Clostridioides difficile (C. diff.) infection (CDI) is a leading cause of hospital acquired diarrhea in North America and Europe and a major cause of morbidity and mortality. Known risk factors do not fully explain CDI susceptibility, and genetic susceptibility is suggested by the fact that some patients with colons that are colonized with C. diff. do not develop any infection while others develop severe or recurrent infections. To identify common genetic variants associated with CDI, we performed a genome-wide association analysis in 19,861 participants  from the Electronic Medical Records and Genomics (eMERGE) Network. Using logistic regression, we found strong evidence for genetic variation in the DRB locus of the MHC (HLA) II region that predisposes individuals to CDI . Altered transcriptional regulation in the HLA region may play a role in conferring susceptibility to this opportunistic enteric pathogen. Clostridioides difficile (C. diff.) infection (CDI), formerly known as Clostridium difficile infection, is the leading infectious cause of nosocomial diarrhea in North America and Europe and is associated with a high global burden of disease1. Once acquired, this reemerging, Gram-positive, spore-forming bacterium secretes a toxin that causes watery diarrhea, sometimes progressing to severe pseudomembranous colitis, toxic megacolon, and sepsis2. In the early 2000s, the emergence of C. diff. strain NAP1/BI/027 led to increased incidence, prevalence, morbidity, and mortality associated with CDI4. This epidemic strain produces more toxin, has a higher resistance to common treatments, and causes more recurrent infections than other common C. diff. strains. Despite aggressive antibiotic treatment  and fecal transplant6, outcomes of NAP1/BI/027 CDI include significant morbidity across all age groups, 5% mortality in individuals older than 65 years of age, and an estimated $1.1 billion dollars per year in healthcare costs2.C. diff. is common among patients in healthcare settings, with an estimated prevalence of 3–26% in adults admitted to acute care hospitals and 5–7% in adults at long-term care facilities7. Progression from C. diff. colonization to acute CDI is generally associated with one or more risk factors8, including new exposure to C. diff., older age, hospitalization or nursing home residency, chemotherapy, severe comorbid illness, proton pump inhibitor or immunosuppressant medication use, or prior use of high-risk antibiotics such as fluoroquinolones or cephalosporins11. Antibiotic use and proton pump inhibitor use are also risk factors for recurrent CDI12. Despite having one or more risk factors, some people colonized with C. diff. either do not develop CDI or successfully clear an initial infection, while other individuals are burdened by severe and/or recurrent CDI. This differential susceptibility may have a genetic component, given that host genetic variation underlies susceptibility for other infections, including enteric infections such as Helicobacter pylori13. Identification of host genetic susceptibility loci could yield methods for prevention and/or treatment of this important pathogen15.Asymptomatic colonization with 16 performed a genome-wide logistic regression analysis of CDI in 646 patients  undergoing stem cell transplantation for multiple myeloma, and found several single nucleotide variants (SNVs) in the RLBP1L1, ASPH, and P7B genes that were associated with higher risk of CDI. Shen et al.17 identified two alleles in in the extended major histocompatibility complex  that were associated with a reduction in CDI recurrence among 704 patients who achieved initial clinical cure with bezlotoxumab treatment in the MODIFY clinical trials. Several studies have also suggested that common SNVs in the promoter region of the interleukin-8 (IL-8) gene may confer increased risk for recurrent CDI by altering neutrophil recruitment during disease pathogenesis19. While these results are collectively suggestive of genetic involvement in CDI risk, the aforementioned studies had small sample sizes and did not always control for major risk factors such as previous antibiotic use or corticosteroid use in their association models. Genome-wide association studies (GWAS) that properly control for known risk factors and include a large number of participants are needed to identify risk loci with sufficient power and reliability. One such study identified 16,464 patients  from the Geisinger MyCode cohort20 using a C. diff. phenotyping algorithm developed by the Electronic Medical Records and Genomics (eMERGE) Network21. While no variants reached genome-wide significance in the full case–control dataset, one variant (rs114751021) in the small nucleolar RNA SNORD117 gene, located in the MHC region, reached genome-wide significance in a subset of cases and controls with recent exposure to antibiotics . Additional validation studies in other large patient cohorts are needed to evaluate the role of genetic factors in CDI risk.Previous studies have identified candidate risk loci for primary and recurrent CDI in small patient populations using a combination of genetic and clinical data. Apewokin et al.22. At the time of this study, the network included a multi-ethnic cohort of roughly 99,000 U.S. participants with linked genetic and EMR data.To identify common genetic variants associated with susceptibility to CDI, we performed joint and ancestry-stratified GWAS and human leukocyte antigen (HLA) fine-mapping using phenotypes extracted from electronic medical records (EMRs) of participants aged two years or older from the eMERGE Network. The eMERGE Network is a National Human Genome Research Institute (NHGRI)-funded consortium of twelve study sites across the United States (U.S.) that supports research for furthering the implementation of genomic medicineC. diff. phenotyping algorithm , which reflected the patient populations of the participating eMERGE study sites. Controls also tended to have higher BMIs than cases . Cases had slightly higher exposure to Class 1 (high-risk) antibiotics than controls (28% vs. 21%), yet they had much less exposure to Class 2 (moderate risk) antibiotics than controls (11% vs. 26%). More cases received chemotherapy outside of the exclusionary time period than did controls. It is worth noting that while 14 cases were identified from Cincinnati Children’s Medical Hospital, no controls were identified from this site. These cases were 57% female, with a median age of 4.0 (IQR 3.0–12.5) years and a median BMI of 16.09 (IQR 14.90–17.00). Approximately 93% of these cases were of European ancestry  and tended to be at high risk for C. diff. infection, with 50% having recent exposure to Class 1 or Class 2 antibiotics and 43% having recent exposure to transplant medications.After all exclusions, there were 1349 cases and 18,512 controls identified via the eMERGE After finding the intersection of self-reported ancestry and genetically determined ancestry, there were 3700 African participants, 14,620 European participants, and 135 Asian participants. Table P = 8.06 × 10–14; rs3828840, P = 9.96 × 10–14; rs35882239, P = 8.18 × 10–12; rs71534541, P = 5.12 × 10–11; rs35222480, P = 9.88 × 10–11) mapped to the intergenic region between the HLA-DRB5 and HLA-DRB1 genes in the beta block of the MHC Class II region. Three of the five most significant SNVs , with minor allele frequencies (MAFs) of 0.17, 0.17, and 0.20, respectively, also mapped to the 3ʹ end of the HLA-DRB6 pseudogene. A review of the NHGRI-European Bioinformatics Institute (NHGRI-EBI) GWAS Catalog23 and dbSNP24 revealed that rs3828840 has been previously associated with multiple sclerosis, an autoimmune inflammatory disease that impacts the central nervous system25.Table 26, a regional LD plot with reference to the index SNV (rs68148149) was generated using P-values from the European logistic regression analysis and using the 2014 1000 Genomes European superpopulation as a reference group , the index SNV had the highest regulatory potential among the most significantly associated SNVs, as annotated by RegulomeDB27. To assess the possibility that the lack of disease association in the African ancestry sample is a result of different regional LD structures, a regional LD plot with reference to the index SNV was generated using the 1000 Genomes African superpopulation as a reference  than in the European superpopulation (R2 > 0.2). On the other hand, lower LD was observed with SNVs in the region spanning HLA-DRB1 and HLA-DQA1 in the African superpopulation (R2 > 0.6) than in the European superpopulation (R2 > 0.8). Differences in regional LD patterns between the European-ancestry and African-ancestry samples could therefore have contributed to the observed differences in gene-disease association patterns, in addition to insufficient detection power.Given the well-known presence of high LD within the HLA regionP = 4.54 × 10–9; rs9270896, P = 6.09 × 10–9; rs9270894, P = 1.12 × 10–8; rs9270895, P = 2.32 × 10–8; rs618095, P = 3.71 × 10–8)  Table , S8. WhiHLA-DRB3, 4 and/or 5 gene 28 , and among individuals with at least one DRB5 gene only, or DR51 haplotype carriers  , and more specifically, carriers of the HLA-DRB1*15:01 allele . No SNVs reached genome-wide significance among participants with at least one DRB3 or DRB4 gene only, suggesting that the HLA-DR51 haplotype in combination with variants in the HLA-DRB1/5 intergenic region may singularly drive genetic risk for CDI in the European ancestry population. However, examining the risk allele frequencies of the index SNV (rs68148149) in cases and controls across DR51, DR52, and DR53 haplotype-enriched groups showed that the risk allele frequency was higher in European-ancestry cases than controls in all haplotype groups, suggesting that the SNV may indeed drive risk in all HLA-DR haplotype groups but that the low frequency in the DR52 and DR53 haplotype groups limits the power to detect the association in these groups  Table . Among D29 and the NCI LDMatrix tool30, it was also confirmed that none of the GWAS-identified SNVs were in high LD (R2 > 0.5) with any classical HLA alleles in either the European ancestry or African ancestry 1000 Genomes superpopulations. The index SNV was in moderate LD with the tag SNV for the DRB1*15:01-DRB5*01:01 haplotype in the European ancestry superpopulation  and low LD with the tag SNV in the African ancestry superpopulation .To assess the possibility that one or more HLA alleles themselves were driving the risk association in the European ancestry sample, rather than the most significantly associated SNVs identified in the GWAS, we performed a separate logistic regression analysis using the HIBAG-imputed HLA genotypes in the European ancestry sample. None of the imputed HLA alleles reached genome-wide significance. Using the classical HLA tags identified by de Bakker et al.