Small organic molecules, lipids, proteins, and DNA fragments can remain stable over centuries. Powerful and sensitive chemical analysis can therefore be used to characterize ancient remains for classical archaeological studies. This bio-ecological dimension of archaeology can contribute knowledge about several aspects of ancient life, including social organization, daily habits, nutrition, and food storage. Faecal remains (i.e. coprolites) are particularly interesting in this regard, with scientists seeking to identify new faecal markers. Here, we report the analysis of faecal samples from modern-day humans and faecal samples from a discharge pit on the site of the ruins of ancient Pompeii. We propose that bile acids and their gut microbiota oxo-metabolites are the most specific steroid markers for detecting faecal inputs. This is due to their extreme chemical stability and their exclusive occurrence in vertebrate faeces, compared to other ubiquitous sterols and steroids. Ancient artefacts can contain markers, such as small organic molecules, lipids, proteins, and DNA fragments, that have been preserved over the centuries. Nowadays, new potent and ultrasensitive analytical methodologies can specifically identify these molecules, even in complex matrices, at submolar concentrations. This allows a more detailed study of the historical bio-ecology of humans4. Researchers have sought to define the most suitable marker molecules for a given archaeological purpose, based on their relationship to specific aspects of ancient lifestyles, such as social organization, daily habits, nutrition, and food storage7. Coprolites (i.e. fossilized stool) are particulary interesting in this regard because they are usually abundant on archaeological sites and are easily collectable. Ancient organic molecules preserved in human coprolites can provide strategic knowledge about human biology and evolution, allowing researchers to extrapolate information about host health, microbiome structure, diet, lifestyle, and social habits. However, it is challenging to properly assign a human origin to a coprolite sample10.Localizing and characterizing organic residues from human activities is a key step in archaeological investigations11. One example is steranes, which are cyclic compounds derived from steroids or sterols. Steranes have been recovered from ancient sedimentary rocks, tracking the diversification and ecological expansion of eukaryotes12. However, sterols occur ubiquitously in plants and animal deposits  and in soils (being produced by soil microorganisms). This ubiquity drastically reduces their specificity as an indicator of a human origin. Researchers have therefore proposed using more steroid molecules, from sexual steroids to bile acids (BAs)11.As markers of faecal input, sterols are relatively stable even in samples that are thousands of years old13.BAs are likely the most specific steroid markers for determining the origin of a faecal input. This is because they occur exclusively in vertebrate faeces and other biological fluids (e.g. bile and blood). The BA family is a complex and heterogeneous class of compounds, whose qualitative and quantitative composition differs greatly among vertebrates, including mammals. BAs undergo several metabolic reactions by gut microbiota (GM) to produce secondary BAs in the intestine. Oxo-analogues, also called keto-BAs, are steroids with one or more oxo groups in the steroid polycyclic core. Oxo-BAs cover a significant proportion (about 20–30%) of BAs in human faeces and are present only in trace amounts in other fluids such as blood. their qualitative and quantitative composition is mainly related to the GM compositionHuman BAs differ from those of other species, so oxo-BA metabolites can increase specificity in identifying faecal input. BAs and oxo-metabolites are therefore very important for the growing field of bioarcheology due to their stability and specificity (they are present in vertebrate faeces only and are species-specific).14 for the faecal analysis of BAs, focusing for the first time on oxo-BAs and their metabolic precursors. Here, we modified this method with a new extraction protocol to detect and characterize coprolites in soil samples from the archaeological area of Pompeii. The aim was to confirm the presence of ancient human faecal inputs in samples taken inside the discharge pit of the latrine of the Roman House of M. Obellio Firmo in Pompeii.A new mass spectrometry method was recently developed and optimizedThis house, placed along the prestigious Via di Nola, was owned by the Obelli family, well known in the city for the public offices held by its members. This ancient building was one of the most important aristocratic residences of Pompeii, provided with two atria and a big garden.Our aim was to check if the BA and GM metabolite profiles of samples from Pompeii correlated with those of contemporary human faeces. We therefore, analyzed samples from healthy subjects using the same method for BA and oxo-BA metabolites. The literature already reports that steroids, sterols and stanones can be used to identify faecal contamination. However, to our knowledge, this is the first time that a complete BA profile has been used to identify the source of coprolites, faecal input, or similar in archaeology , coupled online to electrospray mass spectrometry (ESI–MS), for the simultaneous determination of 27 BAs, including 21 oxo-BAs that are potentially present in vertebrate faeces and synthesized by gut microbiota enzymes. Isotope dilution was used to quantify the BAs with commercially available deuterium-labelled internal standards of deoxycholic acid (DCA) and lithocholic acid (LCA). Our abbreviations for oxo-BAs reflect the structure of the respective metabolic precursor, indicating the position involved in the oxidative reaction (i.e. ‘3oxo LCA’ means ‘LCA with a hydroxy group converted to a carbonylic group at position 3 of the steroid ring’).As expected, the studied molecules were extremely diluted in the soil samples from the Pompeii discharge pit. We therefore extracted and concentrated the sample using solid phase extraction (SPE) on C18 silica cartridges to obtain detectable and quantifiable BA levels. Thanks to the extraction procedure, we could detect BAs naturally present in 1 g of faeces even if they were diluted in 1 kg of soil.Secondary BAs and several oxidized metabolites from the gut microbiota were identified and quantified in the Pompeii discharge pit samples . Moreover, the absolute content was lower in the ancient sample (soil) than in fresh human faeces, suggesting that the ancient stools were widely dispersed in soil.However, our results confirmed the presence of mammalian faeces in the ancient samples, whose specific profile of BAs and derivatives is comparable to that of human stools. DCA was the main BA found in the Pompeii discharge pit samples, together with its three derivatives oxidized by the gut microbiota. LCA was detected in high concentration, as was the metabolite 3oxo LCA. Oxo-BAs made up a significant proportion of the detected molecules, up to 25–30% of the total BAs. The slight differences in the BA profiles of Pompeii discharge pit samples and fresh contemporary human faeces can be ascribed to variability in daily BA excretion, related to nutrition, intestinal transit time, and individual gut microbiota profiles. These factors usually slightly modify the faecal BA pool in terms of quantitative content and, to a lesser extent, qualitative composition.The percent ratio between oxidized species (oxo-BAs) and metabolic precursors (BAs) in Pompeii samples was calculated as:entclass1pt{minimaThis metabolite-precursor ratio was used to normalize and directly compare the results without considering the dilution of the ancient faecal samples in soil, but taking into account the yields of the oxidative reactions by the bacteria hydrogenases in ancient Pompeiians and contemporary subjects.As shown in Fig. t test, P NS, α = 0.05), with a greater variability associated with 3oxo-LCA.No significant differences were found in the quantitative composition . Although the building was excavated in the early twentieth century and some external contaminations cannot be excluded (the structure was still connected with the latrine and with other channels in the garden), the pit can be considered a valid context for this study. In 2018, the subterranean structure appeared still unexplored and it can therefore be assumed that the massive organic deposit was the same present at the time of the eruption in 79 A.D. The underground location and the absence of a direct connection with the outside have presumably preserved the deposit from the stress of the heat and the pyroclastic material. A structural failure of the pit vault made it possible to climb down and reach the deep sediment inside. The waste pit’s function is clear. By applying the proposed analytical method, we aimed to profile the sample’s BA composition in pit discharge soils, allowing us to unequivocally identify the presence of human faecal remains. To the best of our knowledge, this is the first time that researchers have profiled the pattern of BAs and derivatives in ancient Romans.17. This is important because certain animals have coexisted in close contact with humans over the centuries and their remains have been found near human settlements. No less important, BAs are very stable molecules and more resistant to degradation than Δ5-sterols, stanols, and stanones. Thus, BAs can be used where other markers have already been degraded or further metabolized.According to our results, a comprehensive characterization of BAs and the complex pool of related metabolites can reliably attribute ancient faecal specimens to human origin. Indeed, the composition of human BAs differs from that of other animal species. Determining the complete pool of BAs and metabolites is thus a powerful tool for differentiating faecal inputs. In this study, we used more specific markers, such as the percent ratio of relevant oxidized species (oxo-BA) and metabolic precursor (BA) concentrations Fig. . This alBAs have been studied in research fields including medicinal chemistry, environmental chemistry, evolutionary biology, and archaeology. However, this is the first time that ancient coprolites have been characterized for the extraordinary biodiversity of their BAs and BA metabolites in order to determine their likely origin. The composition of BAs and their oxo metabolites is a new approach to increase the specificity of human faecal detection in faecal soil contamination studies. Of the relevant BAs and metabolites, oxo-BA are easily the most stable, therefore our comprehensive and sensitive analytical method for determining these biomarkers is a significant contribution to the growing field of bioarchaeology.18. Despite the similarities, we hypothesize that the slight differences in BA profiles, although not statistically significant, may be related to the different amounts of fat in the diet. Changes in diet and nutritional status may slightly but detectably affect these metabolic products as a result of a well-controlled steady-state BA balance in the enterohepatic circulation. Oxo-BAs and other gut metabolites could then be used as a chemical proxy, allowing the structure and functionality of the