue, Jin Wei, Qing Duan.Project administration: Guang-Liang Chen, Yun-jing Xue.Resources: Jin Wei, Qing Duan.Software: Jin Wei.Supervision: Yun-jing Xue, Qing Duan.Validation: Guang-Liang Chen, Yun-jing Xue.Visualization: Guang-Liang Chen.Writing – original draft: Guang-Liang Chen, Yun-jing Xue.Writing – review & editing: Guang-Liang Chen, Yun-jing Xue.


The clinicopathological characteristics, hearing test results, germline GSTT1, GSTM1, and GSTP1 polymorphisms, and common laboratory parameters were included in the new analysis. Univariate and multivariate statistical tests were applied. According to multivariate logistic regression, the only independent predictor of unilateral ototoxicity (vs. non-affected) was a GSTM1 null genotype , while for bilateral damage, the GSTT1 null genotype  was a predictor. The higher starting serum urea level was characteristic of bilateral ototoxicity; however, the only independent marker of bilateral  ototoxicity was the presence of GSTT1 null genotype . Different processes, involving the GSTM1 and GSTT1 genotypes, respectively, govern the development of acute unilateral and bilateral ototoxicities. Further research is needed to clarify these processes. Based on the above findings, patients whom are at risk may be selected for otoprotective therapies.Preventing the ototoxicity caused by cisplatin is a major issue yet to be overcome. Useful preventive treatments will soon be available. Consequently, the next step is to filter out those patients who are more prone to develop ototoxicity. The aim of this study was to prospectively evaluate potential predictive markers of acute ototoxicity as determined by measures of distortion product otoacoustic emissions (DPOAEs). A total of 118 patients from our previous DPOAE analysis were put under evaluation. Ototoxic cases were divided according to unilateral .GSTT1 null.The only marker of bilateral hearing loss (vs. non-affected) was the GSTT1 null was also the marker of bilateral vs. unilateral ototoxicity.A high-risk group may be selected for new, individualized otoprotective treatment. Germ cell tumors are among the most frequent neoplasms identified in men from 15–44 years of age . In the GSTT1, GSTM1, and GSTP1 polymorphisms, other laboratory parameters and past records of hearing tests were examined. The a priori identification of a high-risk group can served the basis for a better definition of individualized treatment and the targeted use of new otoprotective drugs.The aim of this study was to prospectively investigate for possible predictive markers of acute ototoxicity, which was determined by using distortion product otoacoustic emission (DPOAE) measurements. The study evaluated the modifications in the acute phase immediately after the 1st cisplatin cycle; thus, the findings do not address changes in DPOAEs due to chronicity or permanent damage to the cochlea. Besides A total of 118 patients with testicular cancer were treated with combination of cisplatin + bleomycin + etoposide were considered for the investigation of predictive markers for acute ototoxicity. These patients represent a subcohort  of patients presented in our previous study .The Institutional Ethical Committee and the Hungarian Medical Research Council approved the study (323-101/2005-1018EKU). All patients signed an informed consent.2/5 days.At the treatment site, patient’s clinical records were reviewed for the following factors: past hearing complaints, present hearing complaints, noise injuries, exposure to noise pollution, hearing loss risk factors, and smoking habits. The historical data were also considered in the analysis. The description of DPOAE measurement and the exclusion criteria for patients including presbyacusis, abnormal tympanic cavity pressure, etc. were detailed in our previous study . OtotoxiThe ototoxic effect of cisplatin is known to primarily occur at higher frequencies ; therefoIn addition to patient’s age, histology result (seminoma vs. non-seminoma), disease stage (1 vs. 2–3), and blood pressure (normotension vs. hypertension) at the beginning of treatment were used in the analysis. The following laboratory data were recorded before the treatment were used for the analysis: white blood cell and platelet count, serum level of hemoglobin and hematocrit, and levels of aspartate aminotransferase, alanine transaminase, gamma-glutamyl transferase, urea, creatinine, glucose, bilirubin, Na, K, Ca, and Mg. At the end of the first treatment cycle, the white blood cell and platelet count; serum level of hemoglobin, hematocrit, and urea; and creatinine levels were available for investigation.GSTT1 and GSTM1 null genotypes and the GSTP1 Ile105Val (rs1695) polymorphism were evaluated by multiplex PCR and the PCR-RFLP method, respectively, as