(HLA-)DRB locus of the HLA region may increase risk of infection in European ancestry populations. In this study, European participants who possessed the minor allele among the most significantly associated SNVs had 56% greater odds of having at least one episode of CDI. As the key beta-subunits of MHC Class II surface receptors on antigen presenting cells (APCs), the proteins encoded by DRB genes play a critical role in stimulating the host adaptive immune response against foreign peptides and are therefore excellent candidates for future studies of host immunity to C. diff.31.Using a robust EMR-based phenotyping algorithm, we identified a large, multi-institutional corpus of patients with a history of at least one episode of CDI and controls without CDI. Our results suggest that genetic variation in the DRB genes are particularly variable in copy number and combination. Although there is only one monomorphic DRA gene per (HLA-)DR haplotype, there are five common DR haplotype families composed of different combinations of protein coding DRB genes  and pseudogenes 28. DRB1 is present in all haplotypes, but any given individual may have as few as two protein coding DRB genes (2 copies of DRB1), or as many as four genes  between homologs. The unique combination of DRB genes on each haplotype is remarkably conserved and has been maintained in ancestral DNA since before the divergence of human and gorilla lineages over five million years ago32. Although having a diverse set of MHC II molecules may confer a selective advantage against infection33, each additional DRB gene is nonetheless susceptible to intragenic and/or regulatory mutations in the highly polymorphic HLA region and may paradoxically increase susceptibility to other diseases. In the case of gastrointestinal infections, protective effects of the DRB1*04:05 allele against enteric infection caused by Salmonella typhi or Salmonella paratyphi have been observed in Vietnamese and Nepalese patients34. Conversely, the DRB1 gene has also been implicated in increasing host susceptibility to a number of inflammatory diseases, including Crohn’s disease, type I diabetes mellitus, rheumatoid arthritis, multiple sclerosis (MS), ulcerative colitis and Alzheimer’s disease, primarily in European populations40.The MHC (HLA) Class I and II loci are among the most polymorphic coding regions in the human genome, and DRB1*15:01-DRB5*01:01 haplotype41, and individuals in this group had 200% higher odds of developing CDI on average. These results indicate that the DRB1*15:01-DRB5*01:01 haplotype is involved in conferring CDI risk among individuals with common genetic variants in the tagged DRB1-DRB5 intergenic region  antibodies play an essential role in shaping an individual’s gut microbial community and maintaining a homeostatic balance of microbes within the mucosal immune system49, and the interactions between APCs and CD4+ T-follicular helper (Tfh) cells are key to driving the production of IgA by plasma cells50. Studies in mouse models have previously demonstrated that MHC II polymorphisms directly affect antibody-mediated microbiota composition, and that the unique microbial communities formed under the influence of different MHC genotypes can impact an organism’s susceptibility to opportunistic pathogens like Salmonella enterica typhimurium when treated with antibiotics52. Understanding the unique interactions between commensal microbe antigens presented by APCs, the MHC II molecules encoded by the DRB1*15:01-DRB5*01:01 haplotype, and Tfh cells may provide valuable insights into how host genetics impact the composition of gut microbial communities in individuals susceptible to enteric infection, compared with those who are resistant to infection.One possible explanation for increased CDI risk among these individuals is that differential MHC II gene expression impacts the baseline composition of their gut microbiota, thereby influencing colonization resistance to opportunistic enteric pathogens like C. diff. bacteria. In addition to sculpting the host microbiota, high affinity IgA helps to neutralize bacterial toxins53. Unique interactions between T-cells and C. diff. toxins specifically bound by DRB1*15:01-DRB5*01:01 MHC II molecules may impact the host anti-toxin IgA response differently than other T-cell-MHC II interactions, thus influencing the host’s ability to clear circulating toxins. Recent Phase III, placebo-controlled clinical trials of the monoclonal antibody treatments actoxumab (anti-TcdA) and bezlotoxumab (anti-TcdB) showed that TcdB toxin neutralization alone could decrease CDI recurrence by 38% among patients receiving standard antibiotic therapy for initial or recurrent CDI54. Naturally occurring anti-TcdB antibodies in the placebo group also conferred protection against recurrent CDI, recapitulating the importance of neutralizing TcdB in controlling infection55. However, other studies have failed to replicate these results when comparing healthy controls with CDI patients, suggesting that anti-toxin antibody concentrations may not fully explain susceptibility to initial and/or recurrent infection56.Alternatively, increased CDI risk among these individuals may be driven by differential T-cell mediated responses to the TcdA and TcdB toxins produced by DRB1*15:01-DRB5*01:01 haplotype is allele-specific DNA methylation of the DRB1 and/or DRB5 regulatory regions, given that that targeted bisulfite sequencing has previously identified the DRB1-DRB5 intergenic space as a differentially methylated region57. Disruptions to normal DNA methylation patterns, and to resulting gene expression, have been known to modulate susceptibility to a number of human diseases58. For example, in the case of DRB1*15:01-DRB5*01:01-associated multiple sclerosis, DNA hypermethylation in exon 2 of DRB1 confers protection against the major risk allele and is driven by several SNVs in high LD with one another that overlap with CpG sites59. It is possible that disrupted methylation patterns at or near the regulatory regions of DRB1*15:01 and/or DRB5*01:01 also contribute to differential expression of these MHC II proteins, thus impacting the landscape of the host adaptive immune response via microbiome-mediated and/or toxin-mediated mechanisms. Additional gene expression analyses, such as expression quantitative trait loci (eQTL) analysis, could be used to explore whether the top SNVs regulate expression levels of nearby genes.Although the MHC II region is strongly associated with CDI in this study, the SNVs that confer risk are neither located in coding regions, nor in high LD with SNVs in coding regions, suggesting that the mechanism for altered gene expression may be regulatory. One possible mechanism for altered expression of the C. diff. GWAS conducted by Li et al. using the MyCode cohort, they do support the hypothesis that immune molecules encoded within the MHC region are involved in CDI pathogenesis. Third, C. diff. cases were not stratified by primary and recurrent CDI, and it is possible that the genetic variants driving pathogenesis are different between these two forms of infection. For example, Shen et al. identified alleles in DRB1 and DQA1 that were different from those identified in this study and were protective against CDI recurrence, suggesting that the genetic factors involved in initial vs. recurrent infection could be distinct from one another. Fourth, the length and severity of infection were not considered in the current study, but future analyses would benefit from continuous trait regression analyses to identify genetic variants associated with increased CDI length and/or severity, rather than susceptibility. Additionally, C. diff. cases in this study included individuals with a positive antigen test as their only criterion for infection. The C. diff. antigen test cannot accurately distinguish between toxigenic and non-toxigenic strains and may falsely identify asymptomatic carriers as C. diff. cases. Finally, the specific toxigenic ribotype that each case was exposed to was not included in the analysis, and it is possible that different C. diff. ribotypes are associated with different genetically determined host responses.This study has several important limitations. First, sample size and statistical power were severely limited among non-European ancestry samples, which may have contributed to the lack of significant associations in the African ancestry analyses. It is also possible that within the European sample, the comparatively low frequency of the risk allele in the DR52 and DR53 haplotype groups, compared to DR51, limited the power to detect a true risk association in other DR haplotype groups. Second, replicate studies are needed to confirm the identified association. However, the large, multi-site biobank of linked EMR and genotype data used in this study supports the replicability and reliability of these results, and future association studies would benefit immensely from these types of biobanks. While the gene associations in this study do not align exactly with those identified in the previous DRB1*15:01-DRB5*01:01 haplotype. Interactions between DRB1*15:01-DRB5*01:01 MHC II molecules, C. diff. exotoxins and T-cells may alternately play a critical role in CDI pathogenesis, and additional work is needed to understand whether and how the host IgA response is differentially impacted by the combined effects of haplotype and transcriptional modifications. Finally, future work should address the possibility that allele-specific DNA methylation is a driver of epigenetic transcriptional regulation of the DRB1 and/or DRB5 genes. If this mechanism is experimentally validated, therapeutics that modulate MHC II molecule transcription levels could potentially be developed to decrease the incidence of CDI among individuals who carry the risk genotype.Our findings suggest that genetic variation in the MHC II locus of the HLA region drives susceptibility to CDI and highlights the importance of the adaptive immune response in combating opportunistic pathogens. To better understand how host genetics might confer microbiome-mediated risk for opportunistic enteric infections, future studies should explore the mechanisms of interaction between commensal microbe antigens presented by APCs and the MHC II molecules encoded by the Cases and controls were selected from among the ~ 99,000 participants of the eMERGE Network. Participating sites included the following: 1. The Children’s Hospital of Philadelphia, Philadelphia, PA; 2. Cincinnati Children’s Medical Hospital, Cincinnati, OH; 3. Columbia University, New York, NY; 4. Geisinger, Danville, PA; 5. Mass General