One in six Americans suffers from hearing loss. While treatment with amplification is possible for many, the acceptance rate of hearing aids is low. Poor device fitting is one of the reasons. The hearing aid fitting starts with a detailed hearing assessment by a trained audiologist in a sound-controlled environment, using standard equipment. The hearing aid is adjusted step-by-step, following well-described procedures based on the audiogram. However, for many patients in rural settings, considerable travel time to a hearing center discourages them from receiving a hearing test and treatment. We hypothesize that hearing assessment with the patient’s hearing aid can reliably substitute the hearing test in the clinic. Over-the-counter hearing aids could be programmed from a distance and fine-tuned by the hearing aid wearer. This study shows that a patient-controlled hearing assessment via a hearing aid in a non-clinical setting is not statistically different from an audiologist-controlled hearing assessment in a clinical setting. The differences in hearing obtained with our device and the Gaussian Process are within 3 dB of the standard audiogram. At 250 Hz, the sound delivery with the hearing aid used in this study added an additional reduction of sound level, which was not compensated. Estimates show that less than 20% of these individuals wear hearing aids6. Several factors lead to the high incidence of untreated hearing loss, including the high cost of devices and limited access to professional services8. Previous research has shown that untreated hearing loss correlates with accelerated cognitive decline, resulting in dementia, anxiety, and depression16. To better treat deaf or hard-of-hearing individuals, the government has removed some regulations for distributing hearing aids17. This regulatory change will ease patient access to hearing aids. However, it will also create challenges for hearing aid providers because the devices should be programmable outside of a clinical setting, at the patients’ homes, for example, through telemedicine. Enabling hearing aid access may also increase the patients’ acceptance and use of the devices. The proposed changes for over-the-counter hearing aids also require changing the standard processes for self-fitting18. This clinical study is an effort to provide a method for patients to adjust their hearing aids outside of a clinical setting using their own hearing aids. Virtually all modern hearing aids are fitted using the patient’s audiogram, determined by pure tone audiometry in a controlled clinical setting, by trained personnel, using instruments specifically designed for this purpose22. The audiologist determines the patient’s hearing by incrementally approximating the lowest intensity for a pure tone that can still be perceived at a set of standard frequencies ranging from 125 Hz to 8 kHz23. At each frequency, a staircase “up 5 dB—down 10 dB” approach is used24. It takes an audiologist 10–15 min to complete an audiogram. Hearing aid amplification settings can be assigned using well-known prescriptive standards such as National Acoustic Laboratories (NAL), Desired Sensation Level (DSL), or others using the patient's audiogram26. Despite pure-tone audiometry is an indispensable component of the hearing aid fitting process, eminent limitations of standard audiometry exist:A trained professional must administer a hearing test with specialized equipment, including a sound booth.A statistical estimate of confidence in the result is generally not available with standard audiometry.Standard test procedures do not use all information available in the data by assuming that the hearing thresholds at different frequencies are uncorrelated, leading to redundant measurements to achieve the desired accuracy and longer testing times.Restricting the stimuli to a fixed set of standard frequencies requires additional assumptions to estimate the threshold at other frequencies, particularly when fitting hearing aids with a large number of channels.Hearing loss is the third most common chronic medical condition in the United States, affecting over 30 million Americans, including half of the adults between 70 and 79 years oldin-situ pure-tone audiometry with Bayesian statistical inference. With this, we name “In-situ hearing testing” as the use of hearing aids to administer a hearing test rather than specialized audiometry equipment. This approach uses a Gaussian Process Classification28 to obtain continuous pure-tone threshold curves in less time than a traditional audiogram. The Gaussian Process Classification also provides a measure of confidence in its threshold estimates and requires only the hearing aids and a connected smartphone to conduct. In recent years, in-situ audiometry and Bayesian hearing tests have been separately compared to conventional pure-tone threshold testing, producing results comparable to a standard audiogram33. However, the threshold was tested using the same standard equipment in the clinic to assess hearing. A combined approach that uses the person’s hearing aids and probabilistic models to optimize in-situ hearing tests has not yet been explored.We test a novel approach with our clinical study, addressing the above limitations. The new procedure combines Note that the audiogram is the starting point for the fitting process, and the need for alignment between the audiogram obtained by the audiologist or obtained by the self-fitting procedure is primarily to satisfy the need for documentation. Following this logic, no audiogram would be required for the fitting process. The fitting could start with an arbitrary audiogram, aligning somewhat with the patient's hearing ability. Using such an approach, the fitting will likely take more iterations. We suggest that a good alignment of the results from the self-fit will optimize the fitting procedure.females ! = mean-agemales, were shown not to be significantly different .Patients who had an appointment with the hearing clinic for a hearing assessment and had no severe-to-profound hearing loss were invited to participate in the study. Driven by patient availability, we did not control gender, ethnicity, and race. Among the participants, 37 (70%) were females, and 16 (30%) were males (Table in-situ” with a hearing aid and the Gaussian Process classifier (method M2). While the standard audiogram collected values at 11 distinct frequencies: 125, 250, 500, 750, 1000, 1500, 2000, 3000, 4000, 6000, and 8000 Hz  on the right and 2.8 ± 7.9 dB (N = 35) on the left side. They are close to zero at many frequencies, but lower frequencies tend to deviate more. It should be noted that while audiogram values collected from M1 could take on values below 10 dB HL, for statistical analysis, M1 values were limited to 10 dB HL to match the dynamic range of M2.The distribution of the differences in the pure tone averages is shown in Fig. lillietest(x), where ‘h’ is the test decision for the hypothesis that the data ‘x’ come from a normal distribution with an unknown mean and variance , at the 5% significance level. For our data, the test was h = 0 for the data from the right or left side. The t-test for a mean equal to zero and alpha = 0.05 in MATLAB,  = ttest(x), has the following results on the right side: h = 0, the probability p = 0.41; the confidence interval ci = , and the test statistics, ‘stats’, produced a value for the statistics tstat = 2.12, with the degree of freedom df = 34, and an estimated population standard deviation sd = 7.9. The returned value h = 0 indicates that the t-test accepts the null hypothesis that the sample data comes from a population with a mean equal to zero at the 1% significance level.The difference between the pure tone threshold averages (PTAs), obtained with M1 and M2, are normally distributed with an unknown mean and variance. The sample data was tested for matching a normal distribution using the Lilliefors test. The test is available in MATLAB, h = For the left side, the values are: h = 1, the probability p = 0.004; the confidence interval ci = , and the test statistics, ‘stats’, produced a value for the statistics tstat = 3.13, with the degree of freedom df = 34, and an estimated population standard deviation sd = 7.17. The returned value h = 1 indicates that the t-test rejects the null hypothesis that the sample data comes from a population with a mean equal to zero at the 1% significance level.gplotmatrix; ‘x’ denotes the groups, and ‘group’ the grouping variable. The MANOVA was performed with the MATLAB command  = manova1; ‘x’ denotes the groups, ‘group’ the grouping variable, ‘d’ is an estimate of the dimension of the space containing the group means, ‘p’ is a vector of p-values testing whether the means lie in a space of dimension 0, 1, and so on. The command also returns ‘stats’, a structure with additional MANOVA results. The largest possible dimension is either the dimension of the space or one less than the number of groups. If the i-th p-value is near zero, it indicates that the group means do not lie on a space of i-1 dimensions. The choice of a critical p-value is specified by the value of the input argument alpha (0.05 or 0.01). For our data, p = 2.26*10–6 for the data from the left and p = 3.0*10–12 for the right side; the estimated dimension is 1 for each side, indicating that the data align along a line and are statistically different.Pure tone thresholds at ten different frequencies define the audiogram. Changes in the audiograms then show deviations from normal hearing. A multivariate version of the analysis of variance is therefore essential to determine whether the entire set of means of thresholds is different from one group to the next. Dependencies can be seen in the grouped plot matrix  as calibration, the results of M2 can be shifted by this amount as a calibration factor. The t-test of the adjusted data shows that h = 0, indicating that the data comes from a population with a mean equal to zero at the 5% significance level. In other words, with a modest scalar calibration, the sample data representing the difference between M1 and M2 can be said to come from a population with a mean equal to zero, indicating that the M1 and M2 are similar.The output d estimates the dimension of the group means. d = 1 indicates that the means differ but fall along a line. The two most-left columns and the top two rows of SFigures 1 and 2, corresponding to 250 and 500 Hz, show differences. These data indicate that the lower frequency differences, compared to the control responses in the standard audiometric method, appear more pronounced, confirming what the difference curves in Fig. The credible interval analysis assumed an interval