described in detail by Kiran et al [Blood samples were taken from patients and DNA prepared as described earlier . GSTT1 aan et al .P < 0.05 value was considered statistically significant. A series of logistic regressions were applied to find independent markers of unilateral and bilateral, or any ototoxicity, and, moreover, to differentiate between unilateral and bilateral ototoxicities. All variables, with P < 0.1 of post hoc tests in univariate analysis, were introduced in the respective logistic regression analysis to determine the independent markers of acute ototoxicity. The Hardy-Weinberg equilibrium was tested for GSTP1 polymorphism on www.dr-petrek.eu/documents/HWE.xls. The NCSS program . NCSS, LLC. Kaysville, Utah, USA, ncss.com/software/ncss.) was used for the statistical analyses.All continuous and categorical data were compared in the three subgroups with one-way ANOVA  and chi-squared (or exact) tests, respectively. Post hoc tests were applied to find all pairwise differences between the subgroups. In the case of parameters available before and after treatment, the changes were also analyzed. The distribution of the clinicopathological parameters of the patients are presented in Table GSTP1, there was no deviation from the Hardy-Weinberg equilibrium (P = 0.195). The frequency of the null genotypes of GSTT1 and GSTM1 was 21.2% and 47.5%, respectively, , while this value was 38–43% for the other subgroups.There was a statistically significant accumulation of patients (43%) with the In comparison with the pre-treatment measures, white blood cell count and urea level significantly increased at the end of first cycle, while the hemoglobin, hematocrit, and creatinine levels were decreased (Wilcoxon signed-rank test) in all subgroups, except for the white blood cell count and the urea level in patients with bilateral ototoxicity.P = 0.02 and P = 0.023). Interestingly, this high initial level of serum urea remained unchanged even after the 1st cycle of chemotherapy, while in the other subgroups, a ~ 20% increase was observed.The initial level of urea was higher in patients with acute bilateral ototoxicity than in the other two subgroups ; P = 0.018. The area under the receiver operating curve (ROC) was 0.624.The following variables were introduced in the logistic regression model of 0 vs. 1 involved ear: GSTT1, glucose, and urea levels were used as covariates in the logistic regression model for 0 vs. two affected ears. Only the GSTT1 null was an independent marker of bilateral ototoxicity: OR = 3.6; (1.1–11.4); P = 0.029. The area under the ROC curve was 0.617.The GSTT1, urea, glucose, and “white blood cell count after,” while “urea change” was excluded because of a multicollinearity. Only the GSTT1 null was found to be an independent predictor of acute bilateral ototoxicity compared with unilateral damage: OR = 4.31; (2.8–4.3); P = 0.033. The area under the ROC curve was 0.706.The model for unilateral vs. bilateral involvement included the following parameters: the GSTM1 were used as covariates, logistic regression resulted in no significant variables.If ototoxicity (either or both ears involved vs. non-affected patients) was considered and age and GSTM1, GSTT1, and GSTP1) were investigated. The frequencies of GSTM1 null and GSTT1 null genotypes were similar to those published for a Hungarian population  [GSTP1 genotypes were in Hardy-Weinberg equilibrium. We have observed that a GSTM1 null was associated with unilateral involvement, while the GSTT1 null was associated with a bilateral acute ototoxicity. It is likely that other non-investigated parameters can also contribute to the development of an ototoxicity, because the observed areas under the ROC curves for both markers were ~ 0.6.In this study, we aimed to evaluate the potential predictive markers of cisplatin-caused acute ototoxicity as determined by DPOAEs to give further assistance to oncologists in designing individual therapies for patients at risk. DPOAE measures indicated earlier damage of outer hair cells compared with the outcomes of routine audiometric tests . In our ctively) . The disGST polymorphisms in cisplatin-induced acute toxicity  genotype. If patients with at least one wild type allele were considered (as in our study), the association was not present. Unfortunately, unilateral and bilateral ototoxicites were not assessed.There are few reports, which have studied isplatin . Talach n cycles , 24. Barorphisms . After pn et al.  reportedn et al. . Aside fs et al.  and Khrus et al.  did not s et al. , or patis et al.  may maskorphisms . InteresGSTM1 or GSTT1 null genotype as a protector or facilitator for ototoxicity) relies on different physiological phenomena: early acute ototoxicity may be associated to damage of stria vascularis and