Brigham, Boston, MA; 6. Kaiser Permanente Washington  and University of Washington partnership, Seattle, WA; 7. Marshfield Clinic, Marshfield, WI; 8. Mayo Clinic, Rochester, MN; 9. Meharry Medical College, Nashville, TN; 10. Mount Sinai, New York, NY; 11. Northwestern University, Evanston, IL; and 12. Vanderbilt University, Nashville, TN. Informed consent was obtained from participants by each eMERGE site. The eMERGE study was approved by each participating site’s institutional review board, and all methods were performed in accordance with the relevant guidelines and regulations at each institution.Clostridioides difficile cases and controls were selected using a variety of information contained in the EMR, including International Classification of Disease (ICD) Clinical Modification (CM) codes 9th and 10th editions, lab and medication data, and clinician progress notes. The C. diff. phenotyping algorithm used in this study was designed collaboratively by the University of Washington, Group Health and Vanderbilt as part of the eMERGE Network and was published in the Phenotyping KnowledgeBase (PheKB) in 201261. Case/control selection and exclusion criteria are depicted as a flowchart in Fig. C. diff. antigen or toxin test were selected. Second, those with one or more inpatient or outpatient diagnoses of C. diff. , followed by one or more days of medication for treatment , followed by another inpatient or outpatient C. diff. diagnosis code, were selected. Third, individuals with at least one C. diff. ICD-CM code combined with at least one affirmative mention  of C. diff. infection in a clinical progress note as identified through natural language processing (NLP), were selected. The C. diff. mentions used by the NLP algorithm are listed in Supplementary Table C. diff. infection on separate calendar days in clinical progress notes, identified by NLP, were selected. To exclude severely immune-compromised participants from the test population, participants meeting one of the four above criteria were excluded from being cases if they had a diagnosis of bone marrow cancer in the 2-year period prior to their C. diff. case index date , or within 7 days following their index date. Participants were also excluded from being cases if they had received chemotherapy in the 180-day period prior to their C. diff. index date, or within 7 days following their index date. Using these criteria, 1598 cases were selected.For participants aged two years or older, there were four combinations of EMR data considered for case selection. First, individuals with a positive C. diff. in their records. Since C. diff. toxin tests have sensitivities ranging from 60 to 70%62, a single test does not rule out disease, and multiple tests could signal a concern that disease exists. Additionally, controls must have had at least one hospital admission with a prior exposure to a high-or moderate-risk antibiotic , or within seven days following the index date. These criteria resulted in the selection of 23,061 eMERGE participants as controls.Controls were selected from eMERGE participants two years of age or older who had no known test for and no diagnosis codes for 2 > 0.3)63.We excluded 202 cases and 2723 controls that were missing genotype data. An additional 31 cases and 889 controls were excluded because the genotype imputation quality failed to meet our quality control (QC) threshold  between cases and controls.Cryptic relatedness was assessed in all participants by calculating the probabilities of sharing alleles identical by descent (IBD), where Z0 is the probability of sharing zero alleles IBD and Z1 is the probability of sharing one allele IBD. Families were constructed when sample pairs had Z0 < 0.83 and Z1 > 0.1The following covariates were identified for all cases and controls using structured EMR data: 1. Age at index date (index age); 2. Body mass index (BMI); 3. Sex; 4. Genetically determined ancestry; 5. Nursing home status (y/n); 6. Chemotherapy (y/n); 7. Diabetes mellitus (y/n); 8. Human immunodeficiency virus (HIV) positive status (y/n); 9. Any transplant medications (y/n); 10. Any corticosteroid medications (y/n); and 11. Any medium or high-risk antibiotic exposure (y/n). We used the median BMI record for the age year that matched most closely to the participant’s index age. Nursing home status was determined either by structured data on skilled nursing facility residence, or by mentions of nursing home status in social work and case management notes, as identified by NLP  as the reference set. The majority of samples from the 13 eMERGE sites were genotyped on the Human 660 Quad (eMERGE-I). Other genotyping platforms included the CytoSNP-850K BeadChip, the OmniExpress chip, the Affymetrix 6.0 array, and the Illumina MEGA among others. In this analysis, variants with an allelic R2 ≥ 0.3 and minor allele frequency (MAF) ≥ 0.05 were included. Additional QC filters were applied as described in case–control selection.Genotypes for all participant samples from eMERGE-I, eMERGE-II and eMERGE-III were imputed using the Michigan Imputation Server66. Variants with ≥ 0.05 MAF, missingness of ≤ 0.1 and LD-pruned R2 threshold of 0.7 were included in the multisample analysis. K-means clustering of Principal Component (PC) 1 and PC2 identified three groups  was used to find genetically determined ancestry of each sample. Genetically determined and self-identified ancestry were checked for concordance, and samples were ultimately grouped into African ancestry, Asian ancestry, and European ancestry clusters. IBD was calculated for all pairwise sample comparisons using the plink –genome function, and cryptic relatedness between samples was assessed as described in case/control selection.The set of ~ 99,000 unique imputed samples was analyzed by Principal Component Analysis (PCA) using the PLINK 2.0 software67. All covariates and genotypes were used in the joint analysis of all participants, whereas the PC1 and PC2 covariates for the African and European ancestry-stratified analyses were derived from ancestry specific PCA analyses. An additive genotypic model of SNV genotypes coded as 0, 1 or 2 copies of the minor allele was used. The regional LD plots of the index SNV were created using the LocusZoom web-based tool68. Following the initial stratified analyses, an additional logistic regression-based association analysis was performed in the European sample using the index SNV as a covariate to determine whether this SNV was truly driving the risk association.To identify genetic variants associated with CDI, we performed logistic regression-based association analyses for the case/control curated phenotype using PLINK 1.9069. HLA-DRB3, 4 and 5 gene dosages were inferred based on the HLA-DRB1 alleles present in each individual, as described in Habets et al.70. Calls were quality-filtered for a HIBAG posterior probability of > 0.5.Classical HLA alleles were imputed against four ancestry-specific reference panels  using the HIBAG softwareDRB3 gene; 2. DRB4 gene; 3. DRB5 gene; or 4. any of the above genes in each participant. Haplotype subgroups were further divided into DR15 and DR16 haplotype carriers , and DRB1*15:01 carriers (stemming from the DR15 haplotype). Logistic regression-based association analysis was performed separately in each haplotype subgroup, using the same covariates described in “Methods: GWAS” for the European ancestry sample.To test for haplotype-specific effects of the most significantly associated SNVs, four overlapping participant subgroups were selected from the European ancestry sample based on the presence of at least one of the following: 1. To test for HLA alleles driving the association, case–control logistic regression-based association analysis was performed in the European ancestry population sample for 276 classical HLA alleles, using the same covariates described in “Supplementary Information.Supplementary Table S2.


Previous studies have shown that a small percentage of people in the general population have atypical gender identity and/or sexual orientation.This study aimed to explore variations in gender identity and sexual orientation in university students and determine genetic factors associated with these variations.AR) and a SKAT-O: an optimal unified sequence kernel association test, which is an exome-based rare variant association study. The results of the genetic tests were compared with the Japanese reference data and the results of our 637 control samples.Deviations from complete gender congruence and exclusive heterosexual orientation in 736 Japanese university students were quantitatively assessed with self-assessment questionnaires. Next, we conducted genetic tests for 80 participants who showed relatively low gender identity scores and/or atypical sexual orientation. These genetic tests consisted of repeat number analysis of the androgen receptor gene (We calculated the gender identity and sexual orientation scores of all participants and analyzed the molecular data of 80 selected participants.AR repeat numbers. The SKAT-O showed that rare damaging variants of TDRP and 3 other genes were more common in the 80 participants than in the control group.The gender identity scores of 736 participants were broadly distributed: only ~15% of natal males and ~5% of natal females had the maximum score that corresponds to complete gender congruence. The sexual orientation scores also varied: ~80% of natal males and ~60% of natal females showed exclusive heterosexual orientation. We found no association between gender characteristics and Our data support the view that gender is a phenotypic continuum rather than a binary trait.This study quantitatively assessed the gender characteristics of a large cohort of university students. Moreover, we conducted systematic screening for genetic factors associated with gender variations. The weaknesses of the study were the limited analytic power of the questionnaires, the relatively small sample for molecular analyses, and incomplete clinical information and relatively advanced ages of the control group.TDRP or other genes.This study revealed significant variations in gender identity and sexual orientation in university students, which may be partly associated with variants in  Gender identity and sexual orientation are fundamental components of sexual identity.AR) on Xq12 have been linked to gender incongruence by several researchers.,,Gender identity and sexual orientation are known to be determined by genetic, hormonal, and neuroanatomic factors, as well as by sociocultural and experiential factors.In the present study, we addressed 2 questions: (1) Are there any variations in gender identity and sexual orientation in the general population? (2) Are gender identity and sexual