between + /− 10 dB when estimating the probability distribution of the difference values between M1 and M2 using an uninformative prior. This analysis suggests that most subjects’ M2 measurements are within 10 dB of the M1 measurement.in-situ with hearing aids are equivalent to currently accepted audiometric procedures45. Our results agree with those studies. DiGiovanni and Pratt reported significant differences between hearing thresholds obtained with standard and in-situ procedures38. It is not clear whether the differences of 6–9 dB at 500, 1000, and 2000 Hz are caused by differences in sound level produced by the transducers.Previous studies suggested that hearing thresholds obtained 46 published their results on classifying audiograms by sequential testing using a dynamic Bayesian approach. The procedure included the clinician’s a priori knowledge to efficiently select the next intensity and frequency. Selections were based on a probability table and the results from previous trials. More recent work has shown that a probabilistic model of the response-generating process can provide this information. Hearing threshold estimation becomes a Bayesian posterior inference using the probabilistic modeling approach. Intensity and frequency are sequentially selected by maximizing the information gained during each iteration46, in particular using Gaussian Process models53.This study is not the first report on combining probabilistic modeling and information gain. Özdamar et al.54. He argued that the Gaussian Process is the most straightforward method of defining Bayesian regression and classification models. The strengths of the models include the variety of possible covariance functions to select from. For audiometry, the most favorable kernel is the squared-exponential covariance kernel over linear or periodic kernels because it best represents the data sets. Other strengths of the Gaussian Process relate to the efficiency in data sampling55, the fact that the Gaussian Process provides a posterior distribution of the model’s belief of the underlying function value for a given test point. The model is a two-dimensional binary classifier in the frequency and by a psychometric function in the intensity dimension, which provides uncertainty bands on the resulting threshold estimate. The model’s output provides objective stopping criteria and allows for optimizing the next intensity and frequency selection, which is based on the previous data collected.In his 1998 paper, Neal strongly advocated using the Gaussian Process for regression analysis and classificationin-situ hearing threshold assessment. The average difference between the threshold estimates provided by these two methods was 3.8 ± 7.2 on the right side and 2.8 ± 7.9 dB on the left side. For the fitting procedure, the method provides a reasonable starting point for the actual gain finally preferred and used by the patient. The in-situ method has advantages because the systems used for testing are the same as those for delivery.The present study measured the similarity between the hearing threshold estimates provided by a standard audiogram procedure versus an experimental In-Situ Procedure:It is designed to be self-administered only using wireless-enabled hearing aids and a smartphone.It uses a probabilistic model to estimate the uncertainty in the estimate, which may be used to determine if a test is valid or should be retaken.It uses probability theory to optimize which tones are presented to the subject using prior response information.Output is a smooth, continuous curve representing the threshold estimate for each as a function of frequency.Summary of Experimental All experimental procedures with human subjects followed the institutional research committee's ethical standards and the 1964 Helsinki Declaration and its later amendments. The Institutional Review Boards at Northwestern University Feinberg School of Medicine reviewed and approved all procedures (STU00205797). The subjects gave informed consent before participating in this study.56. Sound levels start at soft, which is barely or non-audible. After assessing their hearing with a standard audiogram, patients who fulfilled the inclusion criteria were offered to participate in the study. The audiologist immediately connected interested patients with this study's designated investigator, as described in the IRB (STU00205797).This study collected hearing threshold curves from the left and right ears in volunteers using two methods for each subject. Method 1 (M1) was a standard pure tone audiogram, which measured the subject’s hearing sensitivity to acoustic tones at different frequencies. The assessment focused on pure-tone air conduction thresholds inside a sound booth with a certified audiologist of the hearing clinic at Northwestern Memorial Hospital (NMH). The audiologist conducted the test in a sound booth and is part of the clinical assessment for which the patient came to the clinic. Each ear was tested separately using various transducers such as headphones or inserted earphones. As it is a behavioral test, its outcome depends on the individual's response. To estimate a person's pure tone hearing threshold, the sound level of the pure tone with a given frequency was changed incrementally until the lowest level was determined that could still be perceived. A set of standard frequencies ranging from 0.125 to 8 kHz was used, and the sound level was modified before each presentation of a short tone stimulus using a staircase “up 5 dB—down 10 dB” approachin-situ testing procedure, where wireless-enabled hearing aids  connected to a mobile app on a handheld device (Apple iPhone 8 Plus smartphone) played pure tones. The mobile app contains software that can run a Gaussian Process classification algorithm to determine the pure tones and their corresponding intensities to play throughout the test. The algorithm then returns and stores a probabilistic estimate of the hearing threshold for the left and right ears. Sound levels were between 10 and 80 dB HL. After each presentation of the pure tone, the subject was asked to press a button on the screen of an iPhone, indicating on which side the tone was heard. The software then determined the subsequent frequency and sound level based on the previously recorded responses. The following tones and corresponding sound levels were sent to the hearing aid via Bluetooth, playing the pure tone at the given sound level. The results were stored locally on the iPhone. The data were erased from the iPhone after transferring the de-identified data to a secured server at Northwestern University. The hearing aid limited the maximum possible sound levels of the hearing aid. The software used here could not override the hearing aid’s safety limits.Method 2 (M2) estimated pure tone thresholds with a self-guided 57.The hearing aid was the HD100 from Soundworld. Our custom-written software sets the pure tone frequencies and their corresponding sound levels via a Bluetooth connection between an Apple iPhone 8 Plus smartphone and the hearing aid during the hearing tests. The software could not override the internal safety measures of the hearing aid. The maximum level of the pure tones delivered was 80 dB hearing level (HL), where HL was referenced to sound levels in dB re 20 µPa obtained from Ref.The Soundworld HD100 Hearing Aid is a printed circuit assembly powered by a rechargeable lithium-ion battery. The main processing unit of HD100 is an open − programmable DSP − based hybrid specifically designed for wireless, high − performance hearing aids. The PCB incorporates a low-power Bluetooth 4.0 radio and all necessary passive components for interfacing with transducers. The Digital Signal Processor (DSP) core processor supports wireless protocols and combines an open − programmable controller with hardware accelerators for audio coding and error correction support. A Filter Engine enables time-domain filtering and supports an ultra − low − delay audio path. The design allows for 16-channel compression, feedback cancellation, 16-channel noise reduction, and several environmental modes. Only proprietary software written and distributed by Soundworld and its affiliates can be installed on the HD100. “Source code” (both firmware and software) is solely controlled by Soundworld and is not distributed to end-users. The HD100 employs two MEMS microphones, allowing for directional detection of sound. Three pushbuttons allow to power the device on and off, modify the volume, and change the program options. The output consists of a Receiver-in-the-Canal (RIC) speaker employed in the user’s ear canal. The HD100 is housed in a weatherized, plastic enclosure and houses the tuned antenna inside.The otoTune app provides a user interface to connect the phone's left and right HD100 hearing aids. Once the devices are connected, another app mode can be accessed, displaying a left and right button on the smartphone’s screen to collect feedback from the subject. Mathematical code was implemented to perform the large computations necessary to compute GP classification on the mobile device’s hardware. Once the evaluation was completed, the results were saved to the smartphone's memory with any personal identifying information removed. The software also provided a mode to view a list of the collected data files, which could then be securely and wirelessly exported to a personal computer to conduct data analysis. All data were transferred to an encrypted hard drive and removed from the smartphone at the study's end.A pure tone was played through the left or right hearing aid. It was labeled “audible” or “non-audible”, depending on the subject pressing a button on the user interface, indicating that a tone was heard on the corresponding side. The 3-tuple represented each trial , and response label .A list of pre-specified tones was played to generate an initial probabilistic estimate of the subject’s hearing threshold curve.The probabilistic estimate was used to select the next tone determined by which area in the space of tones and frequency the algorithm was most uncertain about.A tone at this most uncertain point was played, and the subject provided feedback on whether this tone was heard or not.The probabilistic estimate was computed again to generate a new point that the algorithm was most uncertain about, and that tone was played in the subsequent presentation.The process continued iteratively until additional tones only provided marginal new information based on the change in the uncertainty estimate. The model could be viewed as a binary classifier for any pair of possible tones, with a decision boundary in the frequency-intensity