supporting cells within the cochlea that have the potential to recover, whereas the damage to outer hair cells results in a permanent hearing loss [GSTT1 null proved to be protective for hearing impairment [Based on our previous , 35 and ing loss . In somepairment .GSTP1 polymorphism is very controversial. Both the homozygous mutant [The difference between acute and late ototoxicity was also presented in studies by Talach et al.  and Juras mutant , 26, 32 s mutant , 30, 33 s mutant  or a pros mutant , 29, 31.GSTT1 null was found to be associated with hearing damage (presbycusis) at high frequencies, which was determined by audiometry in 50 adults [GSTM1 null genotype was associated with noise-induced hearing loss as determined by pure-tone audiometry in 889 Chinese workers [GSTM1 null genotype were also prone to noise-induced hearing loss, but a lower noise exposure threshold may be present, because of cisplatin treatment. This presumption should be investigated in further studies.Interestingly, the 0 adults , while t workers . The lat+), and potassium (K+) levels of patients with further bilateral involvement were within the reference ranges . Impaired kidney function can also be excluded because the creatinine and the estimated glomerular filtration rate levels  did not differ from those of other patients. Also, drugs , which may increase the urea levels of patients, were not initiated before chemotherapy.Regarding the high initial urea levels, acute dehydration can be excluded because the hemoglobin, hematocrit, serum sodium , may raise the urea levels . In ordeThere are some limitations of this study. We have not investigated the combination of polymorphisms, as did other authors , 25, becThe role of the studied  polymorphisms in predicting cisplatin-caused early ototoxicity is far from being clarified. In the future, the most important task would be to find a key to differentiate between those early ototoxic cases, which will recover, and those who are going to develop a permanent hearing impairment. It needs to be emphasized that early ototoxicity is prone to recovery in some patients . In contGSTM1 null and 0% had GSTT1 null genotypes. So this could be a GSTM1-dependent slow process. The rapid response is characteristic of GSTT1 null patients, who presented acute and also late (persistent) bilateral ototoxicity: with 67% of them being GSTT1 null. The situation is further complicated or explained by the fact that improvement is only seen in 12% of patients and only in unilateral cases, who exhibited 0% GSTT1 nulls and 67% GSTM1 nulls. Thus, the slow process involves also reversible ototoxicity, and perhaps this is why it seems slow in the development of bilateral damage. Further research is needed to clarify these phenomena.For the association of genotypes with unilateral and bilateral ototoxicity, we have only hypothetical explanations. The asymmetry and the genetic difference of the bilateral organs are also well-known; thus, an association of a genotype with unilateral ototoxicity is possible. Another option may be related to the unilateral noise–induced effect during treatment, which may result in hearing damage, because the ears during treatment are more sensitive to intense noise. However, this sensitivity may be related to some gene variants. Last, but not least, it is hypothesized that two molecular mechanisms with different speeds may cause ototoxicity. Due to the asymmetry of organs (which includes the asymmetry in the expression of enzymes), the slow toxicity becomes first unilateral and then bilateral. Our patients were also tested more than 1 year after treatment. According to our as yet unpublished preliminary results, in cases where the acute unilateral ototoxicity became bilateral, 50% of patients had GSTM1 null and GSTT1 null genotypes proved to be independent markers of unilateral and bilateral acute ototoxicities, respectively. The a priori identification of a high-risk group can serve as a basis for a better definition of individualized treatment and the targeted use of new otoprotective drugs.In conclusion,