orientation driven by the same genetic factors? To this end, we conducted a quantitative assessment of gender identity and sexual orientation for 736 university students. Subsequently, we performed genetic tests for participants with relatively low gender identity scores and/or atypical sexual orientation to determine genetic factors associated with these variations.The participants of this study were recruited from 3 universities in Japan. We announced this project to students after lectures. These lectures were randomly selected from the university programs. We invited all students >20 years of age, and they freely decided whether to participate. Only students who provided written informed consent were included in this study. As a result, 736 students voluntarily participated. They anonymously filled out paper-based questionnaires and provided saliva samples for genetic analysis. Each participant received 1000 yen for his or her efforts in sample preparation. The participants were classified according to assigned sex at birth into the male group (n = 313) and female group (n = 423) . To assess the results of genetic analyses, we consulted Japanese reference data and our in-house data from healthy Japanese individuals.This study was approved by the Institutional Review Board Committee at the National Center for Child and Development and performed after obtaining informed consent from all participants. The questionnaires and saliva specimens were anonymized immediately after sampling.,The gender identity and sexual orientation of the 736 participants were assessed with paper-based self-assessment questionnaires.First, we analyzed the gender identity of each participant—specifically, whether one’s self-recognized gender was man, woman, or nonbinary. Then, the participants were asked to provide answers to 15 items  those whose self-recognized gender was opposite to the assigned sex, (2) those who answered that his or her self-recognized gender is nonbinary, and (3) those whose gender identity scores were below the fifth percentile of all participants of this cohort with the same assigned sex.Next, we searched for participants with a tendency toward same-sex sexual orientation. From the male group, we chose (1) participants whose scores were 5 to 8 or “nonbinary” for at least 1 of the 3 items and (2) participants who selected a score of 4 for ≥1 of the 3 items and did not select a score of 1 or 2 for the remaining items. From the female group, we selected (1) participants whose scores were 1 to 3, 8, or “nonbinary” for at least 1 of the 3 items and (2) participants who selected a score of 4 for ≥1 of the 3 items and did not select a score of 6 or 7 for the remaining items.AR with an ABI Prism 3130 Genetic Analyzer (Applied Biosystems) and GeneMapper 3.7 software (Applied Biosystems). The methods were reported previously.AR alleles on the two X chromosomes. The results of the participants were compared with the reference data of Japanese individuals.Saliva samples were collected between January 2019 and February 2020. Genomic DNA was extracted with the Oragene-Discover kit (DNAgenotek). First, we analyzed the CAG repeat number polymorphism in ,,,http://database.liulab.science/dbNSFP/). Common polymorphisms whose allele frequency in the ToMMo databasehttps://www.megabank.tohoku.ac.jp/) is >1.0% were excluded from further analysis. The results were adjusted through Bonferroni correction, and adjusted P values <.05 were considered significant.Second, we conducted an optimal unified sequence kernel association test (SKAT-O)U test. The frequencies of sequence variants between the participant and control groups were compared with the Fisher exact test. Both analyses were performed with R (version 3.3.2).Statistical differences in the median scores between the male and female groups were assessed by the nonparametric Mann-Whitney P = .31). One from the female group had already been recognized as a transgender person. The results of the 726 individuals are shown in P = 1.8 × 10–10 and P = 3.5 × 10–6, respectively).In 726 of 736 participants (98.6%), self-recognized gender was consistent with sex assigned at birth, whereas 2 of 313 males and 7 of 423 females were assessed as having nonbinary gender . An exclusive same-sex sexual orientation was observed in 4 males and 1 female .Sexual orientation scores were obtained from 313 males and 423 females. Of these, 13 males and 90 females were excluded from data analyses because they had nonbinary gender or uncertain sexual orientation or lacked sexual experience at least for 1 of the 3 items. The results of 633 individuals  are shown in We selected 83 participants who had relatively low gender identity scores and/or atypical sexual orientation. Of these, 29 and 35 had low gender identity scores and atypical sexual orientation, respectively, while 19 showed both features. Among them, 80  were subjected to molecular analyses; the remaining 3 were not examined because the amounts of DNA samples were insufficient for systematic sequencing.AR in the 80 selected participants. The mean ± SD of the repeat number in the male and female groups was 19.2 ± 2.55 and 19.5 ± 2.23, respectively . K.M.: data curation, investigation, writing (review and editing). H.O.-K.: investigation, software, writing (review). M.M.: investigation, writing (review and editing). K.I.: investigation, writing (review). K.N.: investigation, resources, writing (review). K.H.: investigation, resources, writing (review). I.K.: investigation, writing (review). S.T.: investigation, writing (review). Y.S.: investigation, writing (review and editing), supervision. M.F.: conceptualization, funding acquisition, investigation, project administration, resources, writing , supervision. S.S: conceptualization, data curation, investigation, methodology, resources, writing (review and editing), supervision.This study was supported by grants from the Canon Foundation, the Japan Endocrine Society, National Center for Child Health and Development (2022A-1), Takeda Science Foundation, and the Mitsubishi Foundation.None declared.All datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.Figure_S1_qfad057Click here for additional data file.Table_S1_qfad057Click here for additional data file.


To investigate the effect of antibiotic treatment on PSA when deciding on prostate biopsy.A total of 206 patients with an elevated PSA level (2.5–30) were included. Mp-MRI could be done on 129 patients. Patients were given ciprofloxacin  for 4 weeks and PSA measurements were repeated. Systematic prostate biopsy was performed regardless of PSA changes on all patients. Additionally, cognitive biopsies were performed from PI-RADs III, IV, and V lesions.Prostate cancer was detected in 36.4% of patients. 53.3% had Gleason score of 3+3, 46.7% had Gleason score ≥ 3+4. PSA values decreased in 56.3% and in 43.7% and remained the same or increased but cancer detection rates were not different: 34.5% vs. 38.9%, respectively (p = 0.514). PSA change in whole group was significant . No significant PSA decrease was observed in cancer patients , whereas PSA decrease was significant in patients with benign pathology . In patients with PI-RADs IV–V lesions, adenocarcinoma was present in 33.9% and 30.4% with or without PSA decrease, respectively (p = 0.209). Clinically significant cancer was higher in patients with after antibiotherapy PSA values >4 ng/mL regardless of PI-RADs grouping (p = 0.08). Addition of any PSA value to PI-RADs grouping did not have any significant effect on the detection of cancer.PSA change after antibiotic treatment has no effect in detecting cancer and should not delay performing a biopsy. Prostate cancer (PCa) is the most common cancer in male population in developed countries . Serum pSince prostate infections are also observed among the causes of PSA elevation, it is thought that the probability of prostate cancer is lower in PSA levels that decrease after antibiotics. For this reason, there is an opinion that prostate biopsy should not be performed in PSA levels that tend to decrease. In this way, it is tried to prevent unnecessary biopsies.We have investigated the value of antibiotic treatment and PSA change after such patients on deciding prostate biopsy. We also tried to correlate PSA kinetics after antibiotic treatment with the diagnosis of prostate cancer.This study was carried out at Bezmialem Vakıf University Medical School Hospital Urology Department with the approval of Bezmialem Vakıf University Medical School Hospital Ethics Committee dated 11.04.2018 and numbered 5830.Following the approval of the local ethics committee 441 patients who applied to the urology outpatient clinic of Bezmialem Vakıf University Medical School Hospital between January 2017 and January 2018 and received antibiotic treatment due to high serum PSA level (2.5–30 ng/mL) and then underwent TRUS-Bx were included in this study.Patients excluded from this study were those;With suppressed immune system, severe coagulopathy, and those who received treatment for acute prostatitis,Receiving 5-alpha reductase inhibitor therapy due to benign prostatic hyperplasia,With a history of cystoscopy/TUR-Prostatectomy/TUR-Bladder operation in the last 3 months,With a history of chronic prostatitis,With other urinary infections,With recently included urolithiasis,With urinary retention which needed urethral catheterization.Multiparametric magnetic resonance imaging (mpMRI) was recommended for all patients, but could not be done in 77 patients for various reasons . Images of 129 patients who underwent mpMRI were evaluated by a single experienced radiologist and classified across PI-RADs v2. All patients were given ciprofloxacin  treatment for 4 weeks and PSA levels were repeated (PSAab). Prostate biopsy was performed regardless of PSA changes on all patients under local anesthesia and transrectal ultrasound guidance. A standard systematic 12-core biopsy was performed on 77 patients who did not undergo MRI. On 129 patients who underwent mp-MRI, cognitive biopsies were performed from PI-RADs v2 III, IV, and V lesions in addition to standard systematic biopsy as shown in Demographic data such as age, mpMRI reports, digital rectal examination (DRE) findings, pretreatment PSA levels , PSA values checked after treatment (PSAab) prostate volumes calculated on transrectal ultrasound examination and pathology results were recorded. Analysis and comparisons were performed retrospectively.https://www.medcalc.org; 2019). Mean and standard deviation were used to describe normally distributed continuous data, whereas median and minimum-maximum values were used to describe continuous variables which are not normally distributed. Frequencies (n) and percentages (%) were used as descriptive