gut microbiota to be inferred, supporting microbiologists in archaeology and other scientific fields.Beyond that, our data indicate a BA profile in human faeces that is well-preserved over the centuries from ancient Rome to the present day. Indeed, our results show that the Pompeian Romans had a BA and BA metabolite profile similar to that of contemporary Italians. Accordingly, we hypothesize that the presence and activity of the microbial enzymes responsible for BA metabolism was similar for these two groups. Interestingly, diet can affect the BA levels in the gut, specifically the levels of chenodeoxycholic acid  and deoxycholic acid (DCA)3. The sampling was conducted at different distances from the expected latrine site to obtain a more representative sample. A total of 10 aliquots of 10 g each were used for BA analysis. The samples were stored at − 20 °C until the analysis.The latrine was identified in the House of M. Obelio Firmo in Pompeii during the excavation campaign of September 2018. The dimension of the waste pit was 1.80 × 1.10 m, with a sampled volume of soil of 0.84 mAuthentic chemical standards of CA, HCA, CDCA, DCA, DCA 2,2,4,4-d4, HDCA, UDCA,LCA, LCA 2,2,4,4-d4  were purchased from Sigma-Aldrich .Standards of 3,7,12-trioxo-5α-cholan-24-oicacid, 7 α,12 α-dihydroxy-3-oxo-5 α-cholan-24-oic acid, 3 α,12 α-dihydroxy-7-oxo-5 α-cholan-24-oic acid, 3 α,7 α-dihydroxy-12-oxo-5 α-cholan-24-oic acid, 7 α-hydroxy-3-oxo-5α-cholan-24-oic acid, 3 α-hydroxy-7-oxo-5 α-cholan-24-oic acid, 3,7-dioxo-5α-cholan-24-oic acid, 12α-hydroxy-3-oxo-5α-cholan-24-oic acid, 3α-hydroxy-12-oxo-5α-cholan-24-oicacid, 3,12-dioxo-5 α-cholan-24-oic acid, 3-oxo-5 α-cholan-24-oic acid, 3 α,6 α-dihydroxy-7-oxo-5α-cholan-24-oic acid,3 α-hydroxy-6,7-dioxo-5α-cholan-24-oic acid, 3α-hydroxy-6-oxo-5 α-cholan-24-oic acid, and 3,6-dioxo-5α-cholan-24-oic acid were purchased from Steraloids .14.Standards of 7α,12-dihydroxy-3-oxo-5α-cholan-24-oic acid, 12α-hydroxy-3-oxo-5α-cholan-24-oic acid, 7α-hydroxy-3-oxo-5α-cholan-24-oic acid, 7α,12α-dihydroxy-3-oxo-5α-cholan-24-oic acid, 6α-hydroxy-3-oxo-5α-cholan-24-oic acid, and 6α,7α-dihydroxy-3-oxo-5µ-cholan-24-oic acid were synthesized as reported in the literatureStock solutions of each analyte and IS were prepared in isopropanol at a concentration of 1 mg/mL and stored at − 20 °C. These stock solutions were further diluted in isopropanol to obtain working solutions containing all the analytes used for method optimization and calibration curves.Twenty mL of NaOH (0.1 mol/L) and 5 μL of the internal standard solutions (10 mM) were added to each aliquot of 10 g of dried soil samples and incubated for 2 h at 64 °C. The sample was homogenized and centrifuged at 18,000×g for 15 min. The supernatant was collected and extracted using C18  SPE columns. The SPE cartridge was conditioned with 5 mL of methanol and 5 mL of water prior to sample loading. The samples were loaded into the conditioned cartridge, washed with 10 mL of water, and then eluted with 5 mL of isopropanol. The eluate was dried under vacuum, then reconstituted with 100 µL of 40% isopropanol in 15 mM ammonium acetate buffer at pH 8, filtered, transferred to an autosampler vial, and 5 µL injected into the HPLC–ESI–MS system. The recovery of the extraction method was tested with fortified standard soil samples at three concentration levels for each BA.14.BA quantification was carried out with isotope dilution mass spectrometry (ID-HPLC–MS/MS) with deuterium-labelled internal standards, using a method adapted from Franco et al.Liquid chromatography was performed using a 2690 Alliance system  connected to a triple quadruple mass spectrometer  with an electrospray interface, operating in the multiple reaction monitoring (MRM) acquisition mode. The performance of the analytical method was tested according to International Conference of Harmonization (ICH) guidelines [Guidance for Industry 2016].t test was applied with a significance level of 0.05 .Univariate statistical analysis was performed using GraphPad Prism 5.0 software . A paired


When a distractor appears close to the target location, saccades are less accurate. However, the presence of a further distractor, remote from those stimuli, increases the saccade response latency and improves accuracy. Explanations for this are either that the second, remote distractor impacts directly on target selection processes or that the remote distractor merely impairs the ability to initiate a saccade and changes the time at which unaffected target selection processes are accessed. In order to tease these two explanations apart, here we examine the relationship between latency and accuracy of saccades to a target and close distractor pair while a remote distractor appears at variable distance. Accuracy improvements are found to follow a similar pattern, regardless of the presence of the remote distractor, which suggests that the effect of the remote distractor is not the result of a direct impact on the target selection process. Our findings support the proposal that a remote distractor impairs the ability to initiate a saccade, meaning the competition between target and close distractor is accessed at a later time, thus resulting in more accurate saccades. Natural limitations of the visual system mean we must move our eyes to gather visual information about our environment . These eIt has been shown that when a single visual distractor accompanies a visual target, both saccade latency and landing position (or accuracy) are dependent upon their spatial separation. Generally, little effect on saccade latency has been found when a target and distractors are close together (within a sector of about 20–30 degrees of the target), although shortening of latencies has, on occasion, been reported in these circumstances (speeding: inhibition ofreturn), respectively  . The eyeWhen considered in terms of the underlying neurophysiology of the saccadic eye movement system, there is evidence that could be taken to support both hypotheses. The indirect fixation gating hypothesis originally equated the fixate system with fixation neurons identified at the rostral pole of SC , However, there are a number of reasons to be hesitant in interpreting these results as evidence supporting the indirect hypothesis. First, it is possible that the position of the remote distractor used by In order to address these issues, we report two experiments in which observers were required to saccade to a target in the presence of a close distractor. These stimuli were often accompanied by a further distractor at various, remote locations see . FixatioSeven different naive observers participated in each experiment, five female and two male in each. Observers ranged in age from 19 to 21. All observers had normal or corrected-to-normal vision. Ethical approval from School of Psychology, University of Reading was obtained for this study, and all participants gave their informed consent prior to inclusion.2. Eye movements were recorded using a head-mounted, video-based eye-tracker with a sampling rate of 500 Hz , recording monocularly from observers’ right eyes. Stimuli were presented in grayscale on a 21-in. color monitor with a refresh rate of 75 Hz  in sequences developed using Experiment Builder (SR Research). Head movements were constrained with a chinrest, which held participants so their eyes were in line with the horizontal meridian of the screen, at a viewing distance of 1 m. The eye-tracker was calibrated using a standard 9-point grid, carried out at the beginning of the experiment and after any breaks where the observer removed their head from the rest or removed the eye-tracker. Calibration was only accepted once there was an overall difference of less than 0.5° between the initial calibration and a validation retest: In the event of a failure to validate, calibration was repeated.Stimuli were vertically oriented Gabor patches with a spatial frequency of two (target) or four (both close distractor and remote distractor) cycles per degree (cpd), with a standard deviation of 0.3° and a contrast of 90%. All stimuli were presented on a gray background, with a mean luminance of 23 cd/mIn A distractor more remote from the target/close distractor complex could also be present. For Observers were shown examples of target and distractor stimuli. Following this, an introductory block of up to 20 trials was presented to familiarize observers to the timing and spatial configuration of the experimental trials. Observers were instructed to move their eyes “as quickly and accurately as possible” to the target Gabor patch, ignoring distractors. Trials began with a central fixation cross (+) subtending 0.5 degrees of visual angle, presented for a varying duration between 800 and 1,300 ms. The fixation cross disappeared at the onset of stimuli, which were displayed for 1 s. This was followed by a blank screen, for 500 ms, and then the reappearance of the fixation cross for the next trial. Once the observer refixated within a 1-degree area, centered on the central cross, the next trial commenced.2 acceleration criteria (SR Research). Further analysis was undertaken using in-house software developed in MATLAB . Saccade amplitude, latency, and overall direction were derived from the eye movement records for the first saccade in each trial. Amplitude was defined as the horizontal component of the distance between eye start and endpoint . Saccade latency is the interval between the onset of the target and the initiation of the saccade (in milliseconds). Direction was defined as the angular deviation of saccade direction (degrees) taken from the initial fixation position to final endpoint, in polar coordinates. Saccades were excluded from further analysis if saccade amplitude was less than 1 degree  and distance of the remote distractor from target location. In See In order to address whether a remote distractor impacts on the accuracy of saccade targeting indirectly by impacting on the latency of the saccade or whether it also has a more direct role to play in saccade accuracy, we first need to establish whether the remote distractor impacted on saccade latency. Given previous work, we would expect to see that the presence of a remote distractor will slow saccade latencies. In order to analyze the dependence of saccade latency on remote distractor presence and position, the following strategy was designed.The effect of remote distractor presence on median saccade latency was examined for target location both when the close distractor was present and, again separately, when it was not. The presence of remote distractor was not fully factorial, with neither “no remote distractor present” nor “remote distractor present at center” changing as function of remote distractor distance from fixation or remote distractor distance from target. Due to this, the decision was made to examine the no remote distractor present and remote distractor present at center condition separately from the analysis of the effect of remote distractor distance from fixation and target.F = 5.261, MSE = 23.992, p = 0.023, ηp2 = .467; close distractor present, F = 11.897, MSE = 27.5, p = 0.001, ηp2 = .665) but no effect of target location or interaction (Fs < 1). Follow-up contrasts of center versus no remote distractor and other versus no remote distractor showed