space interpreted as the hearing threshold curve.The process to obtain a threshold estimate using GP classification can be summarized by the following steps:, X is the design matrix,p is (the function f has a) Gaussian distribution with mean vector μ and covariance matrix K, μ is the mean function, and K is the covariance matrix.The testing procedure used a Gaussian process classification model for each ear to estimate the probability that a tone can be heard over the space of dB HL intensity and frequency values. The Gaussian Process is outlined in Eq. .1\documeκ  is the squared-exponential covariance function (kernel) evaluated at xi, xj, σ2f is the variance of the (noise free) signal (σ determines the average distance of the function away from the mean), and l is the characteristic length scale, which determines the ripples in the function.The GP requires selecting a covariance function or covariance kernel; a squared-exponential covariance kernel was used in this application, shown in Eq. .2\docume54.L is the log-likelihood, k is a constant.The standard GP model must be modified using a logistic function to perform classification to obtain a function whose output is between 0 and 1, which can be seen in Eq. 54.3\docuPressing the corresponding left or right button during the tone delivery provided the user’s input. Each response was recorded and was 1 for pressing the correct button and 0 otherwise. With the patient’s prior responses, a continuous two-dimensional curve over the space of tones (dB HL x frequency) can be computed at any time during the test. The function range is a value between 0 and 1, interpreted as a probability estimate that the user can hear a tone at that intensity and frequency.The GP also provides an uncertainty estimate for every possible probability estimate, where the uncertainty estimate curve has an identical domain as the probability estimate curve. This uncertainty estimate serves to determine the next tone and intensity. The selection is accomplished by choosing the point on the uncertainty estimate curve over the tones' space with the highest value. It is the tone that the probability model is most uncertain about and will be the tone that is played next, therefore maximizing the amount of information obtained. Once this next tone is played, its response is recorded as heard or unheard and added to the test dataset.Another Gaussian process is then computed to obtain a new probability and uncertainty estimate curve for which a new tone will be selected. The process is repeated iteratively until approximately 35 responses per ear are collected. A final output curve for the left and right ear is obtained from the GP uncertainty curve given the complete dataset by selecting the maximum value from the uncertainty estimate curve across the entire range of frequencies. The algorithm is based on a probabilistic model, viewed as a two-dimensional binary classifier specified by a GP. The GP outputs a continuous estimate; therefore, a smooth function of frequency is obtained.The study was a single-center study. The criteria to include the patient in the study were: (1) the study participant is 21 years or older, (2) the study participant has English language proficiency, (3) the study participant can follow written instructions in English, (4) the study participant had an audiogram in the past six months, including the day of the patient’s visit to the clinic. All study participants with or without hearing loss were eligible if they did not have a severe-to-profound hearing impairment and had none of the exclusion criteria outlined below.The criteria to exclude the patient in the study were (1) the study participant was not able to consent, (2) for the study participant, English is a second language, (3) the study participant was legally blind  the study participant has a severe-to-profound hearing loss (> 70 dB HL), (4) the study participant cannot understand the written and verbal instructions given by the study software or by the study coordinator.The study investigator brought the patient into a quiet examination room. Informed consent documentation was provided to the participant. The study responsible investigator explained the study participant answered questions from the patient regarding the study. Written consent to participate in the research projects was sought and obtained directly from the participants. Documentation consisted of a signed statement of informed consent approved by the Institutional Review Board (IRB). Participants were informed of the tasks and time involved in the study. They were also told that their participation is strictly voluntary and that choosing not to participate would not harm their relationship with their clinical care providers. They were also informed that if they decide to participate, they can withdraw from the research project at any time, for any reason, and without penalty. Participants were given a signed copy of their consent form. A four-digit random number was assigned to each patient.All written and recorded data were de-identified. Upon entry into the study, participants received a randomized code number, used on all material generated by the research protocols and for data presentation in scientific and professional meetings. Computers used on the project were encrypted, password-protected, and access to files required two-factor authentication.in-situ test method.The primary goal of this study was to determine if the audiogram values obtained from the standard clinical procedure were similar to those hearing thresholds collected using the self-guided The study has a cross-over design where each subject was used as their own control. M1 was used as described above and represented the control group. The experimental condition used M2. Statistical analysis included graphing the threshold curves obtained from M1 and M2 along with the difference curves, computing t-tests, running a MANOVA, and an analysis of credible intervals based on pure tone threshold differences at 250, 500, 750 1000, 1500, 2000, 3000, 4000, 6000, and 8000 Hz.The outcome measure was the hearing sensitivity of each patient for a frequency range between 250 and 8000 Hz. The M1 procedure produced up to 11 discrete audiogram frequencies, whereas M2 produced a continuous function of frequency. Therefore, the difference between M1 and M2 were evaluated at the frequencies from M1 subtracted from M2 at those same frequencies to obtain differences.in-situ data for all subjects, (3) compare difference values at 250, 500, 750, 1000, 1500, 2000, 3000, 4000, 6000, and 8000 Hz for all subjects, (4) calculate the average threshold difference and corresponding standard deviations.The analysis included the following steps: (1) Graph the M1 standard audiometric data for all subjects, (2) plot the M2 experimental Since the study approach includes repeated measures , a MANOVA was used. The Lilliefors test was applied to determine whether the sample data derived from the differences measured between M1 and M2 come from a distribution in the normal family. A credible interval analysis of the difference values across frequency. The power of the study was calculated.The sample size for the study was determined before the start of the study using MATLAB-2018b, MATLAB-2019a , G*Power (RRID:SCR_013726), and IgorPro 8 . After plotting and visually inspecting the plots, the actual power was calculated after the completion of the study with the same software. The a priori calculations for the sample size and power assuming a medium effect size (0.47) and an alpha of 0.05 suggested a target enrollment of 55 participants.Supplementary Information.


Single-nucleotide polymorphisms in genes controlling lipid mediator signaling may modify the colorectal polyp prevention activity of aspirin. Further investigation is required to determine whether testing for genetic variants can be used to target cancer chemoprevention by aspirin to those who will benefit most. 2. We investigated whether 35 SNPs in oxylipin metabolism genes such as cyclooxygenase (PTGS) and lipoxygenase (ALOX), as well as 7 SNPs already associated with colorectal cancer risk reduction by aspirin , modified the effects of aspirin and EPA on colorectal polyp recurrence in the randomized 2 × 2 factorial seAFOod trial. Treatment effects were reported as the incidence rate ratio (IRR) and 95% confidence interval (CI) by stratifying negative binomial and Poisson regression analyses of colorectal polyp risk on SNP genotype. Statistical significance was reported with adjustment for the false discovery rate as the P and q value. 542 (of 707) trial participants had both genotype and colonoscopy outcome data. Reduction in colorectal polyp risk in aspirin users compared with nonaspirin users was restricted to rs4837960 (PTGS1) common homozygotes , rs2745557 (PTGS2) compound heterozygote-rare homozygotes , rs7090328 (ALOX5) rare homozygotes , rs2073438 (ALOX12) common homozygotes , and rs104522 (TP53) rare homozygotes . No modification of colorectal polyp risk in EPA users was observed. In conclusion, genetic variants relevant to the proposed mechanism of action on oxylipins are associated with differential colorectal polyp risk reduction by aspirin in individuals who develop multiple colorectal polyps. SNP genotypes should be considered during development of personalized, predictive models of colorectal cancer chemoprevention by aspirin.Aspirin and eicosapentaenoic acid (EPA) reduce colorectal adenomatous polyp risk and affect synthesis of oxylipins including prostaglandin ESingle-nucleotide polymorphisms in genes controlling lipid mediator signaling may modify the colorectal polyp prevention activity of aspirin. Further investigation is required to determine whether testing for genetic variants can be used to target cancer chemoprevention by aspirin to those who will benefit most. Helicobacter pylori infection; ref. A large body of evidence has accumulated from epidemiologic observations, randomized colorectal polyp prevention trials, and long-term follow-up studies of colorectal cancer outcomes after participation in vascular prevention trials, that regular aspirin use is associated with a 15% to 20% reduction in colorectal cancer risk . However2 metabolite, PGE-M, as a predictive risk, or therapeutic response, biomarker of colorectal polyp prevention by aspirin  of personalized aspirin efficacy that could pinpoint individuals who are either sensitive to, or resistant to, the anti–colorectal cancer activity of aspirin . There h aspirin , althoug aspirin .Prostaglandin G/H Synthase (PTGS); also known as Cyclooxygenase (COX)-1 and -2] in post hoc analyses of randomized colorectal