statistics for categorical variables. Normal distribution was evaluated with the Kolmogorov-Smirnov test. For the comparison of proportions chi-square test was used (or Fisher exact test where available). In order to compare two dependent, nonnormally distributed variables Wilcoxon Signed Rank test was used. Statistical significance was set as p < 0.05. As a multivariate analysis to evaluate the risk factors on malignant pathology logistic regression model was used. Lastly, a mathematical model was tried to be developed in order to find out the best model to predict both prostate cancer and significant prostate cancer by using the logistic regression function. The performance at diagnosing of this model has been inspected with receiver operating curve (ROC) analysis.Statistical analyses were performed by using IBM Corp. Released 2019. IBM SPSS Statistics for Windows, Version 26.0. Armonk, NY: IBM Corp and MedCalc Statistical Software version 19.1 ,DRE findings,PI-RADs groups.Benign pathology and PCa rates according to PSA changes as well as Gleason score 3 + 3 and higher-grade cancer rates in patients with PCa were evaluated according to the following parameters and compared using the chi-square test or Fisher’s exact test:Patient age,Initial PSA,PSA density,PSA change ,DRE findings,PI-RADs groups.Initial PSA and PSAab (PSA levels after 4 weeks antibiotherapy) were evaluated separately for the following patient groups using Wilcoxon signed-rank test for all patients, patients with and without PCa on biopsy, with PI-RADs II–IV lesions, with benign pathology and PCa for each PI-RADs group.Significant PCa was defined as ≥ Gleason 3+4, or >3 biopsy cores positive, or at least one biopsy core with >50% involvement. Insignificant PCa was defined as ≤Gleason 3+3 without Gleason pattern 4 or 5 as tertiary pathology, and less than 3 core samples, and no core sample >50% involved.Correlation of benign pathology and any PCa or significant PCa and the remaining patients were evaluated for patient age, PSAi , PSAD (density of PSA), PSA change , DRE  findings, PI-RADs groups using chi-square test or Fisher’s exact test. Statistical significance was set as p < 0.05.To evaluate the risk factors on malignant pathology multivariate analysis was done using the logistic regression model. A mathematical model was developed to predict both PCa and significant PCa by using the logistic regression function. Performance at diagnosing this model has been inspected with ROC (receiver operating curve) analysis.Demographic data of 206 patients participating in the study are shown in PSA values decreased in 56.3% and 43.7% of patients remained the same or increased in 4 weeks of antibiotic treatment. Median PSAi and median PSAab were 6.38 ng/mL (IR: 4.68–6.38) and 5.95 ng/mL (IR: 4.30–8.30), respectively (p = 0.01) as shown in No significant PSA decrease was observed with antibiotic treatment in patients with PCa , however, a statistically significant PSA decrease was evident in patients with benign pathology . A minimal decrease in absolute PSA value was observed with antibiotic treatment in patients with PI-RADs IV lesions . Among patients with PI-RADs IV lesions only and patients with PI-RADs IV and V lesions but with benign pathology, a significant PSA change was observed . As shown in There was no difference in PCa detection rates between patient groups (PSAi: 2.5–4 ng/mL vs. 4.1–10 ng/mL vs. >10.1 ng/mL) (p = 0.830). The incidence of Gleason 3+3 cancer was higher if PSAi < 4.0 ng/mL compared to those with a PSAi >10 ng/mL  as shown in Thirty out of 206 patients had suspicious findings on DRE, PCa was detected in 19 of 30 patients with suspicious DRE findings (7 Gleason score of 3+3 (Gleason group 1) and 12 Gleason score of 3+4 and higher (Gleason group 2+). Both PCa rates and clinically significant PCa rates were found to be higher in patients with suspicious DRE findings  as shown in The incidence of PCa increased parallel to PI-RADs score. When patients were divided into two groups as PI-RADs II and III–IV, incidences of PCa and clinically significant PCa were higher in patients with PI-RADs III+IV+V lesions . Accordingly, when patients were divided into two groups as PI-RADs II+III and PI-RADs IV+V, incidences of PCa and clinically significant PCa were similarly higher in patients with PI-RADs IV+V lesions .No correlation was found between PSA change and PCa diagnosis in PI-RADs II and III groups as shown in In patients with PI-RADs II lesions; PCa was detected in 6.8% of patients whose PSA decreased and 6.8% of patients whose PSA remained the same or increased. In patients with PI-RADs III lesions and PSA decrease after antibiotic treatment, PCa was detected in 17.2%. In cases with PI-RADs IV–V lesions and PSA decrease after antibiotic treatment, PCa was detected in 33.9% vs. 30.4% of cases whose PSA remained the same or increased. In patients evaluated with mp-MRI, clinically significant PCa rates were higher if both PSAi and PSAab were >4 ng/mL regardless of PI-RADs grouping (p = 0.080). PSAD values were significantly different between patients with PCa and benign pathology . However only PSAD ≤ 0.1 was able to predict clinically significant PCa (p = 0.001).Performance of PI-RADs IV+V lesions in predicting PCa was slightly but not significantly higher when PSAD both before and after antibiotic treatment were included in the analysis  as shown in Lastly, to eliminate the effect of insignificant PCa, we grouped our patients as those with a diagnosis of significant PCa (Gleason Score > 3+3) and the remainder of patients (either with an insignificant PCa as a Gleason 3+3 = 6 cancer and those with a benign histology). In patients with an MRI, patients with a significant PCa have a higher mean PSAi , less common unremarkable digital rectal examination findings , a higher PSAD (p = 0.001 and p = 0.005), and a lower rate of PI-RADs 2 and PI-RADs 2+3 lesions . When all patients are included and MRI findings are ignored, mean age of patients with significant PCa was 65.21 (vs. 62.21), are older than 60. These patients also have a higher PSAi , higher PSAab 8.68 ng/mL vs. 6.31 ng/mL, p = 0.001, higher incidence of suspicious digital rectal examination findings  and a higher PSAD .Although it has been used for prostate cancer detection since its first day of clinical use, PSA is an organ-specific not a disease specific marker and it can be measured high in all diseases  or interventions  affecting the prostate. It is also known that PSA levels may increase parallel to the increasing prostate volume . BesidesDRE findings and PSA levels play an important role in the decision making of performing prostate biopsy. Although these two markers are used together, they cannot be found basis to a sufficient sensitive and specific indication for systematic TRUS-Bx. Prostate cancer is detected in 34% of patients who undergo TRUS-Bx because of elevated PSA levels . In otheThe positive effect of antibiotic treatment on PSA reduction has been shown in many studies –16. In sSerretta et al. found in their study that the bigger the rate of PSA decline, the lower the diagnosis of PCa. They also reported that patients with PSA values returning to the normal range after antibiotherapy did not require biopsy . BaltacıIn our study, prostate biopsy was performed on 129 (38%) patients who underwent mp-MRI and PCa was detected in 49 of these patients. PCa was detected in an increasing frequency parallel to PIRADS scoring: 13.6% of cases with PI-RADs II, in 24.1% of cases with PI-RADs III, in 55.5% of cases with PI-RADs IV, and in 64.3% of cases with PI-RADs IV+V, which is consistent with the literature. Park et al. reported the rate of prostate cancer detection as 17% in patients with PIRADs I and II lesions in their meta-analysis  which isTo our knowledge, no correlation between PSA decline and PI-RADs scoring has been studied so far. Therefore, in order to evaluate the effect of PSA decrease in detecting PCa, we also looked at PCa rates according to PSA change in PI-RADs groups in the current study, in patients who underwent mp-MRI, PCa was observed in 38% (n = 27) of the patients with PSA decrease, and in 37.9% (n = 22) of the patients whose PSA did not change or increased. These results show that PCa is still detected at the same rate in patients with suspicious lesions in mp-MRI, regardless of the PSA change after antibiotic treatment .Zhang et al. performed a study including 273 patients with PI-RADs < 3 and 218 patients with PI-RADs = 3 lesions and reported that low PSAD increased the negative predictive value in detecting overall PCa and clinically significant PCa . WoshinoThese findings suggest that the presence of PCa cannot be ruled out even if PSA decreases after antibiotic treatment in any PI-RADs score, in other words, PSA decrease does not affect the detection of prostate cancer, since prostate cancer is found at the same rate in patients with and without PSA decrease. This study, together with the literature data, supports the idea that prostate biopsy is not necessary in patients with PIRADs < III lesions.In our study, a decrease was observed in PSA levels after antibiotic use, but no correlation was observed between the decrease and the incidence of prostate cancer. Therefore, the use of antibiotics to reduce high PSA levels in order to prevent unnecessary biopsies was not found significant and not recommended. In addition to all these, studies conducted in recent years reveal that long-term antibiotic usage  before prostate biopsy, increases the risk of sepsis caused by resistant microorganisms after biopsy .As a conclusion, in daily urology practice, there may be a tendency not to perform prostate biopsy in a large number of patients with high PSA levels who have a decrease in PSA after antibiotic treatment. Results of this study showed that there is no significant difference in the diagnosis of cancer regardless of PSA decrease after antibiotherapy. This led us to think that a decrease in PSA level after antibiotic treatment could not rule out the presence of prostate cancer. Prostate biopsy should be performed in patients with PIRADs IV+V lesions without any antibiotic trial. In patients who do not have mp-MRI and PIRADs classification for any reason, deciding prostate biopsy should not be based on PSA change after antibiotic treatment and, we recommend that patients should be biopsied. Since sufficient information cannot be provided in patients with PIRADs III lesions with the available data, prostate biopsy should be performed in these patients as well. Studies involving a higher number of patients will be instructive to see the relationship between PSA change in patients with PIRADs III lesions after antibiotherapy and presence of PCa more clearly.