L. iners (CST III), which is linked to a decreased fertility rate. Besides this, L. gasseri—which is known to be associated with oocyte DNA fragmentation and decreased sperm mobility—was identified in the seminal fluids, follicular fluids, and embryo culture media of the unexplained infertility group. Prevotella was increased in the seminal fluids of the explained infertility group, along with HPV-positive seminal fluids: an infection commonly associated with infertility, especially male infertility. Prevotella has been described to negatively affect sperm motility. Taken together, these results suggest that the profiling of the reproductive tract microbiome can add new perspectives to human reproduction.Infertile couples undergoing the use of assisted reproductive technology are a good study model to evaluate the microbiological signatures affecting reproductive health. We tested vaginal lavages, follicular fluids, embryo culture mediums, and seminal fluids from 47 couples for their microbiome composition and HPV infection. Twenty-five infertile couples were diagnosed with unexplained infertility, whereas 22 were diagnosed with explained infertility. Lactobacilli were dominant in the vaginal lavages of both patient groups, and the most abundant species was  The impact of the genital tract microbiome on human reproductive physiology is widely studied ,2,3,4,5.Lactobacillus species, may shift the microbial balance of the reproductive environment in favor of pregnancy [Prevotella and high proportions of facultative anaerobic bacteria, of which the most abundant bacteria are Lactobacillus, Pseudomonas, Gardnerella, Finegoldia, Corynebacterium, and Staphylococcus [Prevotella has been observed in samples with defective sperm motility, while an increased relative abundance of Lactobacillus has been observed in samples with normal sperm morphology [L. crispatus has been described after intercourse, as has the fact that a high amount of Gardnerella in the vagina corresponded to higher signs of inflammation of the male genital tract [While female commensal genital microorganisms, such as regnancy ,7, microlococcus ,9,10. Allococcus , the prerphology . Besidesal tract ,13. Furtal tract ,15,16,17In addition to bacterial dysbiosis in male and female reproductive tracts, viral infections may affect fertility ,19. AmonThe increase of infertility worldwide goes hand in hand with the increased demand for ART. For this reason, we wanted to focus on the possible differences in the genital microbiome and HPV presence in couples diagnosed with unexplained infertility compared with couples diagnosed with explained infertility. We assessed the microbial composition of vaginal lavage, seminal fluid, follicular fluid and embryo culture media from these infertile couples attending ART. Indeed, the addition of new microbiological perspectives may deepen the knowledge of the different infectious infertility causes, especially in unexplained infertility in which no clinical and subclinical abnormalities are detected.Chlamydia trachomatis, Neisseria gonorrhoeae and Trichomonas vaginalis), and antibiotic/probiotic therapy within 6 months prior to the sample collection. All of the samples were stored at −20 °C until the sample processing.From March to September 2017, 47 couples  attending the Infertility Clinic at the IRCCS Burlo Garofolo Hospital, Trieste, Italy, were included in the study. All of the participants were informed and signed written consent. The study was approved by the ethics committee of the hospital (RC 26/13) and all of the experiments were conducted according to the principles stated in the Declaration of Helsinki. After the finalization of the conventional diagnostic assessment, the couples were classified by the treating physicians into the following groups: idiopathic or unexplained infertility, and explained infertility . During the transvaginal oocyte pick-up, the follicular fluids were obtained by trained gynecologists, avoiding the contamination from the lower genital tract microbiome. Before the embryo implant, the cervical–vaginal lavages were obtained by the injection of 20 mL sterile physiologic solution. Furthermore, the embryo culture media were saved. The seminal fluids used for the in vitro fertilization were obtained from the men by masturbation. The exclusion criteria were the use of vaginal douching, positivity for sexually transmitted diseases  for the extraction. The final elution volumes for all of the samples was 50 µL. The extractions were performed using the NucliSENS® easyMAG® system , according to manufacturer’s instructions.The seminal fluids were pretreated with an aqueous solution of 0.01% Dithiothreitol (DTT), according to the manufacturer’s instruction, starting from 500 µL of the sample. The cervical–vaginal lavages and the follicular fluids were centrifuged at 5000× ® dye, Fisher Molecular Biology, Waltham, MA, USA) using the degenerate primer 27FYM (5′-AGR GTT YGA TYM TGG CTC AG-3′) and the primer U534R, targeting the V1–V3 region (500 bp) in order to allow for the construction of rich libraries. A nested PCR, targeting the V3 region (200 bp), was performed with the primers B338F_P1-adaptor (B338F 5′-ACTCCTACGGGAGGCAGC-3′) and U534R_A_barcode (U534R 5′-ATTACCGCGGCTGCTGG-3′). Each PCR reaction (sample) contained a unique IonXpress Barcode Adapter attached to the reverse primer. No-template controls were processed with the clinical samples. The PCR reactions were