polyp prevention trials of aspirin has demonstrated a possible interaction between aspirin and PTGS2 SNP rs4648310  use . Investidatasets .2 from the omega-6 polyunsaturated fatty acid (PUFA) COX substrate C20:4n-6 arachidonic acid -mediated synthesis of other oxylipins with antitumorigenic activity such as leukotriene (LT) B4  B4 .HPGD), the enzyme responsible for inactivating PGE2, has been shown to predict colorectal cancer risk reduction by aspirin  and the effect of aspirin on colorectal polyp risk has not been addressed previously in a randomized trial setting.Despite the relevance of other genes controlling oxylipin synthesis for the chemopreventative property of aspirin, the relationship between SNP genotypes in a wide range of genes controlling oxylipin synthesis and levels (including n-3 eicosapentaenoic acid (EPA) also has colorectal cancer chemoprevention activity  at screening colonoscopy in the English Bowel Cancer Screening Programme  of the anticancer activity of aspirin and EPA, as well as SNPs in selected genes already linked to modification of colorectal cancer risk reduction by aspirin, and colorectal polyp outcomes in the seAFOod polyp prevention trial.This study is part of a wider program of investigations using the seAFOod trial biobank and post-trial BCSP colonoscopy outcomes called STOP-ADENOMA (ISRCTN05926847). The study was conducted in accordance with the Declaration of Helsinki. Ethical approval for this study was granted by London and Surrey Borders Research Ethics Committee (19/LO/1655). The seAFOod Trial biobank has been described elsewhere . Blood sDNA was extracted from a single buffy coat extract per seAFOod trial participant using an in-house phenol-chloroform method, followed by washing with absolute ethanol and storage at 4°C in DNase/RNase-free water, prior to spectrophotometric assessment of the DNA quantity and quality. If any sample had a DNA yield <20 ng/μL, a second sample for that individual was obtained from the Biobank for DNA extraction.Genotyping was carried out using the Fluidigm microfluidic SNP genotyping system (Fluidigm), using custom-built SNP Type genotyping assays (Fluidigm) for 92 SNPs (Supplementary Table S1).A multiplexed preamplification PCR was carried out to increase the available template DNA and reduce allelic drop-out associated with low template concentration. DNA samples were run on 96.96 Dynamic Array IFCs (integrated fluidic circuits), which were primed and loaded using the IFC Controller HX (96.96 arrays), as per manufacturer's instructions. In total, 691 individual samples including 10 technical duplicates, 31 participant duplicate samples and 1 participant triplicate sample, were analyzed, representing 648 individual participant DNA samples.Genotyping PCR and measurement of endpoint fluorescent values were carried out using the Fluidigm BioMark HD system. Data were analyzed and genotyping calls made using Fluidigm SNP Genotype Analysis software.Fourteen SNPs failed quality control; one SNP was monoallelic, one SNP was repeatedly called differently in duplicate sample runs, and 12 SNPs had an absence of genotype call >5% (Supplementary Table S1).P < 0.05 with Benjamini–Hochberg correction for multiple testing). However, on inspection of scatter plots produced during Fluidigm genotyping, “true calls” did not overlap with “fails” suggesting that this was not caused by sampling error. Therefore, these SNPs were included in subsequent analyses to avoid missing a relationship with clinical outcomes given that these SNPs could be causally related to colorectal polyp development  explaining absence of Hardy–Weinberg equilibrium.Therefore, genotype data for 78 SNPs were available for analysis inside the seAFOod trial database. Twelve SNPs did not satisfy Hardy–Weinberg equilibrium , as well as 7 SNPs previously associated with differential colorectal cancer risk reduction in aspirin users -funded Fatty Acid Metabolism (FAME) Consortium - Interlinking Diet with Cardio-metabolic Health. Briefly, all SNPs in PTGS and ALOX genes were grouped into linkage disequilibrium (LD) blocks using PLINK v1.9 software  with blood and rectal mucosal levels of PUFAs measured in the seAFOod polyp prevention trial, as well as colorectal polyp risk, are the subject of a separate report.R2 values for paired SNP relationships.Linkage disequilibrium between SNPs in individual genes was analyzed using the LDmatrix tool (NIH) to derive χ2 test or Mann–Whitney U test, as appropriate, to confirm similarity with the original seAFOod trial cohort and ensure continuing balance across the trial treatment allocations.Only seAFOod trial participants, for whom there were trial colonoscopy outcome data, were included in this SNP analysis. Baseline characteristics  of the seAFOod trial participants included in the SNP analysis were analyzed using the Interactions of SNP genotypes with trial interventions were analyzed “at the margins” , consistent with the primary seAFOod trial analysis  polyp number per participant  between colorectal polyp number and intervention for some strata, an interaction test between the SNP and intervention was conducted fitting the negative binomial regression model with additional terms for interaction between the treatment and SNP variables.All SNPs were then used to stratify a negative binomial regression model of colorectal polyp number  and two colorectal polyp outcomes, the positive false discovery rate (pFDR) was described by the q value for each of the models including individual SNPs .A total of 666 trial participants had at least one buffy coat sample vial in the seAFOod trial biobank . There wA total of 542 seAFOod trial participants, for whom SNP genotype data were available, also had colonoscopy outcome data from the seAFOod trial. Reasons for lack of primary  outcome data for some trial participants are detailed in the seAFOod trial CONSORT diagram . The cliPTGS1, PTGS2, and HPGD SNP revealed that PTGS1 rs4837960 common homozygotes (G:G) and PTGS2 rs2745557 compound heterozygotes (G:A)-rare homozygotes (A:A) were associated with a statistically significant reduction in total colorectal polyp number in aspirin users versus nonaspirin users in contrast to the other genotype . All the other PTGS1, PTGS2 and HPGD SNPs did not reach statistical significance for a difference in total colorectal polyp number between aspirin and placebo users according to genotype (Supplementary Table S2). For PTGS1 rs4837960, aspirin use was associated with a reduction in total colorectal polyp risk in homozygotes for the major allele , but not in individuals with one or more minor T alleles , but not common homozygotes .None of the ALOX5 SNP rs7090328 and for three ALOX12 SNPs , for aspirin users . In the negative binomial regression model, ALOX5 rs7090328 was associated with modification of the effect of aspirin, with a reduction in total colorectal polyp risk in homozygotes for the minor allele , but not in individuals with one or more major A alleles , with a Pint value of 0.03 .None of the TP53 tumor suppressor gene . In the negative binomial regression model, colorectal polyp risk reduction by aspirin was significant for rare homozygotes  with a Pint = 0.06, but not for individuals with one or more major alleles .PTGS1), COX-2 (PTGS2), 5-LOX (ALOX5), and 12-LOX (ALOX12) with differential polyp prevention efficacy of aspirin, but not EPA, in the seAFOod trial.Using a panel of SNPs in genes controlling oxylipin signaling relevant to colorectal carcinogenesis and the pharmacology of aspirin and EPA, we report the association of several SNPs in genes encoding COX-1  with another PTGS1 SNP rs3842787, which has been reported to interact with NSAID use for cancer risk  described a possible interaction between rs4648310 and aspirin use (81 mg daily)  and aspirin or EPA use in seAFOod trial participants. rs20417 has been reported to approach statistical significance (P = 0.07) for an interaction with NSAID use for reduction in colorectal adenomatous polyp risk in one case–control study (494 cases and 584 controls), but not in another smaller study . It wilALOX12 SNPs that was associated with differential colorectal polyp prevention activity of aspirin in our study (the intronic SNP rs2920421) has been reported to interact with NSAID use in a case–control study of colorectal cancer risk  database.One of the cer risk . In thatcer risk . In a secer risk . ConsistWe also demonstrated that homozygosity for the SNP  in the coding region of the TP53 tumor suppressor gene was associated with colorectal polyp reduction in aspirin users in the seAFOod trial, in contrast with the group of individuals that had at least one major (G) allele, who did not demonstrate a reduction in colorectal polyp risk associated with aspirin use. At least one C allele at rs104522 has been reported to have an OR of 1.16  for presence of one or more colorectal adenomas compared with homozygous G:G individuals in a meta-analysis of genetic association studies of colorectal adenomas . The rolWe did not observe any gene–supplement interaction for any SNP related to the modest effect of EPA use on colorectal polyp number in the seAFOod trial. The reduction in total colorectal polyp number associated with EPA use in the seAFOod trial was modest and just failed to reach statistical significance . Howevera priori knowledge of aspirin and EPA anticancer pharmacology , if the effect size is only moderate (IRR > 0.6). We also draw attention to the fact that the seAFOod trial population consisted of individuals who had undergone BCSP colonoscopy after a positive FOBt, thereby generating a predominantly male and white ethnicity cohort  interactions from the seAFOod polyp prevention trial. SNPs in COX-1, COX-2, LOX isoforms, and TP53 should be further evaluated as biomarkers of aspirin chemoprevention efficacy, alone and in combination with other polymorphisms reported to predict colorectal polyp and/or colorectal cancer risk reduction by aspirin  colorectal polyp counts according to seAFOod trial treatment allocation by factorial margins and PTGS SNP genotypes.Click here for additional data file.Supplementary Figure 2Supplementary Figure 2 shows the distribution of individual total (combined adenomatous and serrated polyp) colorectal polyp counts according to seAFOod trial treatment allocation by factorial margins and ALOX SNP genotypes.Click here for additional data file.Supplementary Figure 3Supplementary Figure 3 shows the distribution of individual total (combined adenomatous and serrated polyp) colorectal polyp counts according to seAFOod trial treatment allocation by factorial margins and TP53 rs104522 genotype.Click here for additional data file.