Osteoarthritis (OA) is a debilitating disease with significant personal and socioeconomic burdens worldwide.Rosa canina L, Hippophae rhamnoides, and collagen peptide. We tested the pharmacological efficacy of PL02 in a rodent model of OA induced by Monosodium iodoacetate (MIA).To address this, we developed a multitargeted formulation called PL02, which includes standardized extracts of Our results demonstrate that oral administration of PL02 has antioxidant effects by down-regulating NOS, reduces pain-related behavior, and mitigates inflammation by inhibiting IL-1b and TNF-α production, as well as downregulating CGRP1 and COX-II. PL02 also exhibits anti-catabolic and chondroprotective activity by significantly downregulating MMP13 and upregulating BCL2. Additionally, PL02 demonstrates chondrogenic activity by significantly upregulating SOX-9 (a master regulator of chondrogenesis), Coll-I, and aggrecan, which are major components of articular cartilage. Furthermore, PL02 prevents microarchitectural deterioration of subchondral bone.Overall, PL02 is an orally active, multi-targeted therapy that not only alleviates pain and inflammation but also effectively halts cartilage and subchondral bone deterioration. It represents a safe and promising candidate for the treatment and management of OA. This isRosa canina-Scientific name), which are the aggregate fruits of shrubs belonging to the Rosa genus of the Rosaceae family. They are widely recognized for their antioxidant properties and are commonly used as dietary antioxidants , belonging to the family Elaeagnaceae, is another plant of significant importance, although less extensively studied. In India, Hippophae rhamnoides thrives in the high-temperate zones of the Western Himalayas, particularly in Leh, Laddakh, Himachal Pradesh, Uttarakhand, and Sikkim, at elevations ranging from 2,600 to 4,000 meters. This plant has been recognized for its beneficial properties, serving as a natural antioxidant with anti-inflammatory, immunomodulatory, and homocysteine-reducing effects  leaf extract in an adjuvant-induced arthritis (AIA) rat model revealed significant anti-inflammatory activity, suggesting its potential for arthritis treatment  and other related diseases. In the present study, we aimed to address this gap by standardizing and characterizing the extracts of Rosehip and Hippophae rhamnoides and developing a formulation, named PL02, that incorporates these extracts along with collagen peptides. To evaluate the pharmacological effects of the formulation in managing OA, we utilized the MIA-induced animal model of OA. We assessed various parameters including the levels of anti-inflammatory markers, antioxidants, chemokines, cytokines, gene expression, conducted histological analysis of joint tissue, and analyzed the microarchitecture of subchondral bone. Through these evaluations, we aimed to decipher the mechanism of action of the formulation in managing OA.Despite the known individual therapeutic potential of collagen peptide, Rosehip, and 22.1Rosa canina L.) and Seabuckthorn (Hippophae rhamnoides) were a purchased from GMP certified company. Fish Collagen peptide was procured from FSSAI approved company. The XK 16/70 used for collagen desalting and purification was purchased from GE Healthcare Bio-Sciences AB . Serum glutamic oxaloacetic transaminase (SGOT), Serum glutamic pyruvic transaminase (SGPT) Alkaline phosphatase (ALP), Urea, Creatinine kits for biochemical estimation were purchased from Tulip Diagnostics Private Limited. Lipid Peroxide estimation was done using ELISA kit from Immunotag™ G-Biosciences (Miss The proinflammatory and anti-inflammatory cytokines level y cytokines was checked using Bio-Plex 23 panel) Pro™ Cytokine multiplex kit . Primary and secondary antibodies for TNF-α were procured from Cell Signaling Technology, Danvers, MA. Primers for real-time PCR analysis, designed by NCBI Primer blast and synthesized by Sigma-Aldrich .Monosodium iodoacetate (MIA), sodium chloride (NaCl), Sephadex G-15 beads were purchased from Sigma-Aldrich . Plant extracts of Rosehip (2.2Rosa canina L) and Seabuckthorn (Hippophae rhamnoides), enriched with folic acid, omega-3 fatty acids, omega-7 fatty acids, omega-9 fatty acids, vitamin C, and alpha-lipoic acid.The PL02 formulation consisted of a combination of hydrolyzed collagen and a herbal blend. The herbal blend included standardized extracts of Rosehip (Hippophae rhamnoides (Seabuckthorn) extract to 40 parts Rosa canina (Rosehip) extract. In the PL02 formulation, we used a ratio of 80 parts hydrolyzed fish collagen type 1 to 20 parts of the herbal mixture.For administration, the PL02 formulation was orally administered daily at a dosage of 600 mg/kg body weight, dissolved in MilliQ water. In the herbal mixture, we used a ratio of 60 parts 2.2.1To enhance the biological activity of the peptide and remove excess salts, purification was performed using the Amersham Pharmacia Fast protein liquid chromatography (GE AKTA Explorer) system. The system was equipped with a Pump P-920 controlled by a UPC-900 monitor and an IV-908 injection valve for sample injection. The XK 16/70 column connected to the controller was packed with sephadex G-10/15 beads that had been swelled in MilliQ water overnight at 4°C.The collagen peptide was dissolved in MilliQ water, and a stock solution of 500 mg/mL was prepared. The solution was then passed through the column at a flow rate of 2 mL/min. Peak fractionation was set using the UNICORN method wizard, and pump A and B inlets were immersed in sonicated MilliQ water. The eluted material’s absorbance at 214 nm was continuously measured using a Zn Lamp, and fractions were collected using the FRAC-900 fraction collector.The collected fractions were subsequently lyophilized using an Allied Frost Indian fabricated Lyophilizer under a vacuum pressure of 0.02 mbar. This process facilitated the removal of moisture, resulting in the production of a purified peptide product.2.2.2™ Analyzer  equipped with a 355 nm pulsed Nd:YAG laser. The measurements, both in positive and negative ion detection, were conducted in the reflector mode with delayed extraction conditions. The extraction voltage was set to 20 kV, and gated matrix suppression was implemented to prevent detector saturation caused by matrix ions with m/z values lower than 200.The purified collagen peptide powder was subjected to further analysis using the Applied Biosystems/MDS SCIEX 4800 MALDI TOF/TOFFor each mass spectrum, one hundred single laser shots were averaged. The laser was randomly moved over the sample to account for analyte/matrix variations and homogenize the measurements. It is important to note that the level of detection (LOD) of MALDI-TOF-MS for a specific compound is primarily influenced by the chemical background noise. Therefore, increasing the number of laser shots does not significantly enhance the spectral quality .2.2.3The formulation analysis was conducted using the ACQUITY UPLC Q TOF MS/MS system . The chromatography was performed on an Acquity BEH C18 column  at a temperature of 25°C. The mobile phase consisted of three components: (A) 0.1% formic acid, (B) acetonitrile, and (C) methanol, with a flow rate of 300 μL/min. A sample injection volume of 5 μL was used.The gradient elution program was optimized as follows: initially, 90% B and 10% C, which was then changed to 80% B and 20% C within 2 min. This was followed by a range of 50–60% B and C for 1–3 min, 30–70% B and C for 3–6 min, 10% B and 90% C for 1 min, and finally rapidly increased to 90% B and 10% C within 7–10 min. The MS analysis was conducted in positive ion mode. Centroid mode data were collected in the mass-to-charge ratio (m/z) range of 100–1,000 Da, with a scan time of 1 s. For data analysis, the Software Respect for Phytochemicals2.3The experiments were carried out using two rodent models (mice and rats) of both sexes, aged 8–10 weeks in a double blinded manner. Initially, a pilot efficacy study was conducted in mouse models, employing the carrageenan-induced inflammatory model to ascertain the optimal effective dose of PL02. Once the effective dose of PL02 was determined, we proceeded to induce MIA-induced osteoarthritis in mice, as outlined in the n = 10 each) as follows: Sham control group  received saline instead of MIA injection, disease control group  induced with OA and treated with vehicle, positive control group  induced with OA and treated with orally administered Indomethacin, and formulation PL02 treated group . Pain and inflammation were assessed by performing behavioral tests on days 3, 7, 14, 21, and 28. All animal studies were approved by the Institutional Animal Ethics Committee (IAEC#576/21) of the National Institute of Immunology and conducted in accordance with the ARRIVE  guidelines.Wistar rats were randomly divided into four groups  inhalation, following a fill rate of about 30 to 70% of the chamber volume per minute. Blood, vital organs, and left knee joint specimens were collected for further experiments. Blood samples were centrifuged at 12,000 rpm for 10 min, and the serum was separated, aliquoted, and stored at −20°C for subsequent analysis. Synovial tissue and left knee joints were processed for mRNA isolation, lysate preparation, and histological analysis or stored at −80°C for future studies.2.3.1™ mounted on a sensitive probe. On applying pA, connected LCD Readout will display the weight bared by the paw in grams on applying pressure placed in individual cages with a metal grating floor and allowed a 30 min adaptation before the start of the test. Increasing force was applied to the central plantar region of the hind paw to induce reflecting flexion of the joint. A positive response was defined as a rapid withdrawal of the left hind paw or licking of the paw. The test was repeated three times. The first day of the testing provided a baseline measure of tactile sensitivity. Rats in each group were then tested with von Frey filaments on post-injection 3, 7, 14, 21, and 28.Von Frey is regarded as the gold standard technique used to evaluate the pain sensitivity of the hind paw of the animals with knee joint arthritis. Paw withdrawal threshold (PWT) is measured in response to increasing pressure stimuli applied by von frey hairs to the plantar surface . Electro2.3.2The paw diameter was measured after the injection of MIA using electronic Vernier caliper  after MIA injection . The dif2.3.3Inflammation in the paw was measured using a plethysmometer (Paw Volume) Meter . The original device was designed to measure paw volume and its changes (swelling) in rodents aiming to test the efficacy of anti-inflammatory agents . The lef2.4Blood was withdrawn on day 14 and day 28 through the retro orbital region of rat in the non-anti-coagulant tube. The collected blood samples were centrifuged at 12000 rpm for 15 min at 37°C, and serum was separated from the blood and stored at −20°C.2.4.1The measurement of biochemical parameters  Serum glutamic oxaloacetic transaminase (SGOT), Serum glutamic pyruvic transaminase (SGPT), Alkaline phosphatase (ALP), Urea, and Creatinine analysis was performed as per mentioned in the manual of Coral clinical system .2.4.2™ G-Biosciences, Missouri, United States). Lipid Peroxidation (LPO) Assay was performed according to the instruction manual provided by the manufacturer. Serum levels of proinflammatory and anti-inflammatory cytokines were analyzed using the Bio-Plex Pro™ Cytokine Standard kit . After thawing the sample, the serum was diluted with Bioplex diluent (1:4). The plate was prepared following the instruction manual provided by the manufacturer and read using Bio-Plex 200 Systems . The data was analyzed on Bio-Plex Manager  software at low PMT, RP1 instrument settings  tissue of all the treatment groups using TRIzol (Invitrogen). 