differences in the effect of remote distractor location on saccade latency depending on close distractor presence. When the close distractor was not present, the saccade response slowed only when the remote distractor was shown in the other location  vs. none , F = 8.173, MSE = 61.667, p = 0.029, ηp2 = .577), whereas when a close distractor was present, then slower responses were found both when the remote distractor was shown in the center and other location  vs. none , F = 10.486, MSE = 68.119, p = 0.018, ηp2 = .636; other  vs. none, F = 19.601, MSE = 19.601, p = 0.004, ηp2 = .766). This pattern of analysis outcomes shows evidence for a remote distractor effect with saccade latencies being generally longer when a remote distractor was present.Separate two-way analyses of variance (ANOVAs) were performed depending on close distractor presence with target location (3 degrees and 6 degrees) and remote distractor presence  as factors. In both cases, this showed a main effect of close distractor presence . The average for each remote distractor distance condition was then computed. The resulting average remote distractor effect is shown in F = 23.079, MSE = 65.121, p < 0.001, ηp2 = .794; distance from target, F = 10.079, MSE = 105.735, p < 0.001, ηp2 = .627; all other ps > 0.083; close distractor present: distance from fixation, F = 10.05, MSE = 39.682, p < 0.003, ηp2 = .626; distance from target, F = 3.658, MSE = 180.88, p < 0.032, ηp2 = .379; all other ps > 0.365). Further contrasts examining the main effects show similar patterns regardless of close distractor presence. The effect of remote distractor distance from fixation was found to be largest when it was presented close to fixation but reduced to a similar level at greater distances  vs. 6  degrees of visual angle, F = 44.531, MSE = 19.674, p < 0.001; 6 vs. 9  degrees of visual angle, F = 2.385, MSE = 19. 862, p = 0.173; close distractor present: 3  vs. 6  degrees of visual angle, F = 17.593, MSE = 18.499, p = 0.006, ηp2 = .746; 6 vs. 9  degrees of visual angle, F < 1). On the other hand, the effect of remote distractor distance from the target was found to increase as its distance from the target increased, with the largest remote distractor effect found at the furthest distance from the target. This pattern was shown most clearly when the close distractor was not present but was also shown when it was present  vs. 45  angular degrees, F < 1; 135  vs. 90 angular degrees, F = 13.554, MSE = 25.09, p = 0.01, ηp2 = .541; 135 vs. 180  angular degrees, F = 7.08, MSE = 63.5, p = 0.037, ηp2 = .541; close distractor present: 90  vs. 45  angular degrees, F = 5.019, MSE = 5.138, p = 0.066, ηp2 = .455; 135  vs. 90 angular degrees, F = 6.583, MSE = 31.638, p = 0.043, ηp2 = .523; 135 vs. 180  angular degrees, F = 1.171, MSE = 31.638, p = 0.321, ηp2 = .163).There was found to be no main effect of target location and no interactions, but there were main effects of remote distractor distance from fixation and distance from target . The analysis also shows that the remote distractor effect increased the further its distance from the target. This supports other results showing evidence that the remote distractor also interacts directly with target activation by contributing to it when closer, thereby speeding up saccade responses .Having established that a remote distractor slows saccade latencies the extent of which depends on distance from fixation and distance from target, we now move on to examine the effect on saccade amplitude. In order to examine this, we derived the global effect for each condition separately in the following way. The average saccade amplitude when each target was presented alone was determined for each participant separately. These were taken as baselines, and the amplitudes for each condition were determined relatively as an index of close distractor influence  = 46.139, MSE = 243.133, p < 0.001, ηp2 = .885; remote distractor, F = 4.538, MSE = 15.132, p = 0.034, ηp2 = .431; interaction, F = 10.552, MSE = 49.45, p = 0.002, ηp2 = .638). Simple main effects analysis examining the effect of remote distractor presence for each target location shows that there were no significant differences when the target was at 3 degrees  vs. other  was marginal, p = 0.052; other ps > 0.532). When the target was at 6 degrees, on the other hand, significant differences were found between center  and none  and center and other . This shows that the global effect was not affected by remote distractor presence when the target was close to fixation but reduced when presented centrally when the target was presented at a greater distance.As with median saccade latencies, the global effect caused by the presence of the close distractor was examined across remote distractor condition. Initially, by presence: no remote distractor, central remote distractor, and “other” remote distractor position. A two-way ANOVA with target location and remote distractor presence was carried out. This showed main effects of both factors and an interaction  = 2.389, MSE = 44.285, p = 0.048, ηp2 = .285). Separate two-way ANOVAs were then carried out examining each target location separately. For the target at 3 degrees with a close distractor at 6 degrees, there were no main effects of either distance from fixation  = 2.67, MSE = 58.125, p = 0.110, ηp2 = .308) or distance from target  = 2.974, MSE = 36.489, p = 0.059, ηp2 = .331), but there was a significant interaction  = 2.555, MSE = 33.509, p = 0.036, ηp2 = .299). Direct contrasts were carried out to examine the cause of the interaction with distance from fixation considered across each distance from the target and vice versa, but no differences were of sufficient strength to show a significant difference. In the second two-way ANOVA, when the target was shown at 6 degrees along with a close distractor at 3 degrees showed no main effects or interaction  = 2.215, MSE = 87.222, p = 0.152, ηp2 = .27; distance from target and the interaction: Fs < 1).A three-way ANOVA was carried out next to examine the effect on the global effect of having a remote distractor also present that varied in its distance from fixation  and distance from the target  across target location. This showed a three-way interaction  = 46.805, MSE = 489.011, p < 0.001, ηp2 = .886), a main effect of remote distractor  = 4.087, MSE = 29.981, p = 0.044, ηp2 = .405), and two-way interactions between target location and remote distractor presence and latency and remote distractor presence  = 10.196, MSE = 105.502, p = 0.003, ηp2 = .63; F = 8.232, MSE = 38.797, p = 0.006, ηp2 = .578, all other ps > 0.177). Simple main effects analysis showed that the interaction between target location and remote distractor presence was due to remote distractor presence, provoking differences in global effect extent for each target location. There were found to be no significant differences in the global effect extent for targets at 3 degrees (ps > 0.053), whereas there was a significant reduction in global effect for targets at 6 degrees when the remote distractor was shown centrally compared with no remote distractor and when it was shown at the other position  vs. center , p = 0.012; center vs. other , p = 0.032). The second interaction between saccade latency and remote distractor presence was found to be due to their being no significant difference in global effect between short and long latencies when no remote distractor was present (p = 0.656) but both center and other showing significantly reduced global effects for long latency saccades compared with shorter latency responses . Further contrasts for short latency responses alone show no difference in global effect extent regardless of remote distractor presence (ps > 0.997). Longer latency responses show a significantly smaller global effect for centrally presented remote distractor conditions  vs. center , p = 0.015; center  vs. other , p = 0.023), with global effect extent being largest when no remote distractor was present and becoming less as the remote distractor was present in the other location and then in the central location.A three-way ANOVA was carried out with target location, saccade latency, and remote distractor presence. This showed a main effect of target location  = 107.636, MSE = 1,312.688, p < 0.001, ηp2 = .947; latency, F = 8.229, MSE = 465.348, p = 0.028, ηp2 = .578; distance from fixation, F = 4.328, MSE = 70.162, p = 0.038, ηp2 = .419). There was a three-way interaction between target location, distance from fixation, and distance from target  = 2.526, MSE = 87.358, p = 0.038, ηp2 = .296) and a significant four-way interaction  = 2.452, MSE = 38.341, p = 0.043, ηp2 = .29).To explore the effect of remote distractor distance from fixation and from target (an unpacking of the “other” position), a four-way ANOVA was carried out with target location, latency, remote distractor distance from fixation, and remote distractor distance from target as factors. This showed significant main effects for all factors except for distance from the target  = 2.554, MSE = 68.813, p = 0.037, ηp2 = .299, and a three way interaction, F = 2.368, MSE = 48.909, p = 0.050, ηp2 = .283). However, a simple main effects analysis to explore this further showed no comparisons that statistically survived further exploration, showing a lack of general pattern to the effect of remote distractor. The second three-way ANOVA examining the global effect when the target was shown at 6 degrees showed only a main effect of latency  = 13.469, MSE = 237.74, p = 0.01, ηp2 = .693), displaying a reduction in global effect for longer saccades regardless of remote distractor position .To examine this further, separate three-way ANOVAs were carried out for each target location. For the 3-degree target, there were no main effects, but there was a significant two-way interaction of remote distractor distance from fixation and remote distractor distance from target  = 29.929, MSE = 409.940, p < 0.001, ηp2 = .833, other ps > 0.108), whereas when a close distractor was present, main effects of target location and remote distractor presence were found  = 11.906, MSE = 137.27, p < 0.014, ηp2 = .665; remote distractor presence, F = 26.065, MSE = 283.524, p < 0.001, ηp2 = .813, other ps > 0.632). The main effect of target location showed that saccades evoked by targets at 20 degrees had longer latencies. Follow-up contrasts exploring the effect of remote distractor presence were conducted examining center versus no remote distractor, other versus no remote distractor, and center versus other. These show that the presence of both the central and other remote distractor induced a slowing of the saccade indicative of a remote distractor effect, which was largest when it was shown at the center  vs. none , F = 33.948, MSE = 1,432.071, p = 0.001, ηp2 = .850; other  vs. none, F = 51.820, MSE = 162.167, p < 0.001, ηp2 = .896; center vs. other, F = 19.176, MSE = 865.405, p = 0.005, ηp2 = .762; close distractor: center  vs. none , F = 30.167, MSE = 878.167, p = 0.002, ηp2 = .834; other  vs. none, F = 25.324, MSE = 44.071, p = 0.002, ηp2 = .808; center vs. other, F = 21.482, MSE = 778.905, p = 0.004, ηp2 = .782).As with M = 180 ms, SE = 5; 20-degree target, M = 171 ms, SE = 4), F = 22.099, MSE = 130.74, p = 0.003, ηp2 = .786; distance from fixation , F = 9.914, MSE = 767.331, p = 0.02, ηp2 = .623; all other ps > 0.052; with close distractor present: target location , F = 27.566, MSE = 266.424, p = 0.002, ηp2 = .821; distance from fixation , F = 24.545, MSE = 104.762, p = 0.003, ηp2 = .804; all other ps > 0.121).As with As with F = 15.17, MSE = 105.693, p = 0.001, ηp2 = .717). Direct contrasts revealed that the central remote distractor decreased the global effect  vs. center , F = 30.441, MSE = 101.958, p = 0.001, ηp2 = .835; center vs. other , F = 9.105, MSE = 147.509, p = 0.023, ηp2 = .603). In order to examine the effect of remote distractor distance from fixation and from target, the factor “other” was unpacked and a three-way ANOVA was carried out. This revealed only a main effect of target location, with targets 10 degrees from the horizontal evoking a greater global effect than the more widely separated target–close distractor pair  vs. 20-degree target , F = 16.46, MSE = 691.365, p = 0.007, ηp2 = .733, all other ps > 0.217).The global effect caused by the presence of the close distractor was examined across target location and remote distractor condition. A two-way ANOVA showed a significant effect of remote distractor presence only  = 14.119, MSE = 543.757, p = 0.009, ηp2 = .702; remote distractor presence, F = 13.785, MSE = 226.446, p = 0.001, ηp2 = .697, other ps > 0.185). The main effect of saccade latency is due to shorter latencies evoking a larger global effect . Further contrasts examining remote distractor presence show a significant reduction in global effect when the remote distractor was shown in the central position  vs. none , F = 26.301, MSE = 461.172, p = 0.002, ηp2 = 814; center vs. other , F = 8.091, MSE = 630.366, p = 0.029, ηp2 = .574).A three-way ANOVA was carried out to examine the global effect as a function of saccade latency (short and long) remote distractor presence  across target location. This showed a main effect of latency and remote distractor presence only  = 14.791, MSE = 466.786, p = 0.008, ηp2 = .711; saccade latency: F = 21.893, MSE = 745.702, p = 0.003, ηp2 = .785; target location by saccade latency, F = 8.859, MSE = 203.021, p = 0.025, ηp2 = .596, all other ps > 0.093). These reflect the overall pattern of global effect dependencies on target location and latency, with a larger global effect being shown when the target and close distractor are closer together and saccade latencies are short . Exploration of the interaction showed that the effect of latency on the global effect was found regardless of target–close distractor separation , F = 11.755, p = 0.014, ηp2 = .622; 20 degrees , F = 22.669, p = 0.003, ηp2 = .791). The cause of the interaction was a significant reduction in global effect for longer latency responses between targets at 10 degrees and 20 degrees but not the shorter latencies  vs. longer latency to 20-degree target global effect , F = 30.960, p = 0.001, ηp2 = .838). Taken together, these suggest that saccade targeting improves with a longer response time (as would be expected), and this improvement is greater when the target is more widely separated from the close distractor.Unpacking analysis of remote distractor positions across target location, saccade latency, remote distractor from distance from fixation, and remote distractor from target in a four-way ANOVA shows a main effect of target location and latency and an interaction between them . It may be the case that the differences found in the effect of remote distractor distance from fixation and from the target on saccade latency and amplitude reported here can be better captured when considered in terms of this ratio.In order to examine this, the remote distractor effect and global effect from r(26) = –.402, p = 0.042; r(26) = .078, p = 0.706; r(22) = –.846, p < 0.001; r(22) = –.846, p < 0.001; note that both r values for This analysis shows an effect of ratio on remote distractor effect, which mirrors that reported by r(13) = –.792, p = 0.001). There were no other correlations for the other target positions . Contrary to the relationship found for remote distractor effect with the stimuli eccentricity ratio, these results provide little support that the extent of the global effect is a function of the relative eccentricity of the stimuli.The same analysis was then carried out with global effect extent and showed little evidence supporting a relationship of global effect extent with the remote distractor to fixation and target to fixation ratio. Correlation analysis shows a relationship between ratio extent and global effect extent only in Overall, this pattern provides support for the position that the remote distractor primarily impacts activation at fixation and acts to interrupt disengagement. It increases saccade latency but does not directly impact on saccade amplitude. The results examining saccade latency and global effect suggest that saccade amplitude is more directly tied to interactions between the target and nearby distractors and saccade latency rather than any direct effect of the remote distractor.The examination of the relationship between saccade latency and global effect shows little to support the suggestion that the remote distractor impacted directly on the interactions between the target and closer distractor. Rather, the results support a more indirect interaction with the remote distractor impairing fixation disengagement, allowing saccade targeting processes to continue unabated and be accessed later and hence result in more accurate saccades. Both experiments presented here show accuracy improvements  Theglobal effect: Saccades are less accurate when made in the presence of a distractor close to a target.As would be expected, the presence of a distractor close to the target location was found to influence saccade accuracy as estimated based on saccade amplitude in The global effect is usually attributed to distributed spatial coding in the neural populations in the intermediate and deeper layers of SC, which respond to large and overlapping receptive or movement fields . These rOur results show that longer latency saccades show a general reduction in the global effect of the close distractor on saccade accuracy. The relationship between remote distractor, latency, and global effect shows little evidence that the position of the remote distractor plays a role in this; rather, the effect of latency seems to dominate. Of course, not all data support this conclusion. There are a large number of conditions here, and it is to be expected that a completely supportive set of data would throw up some unexpected findings. However, on the whole, data are either supportive of, or at least do not commonly run against, the conclusion that the global effect is dependent both on saccade latency and the relationship between closely located stimuli. They are not dependent on the presence of remotely located stimuli. This, along with previous reports , support(b) Theremote distractor:Saccade latency is slowed when made in the presence of a remote distractor,and the extent of this depends on its position.Results show an increase in saccade latencies in the presence of the remote distractor, the extent of which depended on its position . In geneIn terms of the models put forward in the Introduction, the results largely favor an indirect concept of saccade latency control, one in which the presence of a remote distractor impacts directly on saccade latency and only indirectly affects saccade accuracy. Our results support the suggestion that remote distractor operates through competitive interactions between a fixate system and a move system, which hampers fixation disengagement, thus slowing saccade initiation by increasing activation at fixation , that isIt also worth bearing in mind that the latency of a saccade also depends on many other target and distractor properties, task demands, and stimuli properties . For exa(c) Theglobal effectdoes not depend on theremote distractor effect: Saccade accuracy is a function of its latency rather than remote distractor presence.The relationship between remote distractor effect and global effect was examined by extracting the median split of saccade latency distributions for each remote distractor condition. Despite the overall change in the size of the remote distractor effect depending on remote distractor position, it was found that saccades executed with similar latencies  had similar accuracy . While this was not always the case across all conditions employed here, it was more often found than not and did not run in the opposite direction . These results suggest that distractors displayed close to a target impact directly on competing mechanisms involved in saccade target selection. Their presence contributes to the computation of the saccade landing position (and likely saccade trajectory control) via saccade-targeting mechanisms. These have been generally explained in terms of competitive and cooperative mechanisms via inhibitory and excitatory connections in SC detailed in the global effect section (a), whereas remote distractors play a more indirect role in the control of saccade targeting by impacting on fixation disengagement only and not at all on target–close distractor interactions as detailed in the remote distractor effect section (b). Thus, the impact of remote distractors is shown through longer latency saccades, which have more accurate landing positions than shorter latency responses.In both experiments presented here, we found that accuracy improvements associated with the presence of a remote distractor follow a similar pattern regardless of the presence of a close distracter and regardless of the position of the remote distractor. Previous literature had suggested two possible mechanisms to account for the remote distractor effect: The remote distractor increases saccade latency by invoking activity at fixation, making disengagement more difficult (put forward by Studies of the neural basis of target selection and saccade generation have suggested that the neurons coding potential targets compete as the sensory evidence that supports their location changes or accumulates over time . Models One potential problem with this explanation is that the latency effect caused by the remote distractor changes has been shown to be both dependent on distance from fixation and, on occasion, distance from target (here shown in The results presented in this article mirror those from our previous study  in whichIn conclusion, our results suggest that the involuntary increase in saccade latency induced by a remote distractor inhibits the initiation of a saccade by increasing activity at fixation, thereby interrupting fixation disengagement. This allows the target selection processes to proceed as they would ordinarily. Thus, the delay in saccade initiation induced by the presence of a remote distractor improves target localization by accessing target selection at a later, and more finely resolved, stage of development. We suggest that this may have implications in the design of any visual interface in which accuracy of visual information scanning is an important component. The results of this article suggest that sometimes adding more, potentially distracting information actually improves saccade accuracy with only a small cost in terms of the slowing of saccade initiation.