Organic matter is an integral part of all living things on our planet, but abiotic (not relating to life) organic matter can be found throughout the Solar System within meteorites, asteroids, comets and even on the surfaces of other planets such as Mars. Therefore, one of the most profound questions for humanity concerns how life originated on Earth. In order to answer this, we must first understand how organic matter is formed and altered throughout our solar system and the universe in general. To better understand the origin and evolution of organic matter, the Japan Aerospace Exploration Agency (JAXA) sent the Hayabusa2 spacecraft to collect and return material from the near-earth asteroid Ryugu. Here, a review concerning the organic material within the returned Ryugu samples will be presented. The review will inform the reader about the Hayabusa2 mission, the nature of the organic matter analyzed and the various interpretations concerning the analytical findings. The review puts the findings and individual interpretations in the context of the current theories surrounding the formation and evolution of Ryugu and highlights the issues that remain to be solved.The Hayabusa2 mission was tasked with returning samples from the C-complex asteroid Ryugu (1999 JU3), in order to shed light on the formation, evolution and composition of such asteroids. One of the main science objectives was to understand whether such bodies could have supplied the organic matter required for the origin of life on Earth. Here, a review of the studies concerning the organic matter within the Ryugu samples is presented. This review will inform the reader about the Hayabusa2 mission, the nature of the organic matter analyzed and the various interpretations concerning the analytical findings including those concerning the origin and evolution of organic matter from Ryugu. Finally, the review puts the findings and individual interpretations in the context of the current theories surrounding the formation and evolution of Ryugu. Overall, the summary provided here will help to inform those operating in a wide range of interdisciplinary fields, including planetary science, astrobiology, the origin of life and astronomy, about the most recent developments concerning the organic matter in the Ryugu return samples and their relevance to understanding our solar system and beyond. The review also outlines the issues that still remain to be solved and highlights potential areas for future work. The Hayabusa2 mission was the successor of the Hayabusa mission , which r−3.The Hayabusa2 spacecraft was launched in December 2014 by a H-IIA rocket and after 3.5 years reached its home position ~20 km above Ryugu ,7. AccorIn addition to the remote sensing, several touchdown sites were selected and two were picked for the collection of the samples to be returned to Earth  8,9]. T,9. T8,9]−1, with the surface of Ryugu [The second touchdown site (TD2) was selected for the creation of an artificial impact crater, which would allow access to subsurface materials that should be less affected by space weathering . The artof Ryugu . The impof Ryugu . The TD2of Ryugu . The calAfter completing its remote sensing and sample collection operations, the Hayabusa2 spacecraft returned the Ryugu samples to Earth on the 6th of December 2020, in a hermetically sealed container . SubsequExtraterrestrial organic matter (OM), such as that found within the Ryugu samples and carbonaceous chondrites, is composed of two main fractions. The first fraction is soluble in common lab solvents and not bound to a macromolecular structure and is thus termed either soluble organic matter (SOM) or free organic matter (FOM) ,16. The The OM in extraterrestrial objects is thought to originate in a number of distinct environments, ranging from the interstellar medium (ISM) and the protosolar nebula (PSN) to the progenitor planetesimals of asteroids/comets and their daughter meteorites  16,17,1,19. The Once in the ISM, the organic elements can be present in their atomic form in diffuse atomic clouds or when the clouds are denser in their molecular forms, as is the case for molecular clouds . MoleculMeanwhile, dense molecular clouds offer protection from UV irradiation within their interiors, which prevents the destruction of organic molecules formed within them . Organic2, HCO, H2CO, CH3OH, CH4 and NH3 [2CO, CH3OH and CH4, as well as inorganic molecules that can go on to form OM upon further processing. The buildup of ice on dust grains also creates a matrix that stabilizes certain ions and radicals and allows for solid-phase reactions to occur [Furthermore, the ability of dense molecular clouds to protect their interiors from UV irradiation, allows their gasses to condense to form ice as mantles on dust grains through gas-grain chemistry ,28,29. T and NH3 ,30. As sto occur .2 [2O:CH3OH:NH3:CO = 100:50:1:1) at room temperature, hundreds to thousands of organic molecules are produced [Once the ice mantles have formed, subsequent photo-processing generates ions and radicals via cosmic rays and UV photons, produced by their interaction with H2 ,31. The produced . Accordiproduced ,34 and sproduced ,36. Whilproduced ,38,39. Wproduced ,40, nuclWhen a molecular cloud collapses to form a PSN and eventually a protostellar disk (PSD), the conditions will vary dramatically throughout the disk, with temperatures close to the protostar being hot enough to vaporize rocks, but the conditions at the middle of the PSN and further out along the mid plane being as low as 10 K ,41. As s2 and H2 [While gas phase and gas-grain chemistry reminiscent of the ISM may be a driving force for OM formation in the outer PSN/PSD, a different style of these processes may also be operating within the photosphere throughout the disk. Plasma discharge experiments, referred to as the Nebulotron experiments, investigated the synthesis of OM in the high energy environments of the PSD photosphere ,47,48,4926Al, included in their rocky dust components [When the PSN/PSD enters into the protoplanetary disk phase, streaming instabilities or vortices cause dust, ice and any associated OM to coagulate to form cm-sized pebbles ,51,52. Tmponents . As a remponents ,57,58. Dmponents ,18,59,60The FOM/SOM component of carbonaceous chondrites records many biologically important compounds that could have formed during aqueous alteration . Multipl2/CH3 and C=O/C=C), X-ray absorption near edge structure (XANES) spectra, 13C nuclear magnetic resonance (NMR) spectra and 13C isotopic composition [The MOM/IOM component of carbonaceous chondrites has also been suggested to form during aqueous alteration, with Formose-type reactions, among others, being demonstrated as possible candidates ,60. Indeposition . Therefo15N and δD values for the Gas Giant Jupiter representing some of the lowest values in the solar system (δ15N = −374 ± 82‰ and δD = −833 ± 45‰) [15N = −382 ± 8‰ and δD = −865 ± 32‰) obtained from solar wind values [15N and δD values among solar system objects are recorded for carbonaceous chondrites (bulk δ15N = −23.7 to 359.7 and δD = −225.8 to 763.1) [15N = −10.8 to 2022.6 and δD = −120.5 to 641,000.0) [15N and δD values of comets go up to much higher values than the bulk values from carbonaceous chondrites, there is significant overlap of the values between these two types of extraterrestrial objects at the lower end of the range for comets. To complicate the picture further, Wild2 cometary silicates from the Stardust mission record carbonaceous chondrite-like bulk δ15N values and also contain 15N-rich hotspots similar to those observed in carbonaceous chondrites [The N and H isotope composition among Solar System materials varies greatly , with th3 ± 45‰) ,74. Inted values ,76. Meano 763.1) ,78 and c1,000.0) ,80. Whilondrites ,82. Furthermore, there are far less measurements for comet N and H isotope compositions than for carbonaceous chondrites and many were undertaken using spectroscopy for specific species within cometary coma. As such, measurements for comet N and H isotopes are often associated with large uncertainties . While t15N and δD values for PSN gases were likely very low [15N and δD values of their enriched components. For D, enrichment can occur through many different processes in the ISM, including gas-phase ion-molecule reactions, gas-grain chemistry and photolytic chemistry [As mentioned above, the δvery low ,76. As shemistry ,83. In phemistry , which ihemistry ,80. Wherhemistry ,85.15N enrichment [15N enrichments. This is because there are barriers preventing the exchange reaction, as a result of the requirement for a pair of electrons to be broken and then reformed, which is not the case for D+ or H+ [For N, many of the above processes were previously proposed to result in meaningful isotopic fractionation and thus richment . HoweverD+ or H+ ,86. 15N may be limited. A large fractionation in H isotopes is possible because the mass difference between D and H is very large and this means the mobility of H in the ice is much greater than for D [2, which can escape as a gas. For N, the difference in the mass of its isotopes is small and so the difference in mobility between them is small. Additionally, the abundance of N is much lower than for H, so the initial reactions of N atoms will likely be with H [Similarly, for gas-grain chemistry, the enrichment of an for D . Accordie with H .15N can be passed along [2 self-shielding (photodissociation) is thought to be responsible [14N-14N molecule are adsorbed within that region, generating gas-phase ions and molecules as a result. Interestingly, because the number of 14N-15N molecules is lower, the photons capable of breaking the bond within these molecules are able to penetrate further and thus enrich 15N in any ices or OM formed within the interior facing edge of the exterior of molecular clouds or the outer portion of the PSN [15N. In agreement, an experimental study found that huge enrichments of 15N were achieved during photodissociation of N2 [14N/15N ratios (15N depletion) in the gas-phase overall than their interiors [For photolytic chemistry involving ices, initial enrichments in ed along ,40, but ponsible ,88,89,90on of N2 ,78,79,80nteriors ,88.15N and D enriched ice and dust. The presence of short-lived radionuclides leads to the heating and melting of the ice and the release of any OM contained within them [Upon formation of the Solar System, comets and carbonaceous chondrite progenitor planetesimals would accrete the hin them ,56. At thin them . As such15N enrichment was found and thus N2 self-shielding remains the main process capable of generating enrichment of 15N in carbonaceous chondrites and comets. Finally, another mechanism that has been shown to produce isotopic enrichments in OM directly, is that of plasma discharge reactions ,48,49. AThe asteroid 1999 JU3, which would later be named Ryugu, was classified as a Cg-type asteroid in 2001, based on its strong UV absorption feature shortward of 0.55 um and its flat to slightly reddish slope longward of 0.55 um . Cg-typeMany carbonaceous chondrite