performed using the Kapa 2G HiFi Hotstart ready mix 2× , which has the robust amplification that is necessary for NGS (Next Generation Sequencing), and 400 ng/µL BSA. The amplification of the fragments was accomplished using an initial denaturation step of 95 °C for 5 min, followed by a maximum of 25 cycles for the V1–V3 PCR and a maximum of 13 cycles for the V3 PCR of a denaturation step at 95 °C for 30 s, annealing at 59 °C (V1-V3 PCR)/57 °C (V3 PCR) for 30 s, and extension at 72 °C for 45 s. A final extension step of 10 min at 72 °C was performed. The concentrations of the amplicons were estimated using a Qubit® 2.0 Fluorometer , and roughly equal amounts (~50 ng) of all of the amplicons were mixed in a single tube and diluted to a concentration of 100 pM. The template preparation was performed using the Ion PGM Hi-Q View kit on the Ion OneTouch™ 2 System , and was sequenced using the Ion PGM Hi-Q View sequencing kit  with the Ion PGM™ System technology.In order to characterize the composition and structure of the bacterial communities, we sequenced the V3 region of the 16S rRNA gene. The amplicons were obtained by a real-time EvaGreen PCR  and standardized before any statistical analysis by means of Scikit-learn v0.23.2, in order to: (i) homogenize the different dynamic ranges inherently present within the dataset; (ii) ensure a proper comparison at each taxonomical level. The differences in the alpha diversity were assessed by the Kruskal–Wallis test, while for the beta diversity comparisons, the PERMANOVA  test was used. In order to identify the biomarkers explaining the differences between the groups, we used the linear discriminant analysis (LDA) effect size (LEfSe) method. A t-test was used for the pairwise comparisons on the normalized and standardized dataset. A two-stage False Detection Rate (FDR) of 10% was performed for both the t-test and the non-parametric tests.The dataset has been deposited in the NCBI Sequence Read Archive (SRA) under the accession number PRJNA480201.This observational prospective study included 47 consecutive infertile couples  who, from March to September 2017, attended the Infertility Clinic at the IRCCS Burlo Garofolo Hospital, Trieste, Italy. After the finalization of the conventional diagnostic assessment, the couples were classified by the treating physicians into the following groups: twenty-five couples were diagnosed with idiopathic infertility, whereas twenty-two couples were diagnosed with explained infertility.We performed the sequencing of the V3 region of the16S rRNA gene from 188 biological samples, including the cervical–vaginal lavage, the follicular fluid, the seminal fluid, and the embryo culture medium  from the infertile couples. Seven embryo culture media were not available, and two out of the seven no-template controls analysed did not produce any sequencing output. The sequencing of the remaining samples produced a total of 7,413,839 reads (Q score > 20), and 4511 OTUs  were observed.Prevotella significantly differed . In our cohort, Prevotella spp. was identified in three out of four HPV-positive seminal fluids , there were no significant differences between the samples from the unexplained infertility group and those from the explained infertility group .According to the weighted and unweighted Bray–Curtis distance (beta diversity) metrics, there were no significant differences when comparing the samples from the explained infertility to those of the unexplained infertility group.In order to find out which microbes most likely explained the differences between the groups, we applied the LEfSe  procedure, using the rarefied feature table as the input.Lactobacillus, Porphyromonas, Prevotella, Staphyloccocus, Streptococcus, and the Streptococcus anginosus group differed in the comparison of the samples from the unexplained infertility group with those of the explained infertility group  III [L. gasseri was also identified, corresponding to the CST II. The L. iners amount significantly  differed among the samples (Among the CST) III . A low a samples .The genital tract microbiome of women and men influences the physiology of human reproduction, revealing that a ‘microbiological homeostasis’ is needed for pregnancy establishment .In this regard, a key point is how a resident microbiome can take part in the favorable environment needed for pregnancy, and how it can counteract the action of detrimental factors, such as sexually transmitted infections (STIs). Among these, HPV deserves particular attention, as it is linked to infertility and is the most common STI, to such an extent that—at least once in their life—sexually active men and women will be infected ,34. In oStaphylococcus, Streptococcus, and the Streptococcus anginosus group were shared among these