Diagnosing polycystic ovary syndrome (PCOS) during adolescence is challenging because features of normal pubertal development overlap with adult diagnostic criteria. The international evidence-based PCOS Guideline aimed to promote accurate and timely diagnosis, to optimise consistent care, and to improve health outcomes for adolescents and women with PCOS.International healthcare professionals, evidence synthesis teams and consumers informed the priorities, reviewed published data and synthesised the recommendations for the Guideline. The Grading of Recommendations, Assessment, Development, and Evaluation (GRADE) framework was applied to appraise the evidence quality and the feasibility, acceptability, cost, implementation and strength of the recommendations.This paper focuses on the specific adolescent PCOS Guideline recommendations. Specific criteria to improve diagnostic accuracy and avoid over diagnosis include: (1) irregular menstrual cycles defined according to years post-menarche; > 90 days for any one cycle (> 1 year post-menarche), cycles< 21 or > 45 days (> 1 to < 3 years post-menarche); cycles < 21 or > 35 days (> 3 years post-menarche) and primary amenorrhea by age 15 or > 3 years post-thelarche. Irregular menstrual cycles (< 1 year post-menarche) represent normal pubertal transition. (2) Hyperandrogenism defined as hirsutism, severe acne and/or biochemical hyperandrogenaemia confirmed using validated high-quality assays. (3) Pelvic ultrasound not recommended for diagnosis of PCOS within 8 years post menarche. (4) Anti-Müllerian hormone levels not recommended for PCOS diagnosis; and (5) exclusion of other disorders that mimic PCOS. For adolescents who have features of PCOS but do not meet diagnostic criteria an ‘at risk’ label can be considered with appropriate symptomatic treatment and regular re-evaluations. Menstrual cycle re-evaluation can occur over 3 years post menarche and where only menstrual irregularity or hyperandrogenism are present initially, evaluation with ultrasound can occur after 8 years post menarche. Screening for anxiety and depression is required and assessment of eating disorders warrants consideration. Available data endorse the benefits of healthy lifestyle interventions to prevent excess weight gain and should be recommended. For symptom management, the combined oral contraceptive pill and/or metformin may be beneficial.Extensive international engagement accompanied by rigorous processes honed both diagnostic criteria and treatment recommendations for PCOS during adolescence. Polycystic ovary syndrome (PCOS) is the most common endocrine condition affecting between 8 and 13% of women of reproductive age , 3 depenDiagnosis of PCOS during adolescence is both controversial and challenging due to the overlap of normal pubertal physiological changes  with adult PCOS diagnostic criteria. These challenges have been acknowledged in adult and paediatric consensus statements –10. SpecThe aim of the first International Evidence-based Guideline for the Assessment and Management of PCOS (‘the Guideline’) was to promote the accurate diagnosis of PCOS, optimal consistent care, the prevention of complications and improved patient health outcomes from adolescence to adulthood . The terThis paper focuses specifically on the adolescent recommendations from the Guideline and examines the evidence and rationale supporting these recommendations. We discuss the importance of avoiding missed diagnosis, of delayed, under- or over-diagnosis, and of evidence-based management to address PCOS symptoms in adolescents. Additionally, we expand on approaches to identify girls ‘at risk’ of PCOS but not yet diagnosed, including the need for future follow-up.Best practice evidence-based guideline development methodology was applied –18, as dAs previously outlined, a six-continent international advisory and project board, a multidisciplinary international guideline development group, and consumer and translation committees provided governance of the Guideline processes . The engImportantly, extensive engagement with stakeholders, including women with PCOS, and the multidisciplinary panels informed the Guideline research questions and highlighted the impact of PCOS across the lifespan. It also highlighted the importance of accurate and early diagnosis to optimise education, treatment and the prevention of longer-term complications.Categories of recommendations are described in Table The GRADE quality of the recommendation was determined by the GDG from structured consideration of the GRADE framework , includiThe quality of the evidence was categorised according to information about the number and design of studies addressing the outcome; judgments about the quality of the studies and/or synthesised evidence such as risk of bias, inconsistency, indirectness, imprecision and any other considerations that may influence the quality of evidence; key statistical data; and classification of the importance of the outcomes Table . The quaThe GRADE recommendations affirm that the quality of evidence is a continuum; any discrete categorisation involves a degree of arbitrariness . NeverthFollowing completion of these stages, the draft guideline was distributed to all 37 partnering societies, including paediatric endocrine societies, and to specifically formulated special interest groups in each society providing review and feedback. Evidence was required to support any requests for recommendation modifications. The feedback and responses are available under the Guideline supporting documents (Public Consultation Comments and Developer Responses) . These wThe full Guideline is summarised by Teede et al.  and provThis paper examines the recommendations related to adolescents, including diagnosis, assessment and treatment of emotional wellbeing, and lifestyle and pharmacological treatment for non-fertility indications. We do not cover assessment and treatment of infertility, which can be found in the Guideline. The recommendations provided here include evidence-based recommendations, consensus recommendations and practice points, with most of the evidence-based recommendations in adolescents being related to the treatment of PCOS.Key changes within the Guideline recommendations include the elimination of specific unnecessary tests and the importance of identifying adolescents ‘at risk’ of PCOS. The changes also aim to avoid misdiagnosis or delayed, under- or over-diagnosis. Adolescent consensus recommendations for PCOS diagnosis are based on the best-quality available evidence and its limitations.Irregular menstrual cycles and ovulatory dysfunctionClear definition of irregular menstrual cycles according to time post menarche was the first strong consensus recommendation as there was limited evidence to formulate an evidence-based recommendation Table . When irWhilst the literature search on the question “At what time point after the onset of menarche do irregular menstrual cycles indicate ongoing menstrual dysfunction?” revealed 36 studies , these did not meet the selection criteria for the systematic review framework as they did not report on the duration of menstrual irregularity and/or diagnosis of PCOS. Hence, relevant information from diverse studies was extracted from the literature in a narrative review format. This informed the consensus recommendation based on the evidence on menstrual cycles in healthy girls –32.This recommendation was also informed by previous available guidelines, multidisciplinary expertise, consumer perspectives, international feedback, and the potential for both over and delayed diagnosis when assessing this diagnostic feature in PCOS. This recommendation considered available data on normal physiological events during adolescence such as menstrual cycle interval variations according to time post menarche and the fact that anovulation is a common physiological event in early post menarcheal years –34. Phys2.HyperandrogenismBiochemicalAnother practice point from the Guideline is that ovarian dysfunction can still occur in adolescents or women with regular menstrual cycles and, if anovulation is suspected, serum progesterone levels can be measured to confirm it.Calculated free testosterone, free androgen index or bioavailable testosterone should be used to assess biochemical hyperandrogenism in the diagnosis of PCOS. This was the only strong evidence-based recommendation of low GRADE quality for biochemical testing for hyperandrogenism, including one study in adolescents . This reThere were two conditional evidence-based recommendations of low GRADE quality derived from studies in adult women that reported on the diagnostic accuracy of different hormone markers to detect PCOS –41. One b.Clinical hyperandrogenismImportant practice points included avoiding the assessment of biochemical hyperandrogenism in women on hormonal contraception . Careful interpretation of androgen levels is needed by considering the reference ranges of the laboratory used and normal values derived from a well-phenotyped population, including age and pubertal-specific stages. Additionally, the guidelines noted that, when clinical hyperandrogenism is not detected, biochemical hyperandrogenism should be determined using appropriate high-quality assays.A comprehensive history and physical examination should be completed for symptoms and signs of clinical hyperandrogenism, which in adolescents include severe acne and hirsutism; this was the first strong consensus recommendation because no evidence-based recommendation in relation to clinical hyperandrogenism was available. This recommendation was based on the fact that mild comedonal acne is common in adolescent girls but moderate or severe comedonal acne  in early puberty or moderate to severe inflammatory acne during the peri-menarcheal years is uncommon (less than 5%) and is more likely to relate to clinical hyperandrogenism –45. In aStandardised visual scales are preferred when assessing hirsutism, such as the modified