meteorites contain significant amounts of OM , which mIn the time between the selection of the Hayabusa2 mission target and the rendezvous of the Hayabusa2 space craft with Ryugu, several ground-based investigations were undertaken to try and predict what the asteroid may be composed of. The first of these studies suggested that Ryugu was most similar to a heated CM2 chondrite composition, based on comparisons of the reflectance spectra of Ryugu, obtained from the Very Large Telescope in Chile to that obtained from two heated Murchison (CM2) samples (heated to 900 °C and 1000 °C) . In contAs the Hayabusa2 spacecraft approached Ryugu, it was able to record spectra of the asteroid’s surface. As a result of the remote sensing investigation, the asteroid was reclassified to a Cb-type asteroid, which is similar to Cg-type asteroids but has a flat or linear slope and lacks the strong UV absorption feature of the Cg-type asteroids. Furthermore, the composition of Ryugu was estimated to be most similar to a moderately dehydrated carbonaceous chondrite, due to the absence of a ubiquitous 0.7 um absorption band  and one of the lowest albedos of any solar system object .26Al was still very abundant. In this scenario, OM should have also been altered by the heating and bear similarities to that of heated carbonaceous chondrites.Accordingly, a scenario was envisioned in which the asteroid Ryugu was formed through the catastrophic collision of a much larger asteroid, and which was subsequently gravitationally reaccumulated to form a rubble pile asteroid with a spinning top shape. Moreover, the dehydration was suggested to have occurred from internal heating, likely due to the progenitor planetesimal of Ryugu forming early in the history of the Solar system, when the short-lived radionuclide Interestingly, the study also noted that interplanetary dust particles (IDPs) have a similarly low albedo, but preferred heated carbonaceous chondrites, due to their lack of the 0.7 um absorption band . HoweverAmino acids were included in the first analytical study to report on the OM inventory of Ryugu . The resSubsequently, another study investigated the amino acids in Ryugu, including their abundances and their chirality . Two difHowever, a previous study , using aMeanwhile, the 3D-HPLC-FD technique utilized no initial vacuum drying step and only a very slight alkaline pH of 8. Nevertheless, the 3D-HPLC-FD technique gave a Ryugu glycine abundance that was lower than that reported in the Orgueil study. Previously, another study using essentially the same 3D-HPLC-FD technique reported abundance values for the Murchison meteorite . SimilarIn a separate investigation of the amino acids within the Hayabusa2 return samples , both thThe strange result for the Ryugu hydrolyzed fractions was interpreted to potentially arise due to either the degrading effect of small Fe-rich phases in the extracts or as a result of cations interfering in the derivatization of the amino acids. The authors favored the Fe-rich phases scenario because they did not believe the cations would be sufficient within the hydrolyzed fractions to cause a problem. However, it is curious that Orgueil contains largely the same modal mineralogy as Ryugu samples, yet previous analyses of the hydrolyzed fractions of this meteorite , using aFurthermore, both the aforementioned studies provided invaluable chiral information, which indicates that the amino acids in Ryugu are likely mostly racemic. The exception is several protein-bearing amino acids and a non-protein-bearing amino acid . HoweverFinally, leading on from the qualitative investigation of amino acids in Ryugu reported previously , the qua2 ice) for A0022, compared to C0008  having greater abundances of secondary alteration phases  compared to C0008 (TD2) and evidence for mobilization of its trace elements over the mm scale, which was not seen for C0008. The differences were interpreted as arising from a greater accretion of ice . The amino acid DMG forms through the Eschweiler–Clark reaction and involves the reaction of glycine with formaldehyde and formic acid . Both foAliphatic amines were observed in Ryugu samples and demonstrated a decreasing abundance with increasing chain length, which is consistent with the trend observed for these compounds in other carbonaceous chondrites ,106. TheNitrogen heterocycles (N-heterocycles) were reported previously for carbonaceous chondrites and were thus expected to be present within Ryugu ,119,120.A similar conclusion was drawn concerning the results of another study which investigated the nucleobase and other N-heterocycle contents of Ryugu return samples . The stuThe values for Ryugu are actually much more similar to the unhydrolyzed values for CM2 chondrites and indicate that Ryugu in fact has a uracil content less than or similar to CM2 chondrites. No other nucleobases, apart from some uracil structural isomers and alkylated uracil homologues, were detected and this may relate to the acid hydrolysis step and/or the very low concentrations/absence of these nucleobases in Ryugu. Comparatively, CM2 chondrites were found to contain all the canonical nucleobases found in DNA/RNA . HoweverIn a further study, N-heterocyclic homologue series were reported for a Ryugu particle using UHPLC-OT-MS, nano-liquid chromatography-OT-MS (nanoLC-OT-MS) and DES-OT-MS . The resMonocarboxylic acids (MCAs) are among the most abundant FOM/SOM observed in carbonaceous chondrites ,123,124.Both alkylbenzenes and polyaromatic hydrocarbons (PAHs) were detected in Ryugu solvent extracts . InteresMicrometer scale observations for Ryugu MOM/IOM were first undertaken using scanning electron microscopy (SEM) with energy dispersive X-ray spectroscopy (EDS), Raman spectroscopy and Fourier transform infrared (FTIR) spectroscopy  Figure . The SEM2 atoms [3 orbitals or heteroatoms [Meanwhile, Raman spectroscopy, applied to 16 Ryugu particles, indicated that MOM/IOM was distributed ubiquitously throughout the matrix and revealed a slight difference between the particles in terms of their G-band FWHM and peak center positions. The G-band arises from the in-plane stretching of pairs of carbon sp2 atoms  and is p2 atoms . In Ryug2 atoms ,128. Theeroatoms . When aneroatoms . The dif2/CH3 asymmetric stretching band ratio for the TD1 particle A0035 was anomalously low. The result indicated that the aliphatic material contained within the MOM/IOM consisted of either short chain lengths or highly branched chains. The finding was interpreted as potentially arising from high levels of irradiation [3 groups.Additionally, MOM/IOM was isolated from a TD1 and a TD2 particle, through demineralization via treatment with HCl and HF . The MOMadiation  at the sSubsequently, another study investigated the MOM/IOM within the Ryugu return samples, also using both Raman and FTIR spectroscopy to study the samples at the µm-scale . In term−1) relating to ketones, aldehydes or amines was observed that has not been detected from any meteoritic MOM/IOM previously. Additionally, the CH2/CH3 band intensity ratio was found to be 1.9, which is a lot higher than that recorded for the Murchison (1.2) and Ivuna (1.3) carbonaceous chondrites and also both the anomalous value detected for A0035 (0.77) and the carbonaceous chondrite-like value detected for C0008 (1.17) by the first study [2/CH3 is that Ryugu MOM/IOM should contain aliphatic material with longer chain lengths or less branching than that of most primitive carbonaceous chondrites. However, no indication of what might cause this difference was given. One explanation is that the MOM/IOM may have experienced some heating [The FTIR spectroscopic data for the Ryugu samples from the second study  were fou C0008 1. by the f heating , but thi C0008 1. by the f heating . Alterna heating ,133 or I heating ,135 comp2/CH3 that was lower. While the studies utilized different peak fitting procedures, it may be the case that the Ryugu samples contain a wide diversity of MOM/IOM, which was sourced from distinct environments, including the irradiated asteroid surface, the ISM/PSN and that of the progenitor planetesimal of Ryugu, where aqueous-rich fluids would have been operating.The finding is at odds with that of the first study , which iTransmission electron microscopy (TEM) was employed to understand the OM, petrography and mineralogy of Ryugu particles at the µm to nm scale ,130. TheFor the aromatic class, the STXM response showed that the aromatic-to ketone ratio was higher than for the carbonaceous chondrite-like IOM class. Whereas, for the highly aromatic class a greater diversity of aromatic structures was indicated, due to a broader aromatic peak, compared to the aromatic and carbonaceous chondrite-like IOM classes. Moreover, the particulate and nanoglobular OM was found to be more frequently aromatic or highly aromatic, while the OM dispersed throughout the matrix was mostly present as carbonaceous chondrite-like IOM or diffuse carbon . AdditioWhen comparing the functional group chemistry of Ryugu to primitive carbonaceous chondrites, it was found that Ryugu contained much higher amounts of the aromatic and highly aromatic MOM/IOM present as nanoglobules and particulate matter . WhereasIn terms of the diffuse carbon OM, an association with Mg-rich phyllosilicates and molecular carbonate was observed . In conj15N and D hot and coldspots for OM, where the hotspots yielded values significantly higher than the bulk values and the coldspots significantly lower. It was found that the 15N hotspots or coldspots were the major determiners of the variation in C, H and N isotopes from the bulk values. Furthermore, the deviation in N and H isotopes from the bulk values was found to be both correlated and negatively correlated, with the 15N hotspot in the TD1 particle A0073 being correlated with a D enrichment  and the coldspot in the TD2 particle C0053 being negatively correlated . The results suggested that Ryugu likely accreted OM from various sources, including both ISM and PSN materials and that at least some OM would have been subsequently altered during aqueous alteration, potentially leading to the generation of new MOM/IOM material.Initial isotopic and elemental measurements of OM in Ryugu samples were performed by elemental analysis-isotope ratio mass spectrometry (EA-IRMS) for bulk sample compositions and secondary ion mass spectrometry (SIMS) for in situ measurements . The res15N and D coldspots also appeared to be lower than those found previously in carbonaceous chondrites and this was interpreted as relating to the interaction between the OM and the PSN/ISM N2 gas. The variation in the D isotopic composition of Ryugu MOM/IOM was interpreted as arising due to varying levels of aqueous alteration which can cause hydrogen isotope exchange and hydrolysis of D-rich MOM/IOM structures. Meanwhile, the variation in the N isotopes was explained by the accretion of OM from different sources. Furthermore, the process responsible for the enrichment in D and 15N for hotspots was interpreted as low-temperature fractionation processes within the