samples . In our cohort, Prevotella was identified in three out of the four HPV-positive seminal fluids, and with a higher mean relative abundance (25%) compared with HPV-negative seminal fluids (17%). HPV infection is commonly associated with infertility, especially male infertility [Prevotella species was P. bivia, whereas, in the vaginal lavages of explained infertility group, several Prevotella species were identified (P. bivia produces high LPS concentrations, which may create a toxic vaginal environment [Staphylococcus and Streptococcus, Prevotella is a commensal vaginal bacterium that, if it becomes overgrown, creates a dysbiotic vaginal microbiome [P. bivia was increased in the seminal fluids of the explained infertility group, where it has been seen to negatively affect sperm mobility [When considering the presence of HPV DNA, four seminal fluids and four vaginal lavages tested positive. These samples, compared with HPV-negative samples, showed a significant increase of ertility . Notablyentified . As descironment . As for crobiome , which lmobility .Taken together, our results support the concept that the assessment of the reproductive tract microbiome adds a new microbiological perspective to human reproduction. Male and female genital tracts show peculiar microbiomes that can impair the fertility rate.Prevotella, which affects sperm quality, and/or the presence of viral infection, such as HPV, may have a detrimental effect on human reproduction.The seminal microbiome used for IVF needs to be taken into consideration, especially in the diagnosis of unexplained infertility. In this group, the alteration of sperm microbiome, with the increased amount of Lactobacillus species present in the vaginal lavages. More precisely, L. iners and L. gasseri can negatively impact the fertility rate. Besides this, the characterization of the microbiome of follicular fluids and embryo culture media may be considered as an adjunctive test when no alterations of the sperm/vaginal microbiome have been identified.On the other hand, another aspect to be taken into consideration, regardless of the infertility diagnosis, is the characterization of the


Prochlorococcus and Synechococcus are the most abundant photosynthetic organisms on Earth, an ecological success thought to be linked to the differential partitioning of distinct ecotypes into specific ecological niches. However, the underlying processes that governed the diversification of these microorganisms and the appearance of niche-related phenotypic traits are just starting to be elucidated. Here, by comparing 81 genomes, including 34 new Synechococcus, we explored the evolutionary processes that shaped the genomic diversity of picocyanobacteria. Time-calibration of a core-protein tree showed that gene gain/loss occurred at an unexpectedly low rate between the different lineages, with for instance 5.6 genes gained per million years (My) for the major Synechococcus lineage (sub-cluster 5.1), among which only 0.71/My have been fixed in the long term. Gene content comparisons revealed a number of candidates involved in nutrient adaptation, a large proportion of which are located in genomic islands shared between either closely or more distantly related strains, as identified using an original network construction approach. Interestingly, strains representative of the different ecotypes co-occurring in phosphorus-depleted waters  were shown to display different adaptation strategies to this limitation. In contrast, we found few genes potentially involved in adaptation to temperature when comparing cold and warm thermotypes. Indeed, comparison of core protein sequences highlighted variants specific to cold thermotypes, notably involved in carotenoid biosynthesis and the oxidative stress response, revealing that long-term adaptation to thermal niches relies on amino acid substitutions rather than on gene content variation. Altogether, this study not only deciphers the respective roles of gene gains/losses and sequence variation but also uncovers numerous gene candidates likely involved in niche partitioning of two key members of the marine phytoplankton.Marine picocyanobacteria of the genera  Even thSynechococcus and Prochlorococcus are the two most abundant photosynthetic organisms on Earth  5.1 to 5.3 . Members of clades I and IV have been shown to be cold thermotypes that dominate in coastal, mixed and/or high latitude, nutrient-rich waters, while clades II and III are warm thermotypes, predominating in N-depleted areas and P-depleted regions, respectively  than in Synechococcus/Cyanobium , with genome sizes ranging from 1.625 Mb for Prochlorococcus HLII strain GP2 to 3.342 Mb for Cyanobium gracile PCC 6307 (SC 5.2) and GC% from 30.8%  to 68.7%  and this difference is even