Ferriman–Gallwey score (mFG), with a level ≥ 4–6 indicating hirsutism, depending on ethnicity, and acknowledging that self-treatment is common and can limit clinical assessment. This second strong consensus recommendation was based on the most common visual assessment tool, the mFG , 47. ThiHealth professionals should be aware of the potential negative psychosocial impact of clinical hyperandrogenism. Reported unwanted excess hair growth should be considered important, regardless of apparent clinical severity. This third consensus recommendation was based on a systematic review of quality of life and included a study with 97 adolescents with PCOS .Pelvic ultrasound for PCOS diagnosisPelvic ultrasound should not be used for the diagnosis of PCOS in those with a gynaecological age of < 8 years (< 8 years post menarche) due to the high incidence of multi-follicular ovaries in this life stage. This was a strong consensus recommendation and not an evidence-based recommendation because reliable data regarding longitudinal ovarian morphology are limited. Additionally, the specific 8 years post-menarche cut-off was based on a consensus recommendation by the GDG, consulted with international societies and refined after international peer review. The recommendation also considered 15 studies evaluating the most effective ultrasound criteria to diagnose PCOS in women using various measures and thresholds for ovarian volume and follicle number. However, adult PCOS criteria have been derived from transvaginal ultrasounds, which should be avoided in females not yet sexually active. Three of these 15 studies included adolescents (181 with PCOS and 137 controls), two of which used trans-abdominal ultrasound and one used trans-rectal ultrasound (68 with PCOS and 26 controls) , 55, 56.2.Anti-Müllerian hormone (AMH)Furthermore, a polycystic ovarian morphology may represent a marker of PCOS or may be normal in young women. A population study showed a higher prevalence of polycystic ovarian morphology in very young women without menstrual irregularity and hyperandrogenism  . A smallAMH is a polypeptide of the transforming growth factor-β family secreted by granulosa cells of the pre-antral and small antral ovarian follicles. Hence, AMH has been evaluated in the diagnosis of PCOS, especially in situations where pelvic ultrasound is not feasible , 64.AMH levels should not be used as an alternative for the detection of polycystic ovarian morphology or as a single test for the diagnosis of PCOS. This evidence-based recommendation  was based on 29 studies, including a systematic review  evaluatiAlthough serum AMH levels in adolescents and adult women with both polycystic ovarian morphology and PCOS are significantly higher than in those without these features in all the studies included , 65–70, There is emerging evidence that, with improved standardisation of assays and established cut-off levels based on large populations of different ages and ethnicities, AMH assays will become more accurate in the detection of polycystic ovary morphology.CYP21A2) gene, is the most common type of NCAH seen. The prevalence of NCAH is reported as 1 in 1000 but is even more frequent in certain ethnic groups [The diagnosis of PCOS is a diagnosis of exclusion – all other aetiologies that can cause menstrual irregularities and/or hyperandrogenism must be excluded, regardless of the fact that some aetiologies are less common in adolescents. The most important cause of amenorrhoea in a sexually active adolescent is pregnancy. Menstrual irregularity alone could be due to gonadotropin deficiency caused by functional hypothalamic amenorrhoea, secondary deficiency due to any systemic cause, or to a primary gonadotropin defect. Many conditions can lead to hyperandrogenism; the most common albeit rare condition (outside of PCOS) is non-classic congenital adrenal hyperplasia (NCAH), characterised by a marked elevation of androgen levels. Deficiency of 21-hydroxylase, due to mutations in the 21-hydroxylase . The use of psychiatric agents may worsen obesity and should be used with caution. According to recent but limited data, cognitive behavioural therapy may be beneficial in terms of weight loss and symptoms of anxiety/depression in both adolescents and women with PCOS; however, the data merit further confirmation . SimilarLifestyle interventions  should be recommended in all those with PCOS and excess weight to achieve reductions in weight, central adiposity and insulin resistance. This was a conditional and low GRADE quality evidence-based recommendation for lifestyle interventions. Weight gain is the number one priority expressed by young women with PCOS . AdditioRecommendations for the pharmacological treatment of adolescents with PCOS include the use of the combined oral contraceptive pill (COCP) and/or metformin in those with a clear diagnosis or in adolescents deemed ‘at risk’ of PCOS for the management of symptoms. These recommendations were made following review of the evidence for the efficacy of COCP, metformin and antiandrogens alone or in combination for the management of hormonal or clinical PCOS features in adolescents and adult women with PCOS. These recommendations were also based on the need for interventions and longitudinal follow-up to address PCOS symptoms in adolescents who are ‘at risk’ but do not yet meet full diagnostic criteria. Whilst diagnosis is recommended where both irregular menstrual cycles and hyperandrogenism are present, when only one of these is detected, symptom reduction and future reassessment was deemed preferable to prematurely labelling or over-diagnosing an adolescent as having PCOS. This approach addressed the conundrum that healthcare professionals involved in the medical management of adolescent girls face in potentially over-diagnosing normal pubertal girls, girls with transient menstrual irregularities, or under-diagnosing girls who will likely evolve to a PCOS phenotype. Additionally, medications such as COCP are commonly prescribed in adolescents for contraception and other indications.The following practice points should apply to all medications used in PCOS to inform adolescents and their families and to guide health professionals when considering or recommending pharmacotherapy: (1) an individual’s personal characteristics, preferences and values are of importance when recommending pharmacotherapy; (2) the benefits, adverse effects and contraindications in PCOS and in general populations should be considered; and (3) the fact that COCPs, metformin and other pharmacological treatments are generally ‘off-label’ for the treatment of PCOS should be discussed where allowed, with adolescents and their families to consider the evidence and potential side effects for each option. However, off-label use is predominantly evidence based for other conditions and is allowed in many countries. Off-label prescribing occurs when a medication is prescribed for an indication, a route of administration, or a patient group that is not included in the approval product information document for that medication by the relevant regulatory body. Prescribing off-label is unavoidable and most commonly means that there has not been a submission to request evaluation of the indication or of that patient group for any given medication. The latter is very common in paediatric practice. Off-label use of metformin in adolescents with PCOS has been described .Holistic approaches are required and pharmacotherapy in PCOS should be considered alongside education and counselling, lifestyle, and other options, including cosmetic therapy. Of note, the Guideline did not review the evidence on cosmetic therapies for hirsutism or non-hormonal therapies for acne.COCP (oestrogen and progestin preparations)The COCP alone should be considered in adolescents with a clear PCOS diagnosis or could be considered in those deemed ‘at risk’ but not yet diagnosed with PCOS in both groups for the management of clinical hyperandrogenism and/or irregular menstrual cycles. These two conditional and low GRADE quality evidence-based recommendations were based on limited evidence in adolescents, including two RCTs , 97 and 2.Combined COCP and metforminSpecific types or doses of progestins, oestrogens or combinations of COCP cannot currently be recommended due to insufficient data among women and adolescents with PCOS. Practice should be informed by general population guidelines. This was a conditional and low GRADE quality evidence-based recommendation in relation to the use of specific types of COCP and was based on evidence of COCP use in the general population and World Health Organisation Guidelines as there were no trials evaluating this in women or adolescents with PCOS . A practice point for this section includes that COCPs with 35 μg of ethinylestradiol and cyproterone acetate should not be used as first-line therapy due to the absence of evidence of greater efficacy and the presence of higher risks, including deep venous thrombosis. All COCPs are associated with an increased risk of deep venous thrombosis, but the risk is higher with COCPs containing 30–35 μg of ethinylestradiol and gestodene, desogestrel, cyproterone acetate or drospirenone when compared to the COCP containing 30 μg of ethinylestradiol with levonorgestrel, norethisterone (norethindrone) or norgestimate. Lower-risk COCP preparations should be recommended as first-line therapy .2.Combi2 where the COCP and lifestyle changes do not achieve desired goals. This was a conditional and low GRADE quality evidence-based recommendation based on a single RCT including 79 adolescents followed over 6 months [3.MetforminThe COCP in combination with metformin could be considered in adolescents with PCOS and a BMI > 25 kg/m6 months  and supp6 months . Althoug4.AntiandrogensMetformin in addition to lifestyle interventions could be considered in adolescents with a clear PCOS diagnosis or with symptoms of PCOS before a diagnosis is made. This was a conditional low GRADE quality