ISM/PSN [15N enrichments observed in hotspots within extraterrestrial objects [15N are likely the result of photodissociation processes operating in the ISM or PSN [Further investigation of the isotopic and elemental composition of Ryugu return samples was undertaken via EA-IRMS  and nano ISM/PSN . While s objects ,72. InstM or PSN ,88,89,9015N and δD values for the different studies of Ryugu [When comparing the δof Ryugu ,103,130,of Ryugu , IDPs [1of Ryugu , Wild2 cof Ryugu  and comeof Ryugu  . It is unclear if the progenitor planetesimal of Ryugu was this large. In fact, such a large progenitor planetesimal may be very unlikely due to previous estimates of the size at which a body would facilitate fluid convection. Bodies greater than ~80 km in diameter would likely facilitate fluid convection and this would ultimately lead to elemental fractionation throughout carbonaceous chondrites ,148. Yetid water ,150.An alternative to the catastrophic collision and gravitational reaccumulation model is provided by the cometary nucleus model  10,17,1,152. In Accordingly, the planetesimal progenitor of Ryugu could have formed within the trans-Neptunian disk (TND) and suffered a disruptive event during the massive disk phase, where collisions between trans-Neptunian objects would have been large . The colEvidence for past cometary activity on the cometary progenitor of Ryugu was provided by the occurrence of unaltered olivine and pyroxene clasts and the presence of a clast-like domain in the matrix of a Ryugu particle with a distinct lithology . It was Furthermore, cometary jets are thought to deposit a portion of their material back onto the comet’s surface and this can become sintered in place, trapping any organic or dusty material entrained in the jet . Such a 2/CH3, which is similar to that observed in IDPs and for ISM material, may indicate that Ryugu accreted more comet-like or ISM-like organic matter than most carbonaceous chondrites. Together with the overlap of H and N isotopic values and N/C ratios for Ryugu organic matter and values determined for some comets, the notion of a cometary origin for Ryugu is certainly plausible. Moreover, a cometary origin for Ryugu proposes the advantage of not requiring a catastrophic impact of a rocky asteroid, which may cause significant heating and shock-related features within the resulting material, both of which are not observed In the Ryugu return samples.In terms of organic matter, Ryugu appears to contain CI-chondrite-like organic matter including its amino acids. Accordingly, it has been proposed that CI chondrites, such as Orgueil, may have a cometary origin due to the distinct differences between their amino acids and those of CM2 chondrites . FurtherThe Hayabusa2 mission to the asteroid Ryugu was the first mission to return samples from a primitive C-complex asteroid to Earth and has been a great success, with all of the primary scientific objectives having been completed. The array of techniques that have been applied to such a small sample size is truly exceptional. The Ryugu return samples contain abundant OM, which is similar in nature to that observed within primitive carbonaceous chondrites , but also shows some potential similarity to that thought to be contained within comets or IDPs. Among the most important findings is the discovery that Ryugu experienced heterogenous aqueous alteration and that this affected its organic materials, including some of the building blocks of life. Furthermore, the possibility that Ryugu amino acids are racemic, questions the role of astrophysical environments in the determination of single enantiomers being utilized by life on earth. 2/CH3 ratios need further investigation to better understand what processes and organic matter sources are responsible and if Ryugu is truly comparable in terms of its OM to carbonaceous chondrites observed so far. Therefore, in order to obtain the most from the Hayabusa2 sample return mission, future work should focus on the differences between different Ryugu particles and the ever-growing number of extraterrestrial samples contained within collections on Earth.Nevertheless, while a number of studies have now reported their findings concerning the OM contained within Ryugu, the limited sample size and associated challenges of carrying out analyses on such samples mean there are still many unknowns or ambiguities that need to be elucidated. For example, the reason why Ryugu shares a more similar N-heterocycle (including nucleobase) inventory to Murchison, compared to CI1 chondrites, the nature of the abundances and enantiomeric excesses of Ryugu-hydrolyzed amino acid extracts and the reason for the distinct functional group chemistry of Ryugu MOM/IOM remain to be resolved. Moreover, it still remains unclear whether irradiation has modified the surface OM inventory of Ryugu, or if planetesimal processes, such as aqueous alteration, or accretion of OM from heterogenous sources, are responsible for the variation observed between Ryugu particles in terms of their Raman responses and FOM/SOM contents. Additionally, the large variation in Ryugu MOM/IOM CHIn terms of the formation and evolution of Ryugu, both the catastrophic collision model and the cometary nucleus model provide potentially viable explanations for the origin of Ryugu, but further work is required to better constrain which model is most likely. Such future work should aim to achieve a better understanding of cometary OM and internal structure and include more advanced modeling of both the catastrophic disruption of planetesimals and the dynamic evolution of comets.


Hearing loss (HL) is one of the most common complications of the treatment in head and neck oncology. Most cases of HL are due to the ototoxicity of platinum-based chemotherapy (PBC) - resulting usually in a symmetric bilateral sensorineural hearing loss (SNHL) - or radiotherapy. Radiation-induced SNHL is progressive, permanent, and dose-dependent. Total dose and follow-up time are important factors affecting incidence rates. However, the hearing consequences of proton radiation therapy (PRT), a radiation-type therapy especially used in pediatric malignancies of the central nervous system (CNS), remains unclear and poorly documented. We report here a case of a four-year-old patient with unilateral auditory neuropathy spectrum disorder (ANSD) related to PRT. This case highlights the need for appropriate auditory monitoring in patients undergoing PRT for CNS or head and neck malignancies. Hearing loss (HL) is a common complication of cancer treatments, particularly in head and neck malignancies. They generally stem from a direct impact on the sensory cells of the Corti's organ, caused by the use of platinum-based chemotherapy (PBC) and resulting in an irreversible bilateral high-frequency sensorineural hearing loss (SNHL) ,2. ConveBragg peak, PRT could represent a safe alternative to XRT for pediatric tumors or other neoplasms arising next to critical organs at risk (OARs). Nevertheless, radiobiological uncertainties about the interaction of these charged particles with normal and neoplastic cells persist [We present a case of unilateral ANSD associated with PRT in a young patient with an atypical teratoid/rhabdoid tumor (AT/RT) of the posterior fossa. AT/RT is a rare aggressive central nervous system (CNS) tumor that typically affects children under the age of three and is associated with an unfavorable prognosis . The gol persist . HoweverAt the age of 12 months, this young male patient was admitted to the hospital due to incoercive vomiting. An MRI revealed a 44-mm tumor of the posterior fossa with associated hydrocephalus  was bilaterally normal. Posterior fossa MRI did not detect any vestibulo-cochlear nerve lesion. According to these results, a right PRT-induced ANSD diagnosis was suggested.No auditory improvement was identified in the following audiological evaluations, with this unilateral hearing impairment remaining stable for the past eight years.PBC is a common treatment for childhood cancer. Unfortunately, their use is burdened by short- and long-term side effects, such as hearing impairment. PBC-associated ototoxicity is related to the degeneration of mainly external hair cells as a consequence of oxidative stress and inflammation. HL is symmetric, bilateral, and sensorineural and affects first the higher frequencies . The deficit persists and, although it primarily manifests during treatment, HL can occasionally occur after chemotherapy discontinuation ,7,8. TheInterestingly, XRT, alone or in association with chemotherapy, has also been associated with SNHL, probably as a consequence of radiation-induced cochlear damage (if the cochlea is within the radiation field) . These XPRT is increasingly used in pediatric radiation oncology. The unique radiation dose deposition reduces the volume of healthy tissues receiving medium-to-low radiation doses, thereby reducing the potential risk of long-term toxicities and second primary cancer development in comparison with XRT ,15. HoweANSD is rarely identified in the hearing impairment occurring with head and neck cancer treatment. Rare cases of ANSD have been described after vincristine administration, all of which were bilateral and reversible within a few months after the completion of treatment ,19.In this clinical case, the presence of evoked otoacoustic emissions and cochlear microphonic potential indicates that the function of the cochlea’s outer hair cells was preserved. However, no identifiable waves I, III, and V were recorded in the BAEP, suggesting the diagnosis of ANSD. Most cases of unilateral ANSD are congenital and are detected early through neonatal hearing screening. They are usually associated with cochlear nerve aplasia. In this case, a congenital origin can be ruled out, since the newborn hearing screening using automated BAEP testing was normal. Furthermore, the unilateral and nonreversible nature of the ANSD does not support the hypothesis of vincristine toxicity. Clinical and vestibular examinations have not revealed any involvement of other cranial nerves, including the vestibular nerves. We, therefore, suggest that this clinical case is the first description of a PRT-induced ANSD. Given that audiometric monitoring during oncological treatments often depends on pure-tone audiometry and OEAP, particularly in children, our observation suggests that, especially in the case of PRT of the cephalic segment, an additional BAEP evaluation should be considered, especially when there are symptoms and/or parental concerns. Detecting these deficits is important for enhancing patients' quality of life, especially in children .Auditory deficits are a common complication of cephalic segment oncological treatment. Pathophysiologically, ANSD rarely accounts for posttreatment hearing impairments in these cancer cases. The uniqueness of the case we describe lies in the causal association between proton therapy and the development of unilateral ANSD. To the best of our knowledge, this is the first reported instance of such a case in the medical literature.