1 μg of total RNA was reverse transcribed using the Verso cDNA synthesis kit (Thermo Fisher Scientific). For real time PCR, data acquisition and analyses were performed using the Light Cycler Real Time PCR system (Roche Diagnostics) and SYBR Green I . The relative gene expression levels were normalized against Beta Actin or GAPDH and plotted as fold change to sham control. Primers were designed using the IDT (Integrated DNA technologies) RT primer designing tool. The sense and antisense primers used are given in 2.6in vitro assays of the biological activity of PL02, SW-982 cells were plated at a density of 105 cells per well in a 24-well tissue culture plate. After 48 h cells were rinsed thoroughly with PBS, treated with IL-1RA (15 ng/mL) or PL02  for 1 h and after that stimulated with 5 ng/mL of human IL-1b (Sigma). After 24 h culture supernatants were analysed for human TNF-a levels using Ultrasensitive ELISA kits from eBioscience as per the manufacturer’s instructions.SW-982 cells  procured from ATCC were maintained per the provider’s instructions. The anti-inflammatory activity was evaluated as described by Pasi et al. . In brie2.7On day 28 after dissections organs such as heart, liver, kidney, brain, and left knee joints are fixed with 10% neutral-buffered formalin for 48 h at room temperature. Decalcification of left knee joints specimens was done with 20% formic acid for 3 days and then embedded in paraffin. Sections of the tissue specimens were acquired from the paraffin blocks at 5 μm thickness, deparaffinized, and rehydrated in the order of xylene, absolute alcohol, and 50% alcohol. The rehydrated sections were stained with hematoxylin and eosin (H&E) to observe morphological changes. Besides H&E stain, the sections of left knee joints used toluidine blue to evaluate proteoglycan and glycosaminoglycans. The severity of articular cartilage injury at the proximal tibiae was scored using the Mankin score  with ran2.8Before imaging, fixed left knee joints were further dissected under a dissection microscope to disarticulate them, carefully remove the menisci, and expose the articular cartilage of the tibia and femur. Tibia and femur were subjected to Micro-CT imaging and analysis using μCT . Scans were performed using the Aluminium 0.5 + copper 05 filter with the following parameters: time 14 min, Voxel Size 36 μm, tube voltage 90 kV, and tube current 180 mA. Sub reconstruction of the acquired image was also performed to generate Sub-volume vox files with a size of 18 μm. Analyze 14.0 was used to create serial 2D coronal and axial pictures from 3D reconstructed CT scans . We estimated BMD, BV/TV, BV, Tb.Th, and Tb.Sp using analyse14 software by manually selecting the correct region of interest, i.e., lateral and medial subchondral bone in the tibia and femur epiphysis.2.9t-test. All statistical analyses were performed using Graph Pad Prism 9.0 for Windows .All experiments were repeated at least three times. Data were presented as mean ± standard error for the indicated numbers of independently performed experiments. Statistical significance was assessed by one-way and two-way analysis of variance coupled with a Bonferroni 33.1The analysis and validation of collagen peptides were performed using MALDI-TOF. Hippophae rhamnoides and Rosehip in a chosen fixed ratio, several secondary metabolites were identified  compared to the sham control and MIA groups (p < 0.001) compared to the sham control and MIA-treated groups, but the values were within the physiological range   at each time interval for the MIA and MIA + PL02-treated groups compared to the sham control. However, the MIA + Indo group exhibited significant changes (p < 0.01 and p < 0.001) on day 14 and day 28 compared to the sham control, MIA, and MIA + PL02 groups  . The ave2 groups .p < 0.05 and p < 0.01) on day 7, day 14, and day 28 compared to the sham control, MIA + Indo, and MIA + PL02 groups  to assess motor function at 1, 3, 5, 7, 14, 21, and 28 days.Visible inflammation was observed 2 h after MIA injection and continued to increase until day seven, with no significant changes observed until day 14. From day 14 to day 28, a decrease in inflammation was observed, although it was not statistically significant. TNF-α and IL-1β levels in the serum were measured at 1, 3, 7, 14, 21, and 28 days, and their levels remained significantly higher throughout the 28 days period compared to the sham control . AdditioFor more detailed studies, we evaluated pain, inflammation symptoms, and behavior at 3, 7, 14, 21, and 28 days. Blood samples were collected, and the animals were euthanized on day 28 for molecular and histological analysis. Joint sections from the MIA-induced animals exhibited OA-like features, including surface discontinuity, loss of proteoglycans, disorientation, apoptosis of chondrocytes, and subchondral bone sclerosis   to (298.50 ± 73.44), compared to the sham control group (10.75 ± 6.24) to (123 ± 70.03) and the MIA group (8.80 ± 7.26) to (87.11 ± 30.12). These changes showed even more significant improvements after 28 days of treatment ( 0.0001) .We measured inflammation in the knee area using a Vernier caliper on both knees and noted the difference. On day 1 and day 3 post MIA injection, all groups showed a significant difference in knee width, confirming the development of an osteoarthritis model. However, a significant decrease in the difference between the two knee joint widths was observed from day 10 onwards in the PL02 treated group compared to the MIA and Indomethacin treated groups .p < 0.0001) improvement after 10 days of treatment, increasing from (3.65 ± 0.44) to (5.09 ± 1.46) on day 28, compared to the MIA treated group  of the left paw indicated elevated inflammation in rats administered with MIA. However, the group administered with the PL02 formulation showed anti-inflammatory activity, significantly reducing inflammation on day 28 from (9.19 ± 0.39) to (8.07 ± 0.56) in the rat’s paws, compared to the MIA group .In p < 0.0001) in the paw withdrawal threshold was observed from the 14th day onwards in the MIA + PL02 treated group and the MIA + Indo treated group, compared to the MIA group. The pain significantly decreased further with daily administration of the formulation for 28 days (p < 0.0001)  .p < 0.0001) decreased in the MIA + PL02 group compared to the MIA group. The level of IL-1β also significantly reduced upon PL02 treatment compared to the MIA group. Similar patterns were observed for IL-10 and IL-17 levels, with the PL02-treated group showing lower levels than the MIA group, although the changes were non-significant after analysis. However, the levels of IFN-γ were significantly (p < 0.0001) reduced in the MIA + PL02 group compared to the MIA group.After 28 days of treatment, blood was withdrawn from the rats and used for cytokine assay using a multiplex analyzer . The levp < 0.05) and (p < 0.0001), respectively. Chemokines such as CCL-2 (p < 0.0001 and p < 0.001) after daily administration of the formulation for 28 days in the PL02-treated group, compared to the MIA group. Additionally, to understand the mechanism, a synovial cell line was used to study IL-1β-dependent TNF-α production. Treatment with PL02 showed a dose-dependent decrease in TNF-α production, and at 10 mg/mL, it inhibited IL-1β-induced TNF-α production  in COX-II and CGRP-1 levels was observed in the MIA group compared to the sham control group. PL02 treatment for 28 days significantly reduced their expression (p < 0.001) compared to the MIA and MIA + Indo groups  of NOS-2 (p < 0.0001) of BCL-2  after MIA injection but increased significantly (p < 0.01) upon PL02 treatment. We also assessed the expression level of collagen-II, the primary collagen, and aggrecan, a major proteoglycan in articular cartilage essential for its proper functioning and hydration maintenance. A significant decrease in aggrecan level was observed following MIA injection, with no significant improvement in the MIA + Indo treated animals.Next, we examined the level of the protease MMP-13, which attacks the cartilage matrix and leads to its destruction. MMP-13 expression increased following MIA injection compared to the control group. However, a significant downregulation in the level of MMP-13 was observed in PL02-treated animals compared to MIA . The revp < 0.0001) in aggrecan expression -like changes in the microarchitecture of the tibial subchondral bone. However, we did not observe significant changes in the microarchitecture of the distal/proximal femur bone in the joint following MIA injection. Therefore, we focused on analyzing the tibial subchondral bone in detail.p < 0.05 for both parameters). However, no such increase in bone volume fraction was observed in animals treated with PL02, and it was comparable to the sham control. The trabecular parameter Tb-Th slightly increased after MIA treatment, but the difference was insignificant. On the other hand, there was a significant decrease in trabecular spacing (Tb.Sp) in the MIA-treated group compared to the sham control, which was prevented in the PL02-treated group.p < 0.001).The MIA-injected left knee exhibited a significant increase in subchondral plate thickness  compared to the control left knee. The calculated values for subchondral plate thickness were 140 ± 25 μm and 120 ± 9 μm for the MIA-injected and sham control knees, respectively. In the PL02-treated tibia, a subchondral plate thickness of 115 ± 15 μm was observed, similar to the sham control. Additionally, we observed increased porosity of the tibial subchondral plate in the MIA-injected left knee compared to the sham control and PL02-treated left knee. The %d MIA-Control was 48, while the %d MIA + PL02-Control was 20  is a prime example of a chronic medical condition that requires effective treatment. The impact of the ongoing COVID-19 pandemic has further exacerbated the burden of OA. Despite extensive research efforts worldwide, no therapy