evidence-based recommendation based on a meta-analysis evaluating the effect of metformin versus placebo in women with PCOS. This meta-analysis included 20 RCTs (one RCT in adolescents ) and hign = 14) that evaluated finasteride, but it lacked a direct comparison between groups and did not report side effects [Recommendations suggest the use of the COCP alone with cosmetic therapy for at least 6 months prior to considering antiandrogens. Where COCPs are contraindicated or poorly tolerated, and in the presence of effective forms of contraception, antiandrogens could be considered to treat hirsutism or androgen-related alopecia. The use of effective contraception is essential due to the teratogenic potential of antiandrogens and their impairment of external genital development in male foetuses. This conditional evidence-based recommendation of very low GRADE quality was based mainly on studies in adult women and included the use of flutamide, finasteride or spironolactone alone or in combination with a diet intervention –110. The effects . There w effects .There was no evidence-based recommendation as the systematic search did not identify any studies evaluating any type of models of care. A practice point for this section recommended that interdisciplinary care needs to be considered for those with PCOS where appropriate and available. Primary care is generally well placed to diagnose, screen and coordinate interdisciplinary care considering that women with PCOS face multiple health problems across the lifespan and see multiple health professionals. Differences in healthcare systems and settings, including rural or urban areas and developing countries, as well as primary care system differences in developed countries were acknowledged. The coordination of interdisciplinary care also allows the screening and management of comorbidities associated with PCOS. Four studies have described models of care in adult women with PCOS in terms of barriers, enablers and patient/healthcare provider satisfaction –116. TheThe Guideline recommendations are the result of extensive international engagement and rigorous, evidence-based processes aligned with international best practice. Whilst the Guideline provided recommendations for adult women and adolescents, this paper summarises the evidence and recommendations relating to adolescents as defined by the World Health Organisation, namely as females aged between 10 and 19 years. It also addresses recommendations relating to gynaecological age, and specifically 8 years post menarche, and the use of pelvic ultrasound in PCOS diagnosis as well as recommendations relating to the re-evaluation of girls that do not meet the diagnostic criteria in adolescence but are considered ‘at increased risk’ of PCOS. There are few evidence-based recommendations relating to adolescents in the Guideline highlighting the limited evidence in this age group. Limitations within studies included poorly defined study populations, challenges in diagnosis around puberty, the different diagnostic criteria used and lack of availability of data for “time post menarche” of subjects included in most of the studies. Research recommendations highlighted these gaps and emphasised the need for greater high-quality research of PCOS during adolescence.The controversies of diagnosing adolescents with PCOS are compounded by a reliance on 95th centiles cut-off points for menstrual cycles or hirsutism, which are not diagnostic of pathology and are derived from highly variable populations and relatively poor-quality data. Additionally, there is a lack of available outcome data on the long-term health impact of PCOS in conditions such as diabetes. Moving forward, there is a need for greater research into how the confluence of diagnostic factors can be separated out to establish a clear clinical syndrome and how these factors predict short- and long-term adverse health outcomes, with studies in this area now underway.Diagnosis relied on strong consensus recommendations and aimed to avoid delayed, under- or over-diagnosis of PCOS during adolescence. The criteria included a clear definition of irregular menstrual cycles according to time post menarche and clinical hyperandrogenism, including severe acne and hirsutism and/or biochemical hyperandrogenism, after exclusion of other conditions that mimic PCOS. The use of pelvic ultrasound for diagnosis in females less than 8 years post menarche should be avoided due to the overlap with normal pubertal physiology, the lack of specificity of polycystic ovarian morphology for PCOS diagnosis in this age group and the avoidance of transvaginal ultrasound in those not yet sexually active. AMH was not recommended for a diagnosis of PCOS due to a lack of evidence and apparent lack of specificity. Additionally, special considerations need to be given when assessing adolescents for PCOS; importantly, adolescents’ and their family’s preferences and cultural norms should be included in decision-making.The Guideline recommends recognising adolescents ‘at risk’. The potential for over-diagnosis is minimised by the narrowed diagnostic criteria; however, there is also respectful recognition of the need to avoid delayed and missed diagnosis and a poor diagnosis experience. As such, reassessment is recommended at 3 years post menarche in relation to menstrual irregularity and at 8 years post menarche in relation to the use of pelvic ultrasound to review the polycystic ovarian morphology. This approach recognises a group of adolescents with only one symptom cluster as ‘at risk’ of PCOS, enabling a focus on symptom management whilst avoiding premature labelling and recommending subsequent follow-up.Following a diagnosis of PCOS, healthcare providers should be aware of the likely increased risk of anxiety and depressive symptoms that will benefit from appropriate screening and management. General Guidelines for the assessment and management of these conditions in children and adolescents should be used as there is no specific data referring to adolescent girls with PCOS. Emotional wellbeing is a poorly evaluated area in adolescent girls with PCOS. Clearly, more research is needed to determine the pathophysiology, whether the symptoms start early in adolescence, the optimal treatment and whether these symptoms will influence engagement with management strategies as observed in adult women with PCOS.Multicomponent lifestyle interventions  are the first line of treatment, vital in preventing excess weight gain as well as targeting weight management from adolescence. Pharmacologic management for non-fertility indications in PCOS included evidence-based recommendations with data in adolescents. The treatment options for girls considered to be ‘at risk’ of PCOS and those with a clear diagnosis of PCOS include the COCP and metformin. The COCP is recommended for the management of irregular menstrual cycles and/or hyperandrogenism, whereas metformin alone or in combination with the COCP is recommended to manage weight and metabolic comorbidities.Recognition of a group who are at risk of PCOS but do not meet all diagnostic criteria allows the treatment of adolescent girls with symptoms yet acknowledges the need for future reassessment at or before 8 years post menarche should PCOS features persist. It also highlights the need for appropriate transition from paediatric to adult care.The strengths of the Guideline include the extensive international engagement and rigorous processes aligned with international best practice, including systematic evidence review and synthesis  and European Society of Human Reproduction and Embryology (ESHRE) criteria), extensive consultation and partnership at all stages with those affected by PCOS and interdisciplinary health professionals and experts, GDG meetings, integration and response to feedback from international consultation across 37 societies, and the latest updates in key evidence raised during public consultation.A limitation of the Guideline is the lack of a formal analysis of cost effectiveness or economic feasibility; however, the potential impact of cost was considered in the GRADE process. Furthermore, the Guideline followed priorities that were mostly derived from women with PCOS, not adolescents with PCOS (this will be addressed in the next update of the guideline), whilst the group of multidisciplinary healthcare professionals included paediatricians.Overall, the evidence considered by the Guideline around adolescence was of low to moderate quality and highlighted the need for future high quality research, including longitudinal studies, to identify the early predictors and natural history of PCOS in adolescents and to allow for timely diagnosis and appropriate management during adolescence and in the transition to adult care. Most importantly, the Guideline refined the diagnostic criteria based on the best available evidence and consensus input from all stakeholders and should allow a more accurate diagnosis of adolescent girls with PCOS.The aim of the comprehensive research translation programme that accompanied the release of the Guideline was to improve the timely and accurate diagnosis and management as well as to augment the consistent implementation of evidence-based care worldwide. A recent Cochrane review demonstrates that obtaining patient-provided information improves adherence with guidelines and evidence-based care . Hence, Extensive international engagement and rigorous processes have refined the diagnostic criteria to avoid the delayed, under- or over-diagnosis of PCOS and have clarified the treatment of PCOS during adolescence. The Guideline is supported by a multifaceted international translation programme with co-designed resources to educate health professionals and empower adolescents and women with PCOS, including an integrated and comprehensive evaluation programme. However, the evidence synthesis and guideline process highlighted the need for further research on PCOS during adolescence.