Cartilage conduction hearing aids (CC-HAs) are a novel type of hearing aid relying on cartilage conduction, the so-called third auditory conduction pathway. However, CC-HAs have only recently entered routine clinical use, and therefore data on their usefulness are lacking. The purpose of this study was to examine the possibility of assessing whether individual patients would show good adaptation to CC-HAs. Thirty-three subjects  underwent a free trial of CC-HAs. Age, disease category, and the pure-tone threshold of air and bone conduction, unaided field sound threshold, aided field sound threshold, and functional gain (FG) at 0.25, 0.5, 1, 2, and 4 kHz were compared between patients who subsequently purchased and did not purchase the CC-HAs. Overall, 65.9% of the subjects purchased CC-HAs after the trial. In comparison to non-purchasers, those who decided to purchase CC-HAs showed better pure tone hearing thresholds at high frequencies for both air conduction (2 and 4 kHz) and bone conduction , as well as for aided thresholds in the sound field  when using CC-HAs. Therefore, the high-frequency hearing thresholds of subjects trialing CC-HAs might be helpful for identifying those who are likely to benefit from them. The cartilage conduction pathway was first advocated as a third auditory conduction pathway by Hosoi in 2004 . The carIn Japan, CC-HAs have been in daily clinical use since November 2017, ahead of any other country in the world . CC-HAs At our institution, CC-HAs trials and fittings have been available since November 2017. In this study, we evaluated the factors that influenced the decision to purchase CC-HAs, including age, the pure-tone threshold of air and bone conduction before the CC-HA trial, and functional gain. In particular, the effect of bone-conduction hearing threshold on the likelihood of CC-HA purchase has not been assessed previously. The purpose of this study was to investigate the possibility of assessing patient adaptation to CC-HAs based on their demographic characteristics and hearing test results, etc.This study was approved by the Ethics Committee of Tohoku University Graduate School of Medicine (2022-1-1165), and informed consent was obtained in the form of opt-out on the website. All studies were conducted in accordance with the guidelines of the Declaration of Helsinki (1991). Thirty-three patients who requested trials of CC-HAs at our institution between November 2017 and July 2022, whose pure tone hearing thresholds with air and bone conduction were testable prior to the CC-HA trial, were included in this study. Of these patients, 20 were male, and 13 were female, with a mean age ± standard deviation (SD) of 33.12 ± 25.55 years (range 4–83 years). Audiological thresholds were measured by expert audiometric technicians in a standard sound-attenuated room using a commercially available audiometer . Pure-tone thresholds were obtained with over-ear headphones to assess AC (125 Hz to 8 kHz) and with a calibrated BC vibrator to assess BC (500 Hz to 4 kHz). Sound-field thresholds (SF) were measured to assess CC-HA-aided and unaided thresholds using warble tones delivered from a loudspeaker located 1 m from the subject at 0° azimuth. For patients with unilateral hearing loss fitted with a CC-HA in only the affected ear, we delivered 70 dB masking noise to the other ear through the headphones to prevent that ear from hearing the test sounds.An HB-J1CC  was used for all fittings. Transducers were chosen among ear-chip-embedded, ear-chip-attached, and simple types based on ear condition. The ear tip was made based on an ear mold, allowing for tight attachment to the ear and optimal stability of the transducer. On the other hand, the simple type used double-sided tape for fixation and thus could be applied for any ear condition, regardless of any ear abnormality. Subjects were allowed to try their fitted CC-HAs without charge at a follow-up visit two weeks to one month later. At the follow-up visit, they were asked to assess the utility and comfort of the CC-HAs in their daily activities using the speech, spatial, and qualities of hearing scale (SSQ) questionnaires  and to uThe results obtained were compared between purchasers and non-purchasers in terms of age, disease category, SSQ score, the pure-tone threshold of air and bone conduction, the unaided and aided sound field threshold, and functional gain (FG) at 0.25, 0.5, 1, 2, and 4 kHz.p < 0.05.Mann–Whitney U test, chi-square test, and analysis of variance (ANOVA) were employed for statistical analyses using GraphPad Prism 7.0 . Bonferroni tests were used for post hoc comparisons in ANOVA. The statistical significance level was set at This study evaluated the results of the CC-HA trial in 41 ears (33 patients). CC-HAs were purchased for 27 ears (19 patients) and not purchased for 14 ears (14 patients), giving an overall purchase rate of 65.8% for ears that trialed the CC-HAs. p < 0.05; Mann–Whitney U-test).p = 0.033) and purchase rate  = 14.66, p < 0.001). The interaction between frequency and purchase rate was significant  = 3.366, p < 0.05). Post hoc comparisons showed that purchasers had significantly better air conduction thresholds than non-purchasers at 2 kHz and 4 kHz (p < 0.05 post hoc Bonferroni test). For the bone conduction thresholds in p < 0.001) and purchase rate  = 52.24, p < 0.001). The interaction between frequency and purchase rate was not significant  = 1.651, p = 0.164). Post hoc comparisons showed that purchasers had significantly better bone conduction thresholds than non-purchasers at 1 kHz, 2 kHz, and 4 kHz (p < 0.01 post hoc Bonferroni test).For the air conduction thresholds in p = 0.038) and purchase rate  = 15.15, p < 0.001). There was no significant interaction between frequency and purchase rate  = 0.574, p = 0.68).p < 0.05 post hoc Bonferroni test). For the aided thresholds in the sound field in p = 0.0036) and purchase rate  = 42.90, p < 0.001). The interaction between frequency and purchase rate was not significant  = 0.876, p = 0.480). Post hoc comparisons showed that purchasers had significantly better bone conduction thresholds than non-purchasers at 1 kHz, 2 kHz, and 4 kHz (p < 0.05 post hoc Bonferroni test).Post hoc comparisons showed that purchasers had significantly better unaided thresholds in the sound fields than non-purchasers only at 4 kHz (p = 0.036) and purchase rate  = 42.90, p < 0.001), but no significant interaction between frequency and purchase rate  = 0.876, p = 0.48). Further post hoc comparisons showed that purchasers did not have significantly higher FG than non-purchasers at any frequency (p < 0.05 post hoc Bonferroni test).p < 0.05; Mann–Whitney U-test). On the other hand, SSQ spatial did not differ significantly between purchasers and non-purchasers.This study aimed to evaluate the efficacy of CC-HAs for hearing-impaired patients who were unable to use conventional air- or bone-conduction hearing aids, and the factors that contribute to the decision to purchase them. In particular, we were interested in whether test results obtained prior to the start of CC-HA use, such as those of pre-trial pure tone audiometry, could be used to assess whether patients would adapt well to CC-HAs.The overall rate of CC-HA purchase in this study was 65.9% 27/41 ears), which is within the ranges reported previously /41 ears,. In the In the present study, CC-HA purchasers were significantly younger than non-purchasers. Regarding the relationship between purchase rate and age, it has been reported that purchasers are significantly younger than non-purchasers among patients with hearing loss due to unilateral atresia auris ,10. ThisIn this study, more than 80% of the subjects trialing CC-HAs had aural atresia/ear canal stenosis. In previous studies, CC-HAs have been used most frequently in patients with congenital atresia or acquired atresia due to surgical treatment, such as ear canal cancer, and have been reported to improve hearing ,14,15,16With regard to pure tone hearing thresholds, these were significantly better in purchasers than in non-purchasers at frequencies of 2 kHz and 4 kHz for air conduction and at 1 kHz, 2 kHz, and 4 kHz for bone conduction. This means that for both air conduction and bone conduction, the pure tone hearing thresholds at higher frequencies were significantly better in the individuals who purchased CC-HAs than in those who did not. Previous reports have often compared CC-HAs unaided and aided with sound field thresholds ,7,9,10, For aided thresholds in the sound field, these were significantly better among CC-HA purchasers than among non-purchasers at 1 kHz, 2 kHz, and 4 kHz. Previous reports suggested that purchasers had significantly better sound field assistance thresholds than non-purchasers at lower frequencies of 0.25 kHz and 0.5 kHz, in contrast to the present results. However, one study comparing the transmission efficiency of cartilage conduction (CC), air conduction (AC), and bone conduction (BC) revealed that the threshold increases were significantly better for BC than for CC at frequencies of 1 kHz and 2 kHz . TherefoNo significant difference in FG was found between purchasers and non-purchasers at any frequency, suggesting that threshold increases in the two groups were similar. Previous reports have also indicated that CC-HAs improved hearing thresholds at all frequencies, regardless of the purchase outcome of CC-HA trials ,7,17. InIn the present study, we evaluated hearing aid use using the SSQ questionnaire to assess the usefulness and comfort of the CC-HAs. Purchasers had significantly higher SSQ speech and SSQ quality than non-purchasers. On the other hand, SSQ spatial was not significantly different between purchasers and non-purchasers, but there was a tendency for advantages among purchasers. Although the evaluation of CC-HAs using questionnaires has been studied in the past using “Evaluation of hearing before and after wearing a hearing aid, ” there aThe limitations of this study included its small sample size, lack of speech audiometry assessment, and absence of any comparison between CC-HAs and other hearing aids . It has already been reported that speech audiometry assessments are improved, as well as the hearing threshold ,17. The This study investigated the factors influencing the decision of patients to purchase CC-HAs on the basis of trials performed in our department. Overall, 62.2% of the subjects purchased CC-HAs after the trials. Purchasers had better air-conduction and bone-conduction thresholds for pure tone hearing thresholds than non-purchasers at high frequencies, as well as for aided thresholds in the sound field when fitted with CC-HAs. Hearing-impaired patients with better pure tone hearing thresholds at relatively high frequencies may be better candidates for CC-HAs. However, there are still few reports investigating the clinical adaptation of CC-HAs. Further comparisons with other types of hearing aids, such as AC-HAs and BC-HAs, are needed.