more marked using GC3%, i.e., the GC content at the third codon position . By contrast, the warm-adapted clades II and III displayed significantly higher values , while the highest GC3% was found for members of the brackish strains of Synechococcus clade VIII and SC 5.2 . Thus, although the strongest GC3% variation was associated to genome reduction in Prochlorococcus, some of the GC3% variations might be related to the ecological niches occupied by these organisms and notably to thermal and variable salinity niches , reflecting a very ancient genomic diversification between the three SC (see below). Because of this clear discontinuity, we propose to split Synechocococcus and Cyanobium into three distinct taxa: Ca. Marinosynechococcus (SC 5.1), Cyanobium (SC 5.2) and Ca. Juxtasynechococcus (SC 5.3). Prochlorococcus strains from different LL clades also fell below the 65% AAI discontinuity, highlighting the large divergence within this group. It is noteworthy that strains within SC 5.2 displayed a particularly low 16S rRNA identity compared to strains within SC 5.1, likely due to the low number of sequenced genomes relative to the wide diversity of this lineage, while in contrast the only two Synechococcus SC 5.3 genomes of our dataset were very closely related.A plot of the relationship between 16S rRNA identity and AAI for the different pairs of genomes  additionle clade  should b1, in which predicted genes were grouped into clusters of likely orthologous genes (CLOGs) by all-against-all sequence similarity. This clustering allowed us to determine the core genome, i.e., CLOGs present in all strains, and the pan-genome, i.e., all CLOGs present in at least one strain, at various phylogenetic depths  and Synechococcus  independently, indicating that we still missed an essential part of the genetic diversity within both genera that is yet to be sequenced from the field. A major asset brought by the 34 newly sequenced Synechococcus genomes is the availability of several genomes per clade, which allowed us to estimate the relative sizes of the core set of CLOGs at different taxonomic levels , the accessory genome, i.e., the non-core CLOGs shared at least by two strains, and, and unique genes, i.e., CLOGs present in a single strain  contained by far the largest gene number of the genome dataset , with a large proportion of unique genes (31.2%). Noteworthy, a significant proportion of CLOGs was present in all strains of a given clade  and could thus potentially be involved in the adaptation of these taxa to specific environmental conditions. However, it should be noted that only a sub-set of these CLOGs were truly specific to each clade , a higher number of events were generally observed on terminal branches as well as an excess of gains compared to losses, with up to 1,662 gained genes on the branch leading to Synechococcus BIOS-E4-1 (SC 5.1) and 831 on the one leading to Prochlorococcus MIT0701, for 105 and 108 lost genes, respectively.To better understand the evolutionary processes that led to the diversification of gene content within marine picocyanobacterial genomes, we estimated by Maximum Likelihood the number of gene gain and loss events on each branch of a reference phylogenetic tree built from a concatenation of 821 single core proteins . As prevadiation . GloballSynechococcus SC 5.1, respectively, while internal and terminal branches of Prochlorococcus HL gained 1.45 and 4.5 genes  involved in cyanate transport  Syn_GI022, a module found in many SC 5.1 strains with the notable exception of clade II strains, which encompasses a large gene cluster involved in glycine betaine synthesis (gbmt1-2) and transport (proV-W-X), located in some strains next to another gene cluster involved in the biosynthesis of glucosylglycerate [gpgS-gmgG-gpgG;  and to share genes with both ecotypes  in the phyletic pattern tree than in the core tree . This corresponds to a fixation rate of 78 and 18 amino acid changes in core proteins per My for Prochlorococcus HL and Synechococcus SC 5.1, respectively, while one gene is fixed once every 2.6 My for Prochlorococcus HL and once every 6.3 My for Synechococcus SC 5.1. While these numbers show that substitutions played a major role in genomic diversification, the question remains as what part of this diversification is related to an adaptive process.We also compared at each node the fixation rate of amino acid substitutions in core proteins  to the fixation rate of genes . 