The splenic cyst is a rare disease with unknown etiology. The inner wall of the cyst has lining epithelium. The cyst can be unilocular or multilocular. According to pathology, it can be divided into four types: epidermoid cyst, dermoid cyst, cystic lymphangioma, and cystic hemangioma. Ultrasound examination is often the first choice for splenic cysts because of its nonradiation, low cost, and convenient examination. The images are mostly cystic masses with clear borders and dark areas without echoes, after the detection of splenic space-occupying lesions by ultrasonography, CT, and MRI. Here, we report robot-assisted partial splenectomy for a splenic cyst. Imaging diagnosis of abdominal CT enhancement: the cystic space-occupying of the spleen is considered. We should improve the preoperative examination and exclude operative contraindications. During the operation, there was about 8 cm of the upper pole of the spleen, and the boundary was clear. There was no obvious abnormality in the exploration of the abdominal viscera. The operation was successful. The operative time was 115 minutes, and the blood loss was 20 ml. On the first day after the operation, the patient took a liquid diet. The time of first anal exhaust was on the second day after operation. The patient was discharged at the fourth day. Postoperative pathology revealed epidermoid cyst. The therapy strategy of the splenic cyst is ambiguous. Better understanding of the splenic segmental anatomy and surgical skills has made minimally invasive partial splenectomy a preferred treatment for splenic cysts. In this paper, we report a case of splenic epidermoid cyst managed successfully by robot-assisted partial splenectomy. Partial splenectomy includes irregular partial splenectomy and regular partial splenectomy. The latter is based on the anatomical basis of sectional blood supply in the spleen. After pretreatment of the two- or three-grade splenic pedicle ligature, the corresponding splenic segment, splenic lobe, or half spleen can be resected according to the ischemic line of the spleen surface. Regular splenectomy is much more difficult than total splenectomy and irregular splenectomy. The main technical points of regular partial splenectomy are as follows: 1) fine dissection and ligation of the second and third splenic pedicle vessels close to the spleen hilum, where each bundle is as small as possible, while observing the spleen blood circulation; (2) moving 0.5-1.0 cm to the healthy side in the relatively nonvascular plane of the spleen and cutting the spleen from a part shallow to a deep part with an ultrasound knife, ligating the blood section ; it can be applied to various bleeding measures such as bleeding on the section, wet dressing with warm saline gauze, 8-word suture, adhesives, microwave, and radiofrequency; and (3) peritoneum or splenic capsule transplantation [ fine disIn recent years, laparoscopic splenectomy has gradually replaced open splenectomy as an important method for the treatment of splenic cysts with the accumulation of laparoscopic surgery experience and the improvement of technology. Compared with the operation time and prognosis, laparoscopic splenectomy has less bleeding and shorter hospitalization time. Compared with laparoscopy, the 3D operation vision and mechanical arm of Da Vinci robotic surgery system overcome the limitation of flexibility in vision and operation instruments, which can greatly enhance the controllability, stability, and accuracy of surgery. Although its application in clinical practice, especially in splenic surgery, is in its infancy, it has been reported that robot-assisted partial splenectomy significantly reduces the anatomical time of the splenic pedicle and overcomes the difficulty of hemostasis and spleen translocation in traditional laparoscopic partial splenectomy and reduces the amount of bleeding . In a reThe patient, a 28-year-old woman, was admitted to the Department of General Surgery, Sir Run Run Show Hospital, for half a month of splenic mass found by physical examination. Physical examination showed clear, spiritual, nonyellow skin and sclera, no swelling of superficial lymph nodes, no difference between cardiopulmonary examination, abdominal soft, no obvious mass, normal bowel sounds, no tenderness and rebound pain in the whole abdomen, negative mobile voice, negative Murphy's sign, negativity of renal percussion pain, no edema in both lower extremities, and negative pathological signs. In the imaging diagnosis of abdominal CT enhancement, the cystic space-occupying lesion of the spleen is considered . We shouThe patient was placed in an incomplete right lateral decubitus position. A pneumoperitoneum of 12 mmHg was established using a Veress needle through a left paraumbilical incision, and an optical port (5- to 12-mm trocar) was introduced afterward. Under visual control, two robotic 8 mm trocars were placed in the left hypochondriac region and the epigastrium. An additional 5- to 12-mm accessory port was placed in the left lumbar region on the middle axillary line for the side assistant surgeon. The surgical cart with the robotic arms was positioned on the patient's left side at a 45° angle to the table's longitudinal axis.As reported by the European Association for Endoscopic Surgery clinical practice guidelines, in preoperative imaging, splenomegaly was defined with a maximum splenic diameter of more than 15 cm .Dissection was performed with the robotic EndoWrist® Fenestrated Maryland Bipolar Cautery on the left hand and HarmonicTM Curved Shears on the right hand. The trocar position is presented in The specimen was removed in an Endo Catch™ II 15 mm specimen pouch through the accessory port incision. Hemostasis on the transection surface of the splenic remnant was completed in two cases with a TachoSil® hemostatic sponge.The drain tube was removed after 1 day. The patient was discharged on postoperative day 4 with no postoperative complication. And the final pathological findings showed epidermoid cyst. We performed a follow-up visit of 1 year, and no recurrence or abnormal platelet count was found.With the popularization and application of ultrasound, abdominal CT and magnetic resonance imaging in clinical practice, and the awareness of public health examination, the number of splenic cysts diagnosed in recent years has increased significantly. According to the etiology, splenic cysts can be divided into two categories: nonparasitic cysts and parasitic cysts. In nonparasitic cysts, splenic pseudocysts and splenic true cysts can be classified according to the presence or absence of lining epithelium in the cyst wall. Pseudocysts are more common. Most of them are formed after trauma secondary to subcapsular hematoma of the spleen. They are also called secondary splenic cysts. The wall of the cyst is only a fibrous tissue without lining epithelium. The cyst contains blood or serous fluid. The splenic cyst is a rare disease with unknown etiology. The inner wall of the cyst has lining epithelium. The cyst can be unilocular or multilocular. According to pathology, it can be divided into four types: epidermoid cyst, dermoid cyst, cystic lymphangioma, and cystic hemangioma. Ultrasound examination is often the first choice for splenic cysts because of its nonradiation, low cost, and convenient examination. The images are mostly cystic masses with clear borders and dark areas without echoes. After the detection of splenic space-occupying lesions by ultrasonography, CT, MRI, and other imaging examinations can further understand the size, shape, and type of splenic cysts. The number, characteristics of cyst cavity and wall, and adjacent relationship with surrounding organs are of great value.Surgical treatment is the main method for a splenic cyst. Clinically, nonparasitic splenic cysts less than 2.0 cm in diameter and without any symptoms can be observed regularly. When the diameter of splenic cyst is more than 5.0 cm, the follow-up of the outpatient department increases rapidly or complications such as compression of surrounding organs, rupture, and infection occur; timely surgical treatment is advocated. At present, the main surgical methods depend on the size and location of the cyst, including fenestration and drainage of splenic cyst, total splenectomy, splenectomy with preservation of accessory spleen, hemisplenectomy, and partial splenectomy. As the main immune organ of the human body, the spleen plays the role of regulating immune function and clearing senile blood cells. If total splenectomy is performed, the chance of overwhelming postsplenectomy infection will increase, and complications such as platelet elevation and venous thrombosis will be more likely to occur. Therefore, it is advocated that surgery should preserve the function of the spleen as far as the patient is concerned, such as the presence of a cyst on the upper pole of the spleen or the presence of a cyst at the lower part of the spleen. The diameter of the spleen is less than 50% of the total volume of the spleen. The partial splenectomy or partial splenectomy can be performed according to the specific conditions of the patients.Splenic cysts are classified into true cyst (primary) and pseudocysts (secondary), on the basis of the presence of an epithelial lining inside the cyst . SplenicIn recent years, mounting splenic cysts are diagnosed due to the development of modern diagnostic technology. And multiple surgical approaches have been applied in the therapy, especially spleen preserving minimally invasive procedures based on splenic vascular anatomy, which can preserve more than 25% of splenic parenchyma . PartialLaparoscopic splenectomy has gradually replace open splenectomy for its safety and minimal invasion. However, a systematic review involving 20 articles elucidated that no statistical differences were observed in many parameters, such as blood loss, time of drainage removal, and incidence of complications between laparoscopy and laparotomy [As a new mini-invasive technique, robotic surgery has shown its superiority in many disease therapies, which is gradually applied in splenology. A robotic surgery system allows for an exquisite dissection due to the high definition and stereoscopic vision and tremor reduction along with perfect maneuverability. These characteristics make it possible to perform complex and advanced surgical procedures. Some surgeons have published their experience with the robot-assisted PS , 10.With regard to the laparoscopic technique, most data focus on laparoscopic decapsulation, while in the literature the feasibility of laparoscopic partial splenectomy for splenic cysts has been well established , 11.A total of 21 cases of the nonparasitic splenic cyst were compared with laparoscopic partial splenectomy. The results showed that intraoperative hemorrhage in the robotic group was significantly less than that in the laparoscopic group. There were no postoperative complications in the robotic group, while 2 cases occurred in the laparoscopic group . In this case, the robot-assisted regular partial splenectomy was performed to prevent the patients from having low comprehensive immunity and more precise excision of the lesion. The procedure was smooth, the field of vision was clear, and the bleeding was less. In practice, it is found that the magnification effect of the robotic surgical system is stronger than that of laparoscopy. It has more advantages for the fine anatomy of the two-degree splenectomy vascular pedicle and can better perform regular partial splenectomy. As a new minimally invasive technique, it has more room for development, though there are still many problems. With problems and controversies, but with the continuous updating of surgical instruments and equipment, the continuous progress of basic and clinical research of spleen, the accumulation of surgical experience, and the improvement of surgical skills, this method of operation will be applied more and more.While it might be difficult technically to manage partial laparoscopic splenectomy and bleeding from the cut edge of spleen, it can be performed safely with the understanding of the spleen's vascular anatomy. The spleen is divided primarily into two lobes on the basis of vascular distribution. In our case, the demarcation line was formed when the upper lobe vessels were ligated. It is easily accessible without loss of blood for partial splenectomy.The robot-assisted technique may be the most appropriate for partial splenectomy, as it enables great dissection. The value of partial splenectomy for selected benign diseases in preserving the immune function has been well demonstrated. The comparison of complete and partial laparoscopic splenectomy in children They found that laparoscopic partial splenectomy was as successful as complete laparoscopic splenectomy, although more pain, longer time for oral intake, and longer hospital stay were present in the partial category. The technical challenge associated with partial laparoscopic splenectomy is another factor to consider. The robotic approach is able to overcome the limitations inherent in laparoscopy, especially for hilar dissection and vascular isolation. A partial splenectomy requires effective splenic branch dissection, and in our experience, 3D vision robotic technology and the wrist-like equipment allow good vascular control. In comparison, by applying pledgets, the hemostasis of the cut segment after the parenchymal transection can be performed well. The robot can also be helpful for this challenging task.In conclusion, robot-assisted PS is a promising option for the treatment of splenic cysts, which provides an alternative method to the standard laparoscopic approach especially in difficult cases. However, more series and prospective studies are required to further compare the effects of laparoscopic and robotic approaches.