has been developed that can effectively halt, delay, or reverse OA progression or provide long-lasting symptomatic relief. Consequently, surgical interventions like joint replacement are often pursued by patients seeking improved pain management and quality of life. There is an urgent need to develop a novel, safe, and multitargeted therapy that can address OA symptoms in the long term and prevent or reverse joint tissue loss.To tackle this challenge, we have utilized our knowledge and expertise in traditional medicine and bone joint physiology to develop a multitargeted formulation called PL02 (patented trademark). The botanical ingredients used in this formulation are already known for their individual health benefits and are widely sold as nutraceuticals worldwide , 26, 50.Our study aimed to establish a non-traumatic animal model that closely resembles the tissue-level pathophysiology of human osteoarthritis (OA). This model would enable us to investigate the effects of disease-modifying therapies on both cartilage and subchondral bone. To achieve this, we utilized a low-dose monosodium iodoacetate (MIA)-induced rat and mouse model of OA, which effectively replicates the articular cartilage and subchondral bone pathology observed in human OA . CompareAfter optimizing the dosage, we conducted acute and subacute (28 days) toxicity studies of PL02 and found no evidence of any toxic effects based on serological and histopathological evaluations. Subsequently, we assessed the efficacy of PL02 in addressing various OA symptoms, including inflammation and pain. Within 1 week of treatment, PL02 demonstrated significant pain relief and reduction of inflammation, surpassing the effects of indomethacin. The superior anti-inflammatory and analgesic activity of PL02 can be attributed to its polyphenols and other secondary metabolites. Notably, alpha-lipoic acid in the formulation played a significant role in controlling inflammation by reducing serum levels of IL-1β, IL-6, TNF-α, IL-17, and IL-23 . AdditioTo evaluate the anti-inflammatory properties of PL02, we measured the knee joint width as an indicator of edema, which is a manifestation of inflammation. Our data from mice and rats showed a significant reduction in knee joint difference (edema) after 14 days of oral PL02 treatment. This finding suggests that PL02 effectively prevented inflammation-associated cell infiltration and fluid accumulation in the knee joint. Furthermore, we assessed thermal and mechanical hypersensitivity as additional indicators of inflammation. The PL02-treated group exhibited a higher threshold of mechanical hypersensitivity, as determined using the Von Frey system, compared to the MIA and MIA + Indo groups. The observed decrease in edema and increase in mechanical threshold provide further evidence of the superior anti-inflammatory and analgesic properties of PL02 compared to Indomethacin.To gain insights at the molecular level and support our behavioral findings, we examined the levels of pro-inflammatory cytokines in both serum  and mRNA expression in joint tissue . The data obtained confirmed the symptom profiles and demonstrated that PL02 significantly ameliorated inflammation and pain by modulating cytokine levels in the serum and joint tissue, surpassing the effects of the MIA or MIA + Indo group. This enhanced anti-inflammatory effect is likely attributed to the synergistic actions of several secondary metabolites present in PL02, as previously described.Based on the remarkable efficacy of PL02 in alleviating MIA-induced OA symptoms, we delved into understanding the underlying molecular mechanisms. Among the various cytokines, IL-1 has been identified as a potent inducer of cartilage and bone damage, particularly when compared to TNF-α. IL-1 and TNF-α play key roles in inducing matrix-degrading enzymes that lead to rapid and extensive cartilage damage. Therefore, the inhibition of IL-1-induced inflammatory cascades by PL02 has a more pronounced effect on cartilage and bone damage, aligning with the therapeutic goals in OA.The oxidative stress theory provides an explanation for the key pathogenic events in OA, which involve the degradation of extracellular matrix (ECM) and apoptosis of chondrocytes. Inflammation triggers the generation of reactive oxygen species (ROS) through the activation of multiple signaling pathways. ROS functions both as a signaling molecule and a mediator of inflammation and senescence . By actiCompounds possessing antioxidant properties have demonstrated the ability to neutralize ROS and interrupt the chain of ROS-mediated damage to cartilage and the synovial capsule . In our The degradation of articular cartilage and bone matrix components is a characteristic feature of osteoarthritis (OA). Early events involve the loss of type II collagen and the proteoglycan aggrecan, leading to structural and functional deterioration of the cartilage. MIA-induced OA is characterized by significant cartilage destruction, with a single injection causing over 33% loss of cartilage tissue. A major challenge in modern medicine is to prevent or regenerate cartilage to improve joint mechanical properties. Fortunately, various secondary metabolites derived from plants have shown promise in halting cartilage tissue damage .For instance, the flavonoid Diosmin, found in citrus fruits, has been shown to upregulate type II collagen, the main component of the cartilage extracellular matrix. Diosmin has also been associated with increased activity of the chondrocyte-specific differentiation marker SOX-9. Clinical trials are even underway to investigate its analgesic properties , 57. Matp < 0.0001) and aggrecan (p < 0.001), along with a significant decrease in MMP-13 (p < 0.01) in the PL02-treated group compared to the disease control. These findings provide essential evidence supporting the disease-modifying potential of PL02 in protecting against cartilage degradation following MIA injection in rats. Moreover, the histopathological analysis further supports our claims.Following treatment with PL02, there was a significant decrease in MMP13 expression and a significant increase in levels of aggrecan and collagen II. As PL02 contains abundant quantities of these beneficial secondary metabolites, we observed a substantial increase in the expression of SOX-9 . In contrast, MIA injection resulted in proteoglycan loss, diminished viable chondrocytes, and disrupted proliferation zones . HoweverThe ECM plays a vital role in providing structural and biochemical support to neighbouring cells in cartilage, serving various functions in chondrogenesis . It is wIn our study, we observed an increase in the expression of the anti-apoptotic protein BCL-2 following PL02 treatment. This finding suggests that PL02 may suppress chondrocyte apoptosis by inhibiting inflammation-induced ECM degradation. As a result, both macroscopic and histological examinations provided evidence that oral administration of PL02 effectively protected cartilage from destruction.The subchondral bone serves as both a supplier of nutrients and a mediator of physiological and non-physiological shock absorption, providing support to the overlying cartilage . Any disRecent pre-clinical and clinical studies have revealed that changes in bone structure may precede and contribute to cartilage pathology, and that OA progression is associated with temporal changes in bone structure –19. EarlTherefore, it was essential to investigate the effects of PL02 on the microarchitecture of the subchondral bone in the femur and tibial head at the knee joint. Our observations revealed more pronounced changes in the tibia compared to the femur, with significant differences observed in the medial compartment compared to the lateral compartment in the tibia. Detailed analysis demonstrated a significant increase in bone sclerosis, plate thickness, porosity, bone volume fraction, and trabecular thickness, along with a decrease in trabecular spacing in the MIA-induced OA group. This confirms that the MIA-induced model used in our study accurately mimics the pathological progression observed in human OA in the tibial subchondral bone, consistent with findings reported by Mohan et al. .Interestingly, treatment with PL02 prevented disease progression in the subchondral bone. No thickening or sclerosis of the subchondral plate was observed.While this study presents promising findings, it’s crucial to acknowledge several technical limitations that could have influenced the outcomes. Firstly, our approach involved analyzing joint tissue for gene expression to correlate with serum data. We aimed to comprehensively assess the anti-inflammatory and analgesic effects of PL02 by combining serum markers, behavioral observations, and gene expression levels. Additionally, lipid peroxidation is an established mechanism of cellular/tissue injury across plants and animals, serving as an oxidative stress indicator . Our obj5The understanding of osteoarthritis (OA) has evolved to recognize it as a complex disease involving multiple components of the joint, beyond just the cartilage or synovium. This multifaceted nature of OA makes it a highly heterogeneous condition, and therapies targeting a single tissue or aspect of the joint may not be sufficient.In this context, PL02 emerges as a promising intervention. It effectively reduces pain and inflammation while providing protection against cartilage damage. Furthermore, PL02 addresses crucial aspects of the disease by preventing chondrocyte apoptosis and regulating subchondral bone turnover, as depicted in The results of our study indicate that PL02 significantly modifies the progression of OA, offering potential benefits in preventing the need for joint replacement, even in advanced stages of the disease. This novel approach recognizes the complexity of OA and highlights the importance of comprehensive therapeutic strategies for its management.The original contributions presented in the study are included in the article/All animal studies were approved by the Institutional Animal Ethics Committee (IAEC#576/21) of the National Institute of Immunology and conducted in accordance with the ARRIVE  guidelines. The study was conducted in accordance with the local legislation and institutional requirements.PU and SG identified the ingredients for the formulation, designed the research, analyzed the final data, and wrote the manuscript. PU, AN, and DK conducted the majority of the experiments, including articular injections, collection of animal samples, molecular studies, and data analysis. AN and DK performed bone scanning using μCT and analyzed the data under the supervision of SG. VA facilitated the purchase of the formulation for commercialization, provided assistance with label claims, and protected it with a trademark. All authors contributed to the article and approved the submitted version.