201× more amino acid variants than genes were fixed per My in Prochlorococcus  possessing this gene. All members of clades I and IV also lack the genes phoB and phoR coding for the two-component system involved in P sensing and the regulation of P metabolism, as previously observed on fewer strains , while members of SC 5.2 share 31 specific genes, including another mscS gene copy (CK_00003081) as well as genes encoding a putative chloride channel (CK_00042275) and a NAD-dependent malic enzyme, a protein known to be enhanced under salt stress in plants , Cyanobium (SC 5.2), and Ca. Juxtasynechococcus (SC 5.3). This proposal notably solves the inconsistency to have a mix of strains named Cyanobium spp. and Synechococcus spp. within SC 5.2, which should clearly all be called Cyanobium spp. For the universal acceptance of the revised taxonomy of this group and cyanobacteria at large . Similarly, adaptation to phosphorus-depleted oligotrophic areas might have driven the differentiation of Synechococcus clade III, as revealed by the occurrence of P- and other nutrient-uptake genes specific to this clade. Interestingly, co-occurring ESTUs IIIA, WPC1A, and SC 5.3A only share a few common genes potentially involved in the adaptation to this limitation. Instead, these ESTUs seem to have independently acquired different sets of genes to improve P-uptake and/or assimilation and potentially use different sources of organic phosphate  and warm (HLII) environments , the most parsimonious explanations for these many shared substitutions would be either an adaptive convergence or an ancient homologous recombination between ancestors of these clades. In this context, it is interesting to note that mutations were found to arise in just a few generations in a clonal Prochlorococcus strain as an adaptation to selective conditions such as UV radiation  to a particular niche. We could advocate achieving this via comparative genomics, but this usually necessitates hundreds to thousands of closely related genomes  and transferred three times on 0.3% SeaPlaque Agarose  to clone them and reduce contamination by heterotrophic bacteria. A first set of 25 Synechococcus genomes (including WH8103) were generated at the Genoscope  by shotgun sequencing of two libraries: a short-insert forward-reverse pair-end (PE) library (50–150 bp) and a long-insert reverse-forward mate-pair library (4–10 kb), both sequenced by IlluminaTM technology. Additionally, seven other genomes were sequenced at the Center for Genomic Research  by shotgun sequencing of 250 bp reads. Single or PE reads were first assembled into contigs using the CLC Assembly Cell© 4.10 . Synechococcus contigs were identified based on their different coverage compared to heterotrophic bacteria, scaffolded using WiseScaffolder and 28 out of 31 genomes were closed by manual finishing as described in dnaN start, corresponding approximately to the origin of replication. Automatic structural and functional annotation of the genomes was then realized using the Institute of Genome Science (IGS) Annotation Engine3 (Synechococcus genomes were deposited in GenBank under accession numbers CP047931-CP047961 (BioProject PRJNA596899), except Synechococcus sp. WH8103 that was previously deposited to illustrate the performance of the pipeline used to assemble, scaffold and manually finish these genomes as well as the three KORDI genomes that have been deposited in Genbank in August 2014 , which was used to extract lists of genes shared at different taxonomic levels. Core genomes were defined at the genus, sub-cluster and clade levels when more than three genomes were available for a given taxonomic level  were calculated following the method described in petB phylogenetic tree was built using PhyML 3.1  with a ratio of nucleotides from gained CDS to total coding nucleotides higher than 50%. A network approach was then applied on all predicted islands to compare the gene content of these islands between all strains. Jaccard distances based on shared gene content were calculated between islands and an edge was drawn to connect two islands if their distance was higher than 0.1 . Network modules detection was then performed using the modularity algorithm . Furtherreltime algorithm  can be found in the article/ Synechococcus strains. FH, MR, FDP, DC, JN, and MO extracted the DNA. KL, J-MA, PW, FDP, DC, JN, FP, LG, MH, and GF participated in sequencing and/or assembly of the genomes. MH, GL, EC, AB, LB-G, and GF developed and ran the automatic clustering and annotation pipelines. HD, UG, FP, and LG participated in the expert manual annotation of the genomes. HD, GF, UG, and LG generated and processed the data. UG, HD, JH, and DE produced the genomic island networks. HD, GF, UG, DE, DS, FP, and LG analyzed the results. HD, UG, JH, GF, and LG made the figures. All authors contributed to the preparation of the manuscript. HD, LG, FP, and DS wrote the manuscript. All authors read and approved the final manuscript.FH, MR, FDP, MO, DC, JN, and CS purified newly sequenced The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.