Zea mays, L.), soybean , and rice , which vary in linkage disequilibrium (LD) decay rates and have contrasting genetic architectures.Genomic selection is a powerful tool in plant breeding. By building a prediction model using a training set with markers and phenotypes, genomic estimated breeding values (GEBVs) can be used as predictions of breeding values in a target set with only genotype data. There is, however, limited information on how prediction accuracy of genomic prediction can be optimized. The objective of this study was to evaluate the performance of 11 genomic prediction models across species in terms of prediction accuracy for two traits with different heritabilities using several subsets of markers and training population proportions. Species studied were maize . Maize, which has the shortest extent of LD, showed the highest prediction accuracy. Amongst all the models tested, Bayes B performed better than or equal to all other models for each trait in all the three crops. Traits with higher broad-sense and narrow-sense heritabilities were associated with higher prediction accuracy. When subsets of markers were selected based on LD, the accuracy was similar to that observed from the complete set of markers. However, prediction accuracies were significantly improved when using a subset of total markers that were significant at We conclude that prediction accuracy for genomic selection can be improved by using the Bayes B model with a subset of significant markers and by selecting the training population based on narrow sense heritability.The online version contains supplementary material available at 10.1186/s12870-022-03479-y. Plant breeders observe the phenotypes of crops to choose desirable offspring in an aim to genetically improve target traits. Selection during the breeding process is a crucial step in crop breeding, with historical and current conventional plant breeding depending on phenotypic selection. The Selection-index (SI) method  selects Genomic selection provides a potential advantage in crop breeding by accelerating the genetic improvement of crops per unit time through the reduction in cost per breeding cycle and shortening of the generation interval . AdditioNumerous genomic prediction models have been developed for predicting phenotypes using large sets of genetic markers   because Several factors affect the accuracy of genomic prediction including the size and genetic diversity of the training population, trait heritability, marker density, gene or marker effects, and the extent and distribution of LD between markers and QTL. Therefore, there is an ongoing need to understand how accuracy of different models reacts among crop species that vary in the LD decay as well as how prediction accuracy is affected by marker number, training population proportion, and trait heritability. The main objective of this study was to compare the accuracy of numerous genomic prediction models for several traits that differ in heritability for three crop species with different LD decays rates and contrasting genetic architecture, as well as testing the effect of several methods of sub-setting total marker number and the interaction with training population size.13C ranged by 1.46%. In maize, the DT ranged by 30.5 days and the EH ranged by 128 cm. In rice, the PPP ranged by 1.89 panicles and the SPP ranged by 2.19 seeds  of the association between a marker and given trait by FarmCPU [Tables S2, S3, and S4  or by marker significance (four threshold subsets) and training-to-testing population proportions. We found a difference in prediction accuracy for all traits in soybean, rice, and maize using different subsets of markers at three training-to-testing proportions Fig. . Compariδ13C at all cross-validation levels. For SPP in rice, BayesB yielded the highest accuracy for all cross-validation levels. For PPP in rice, the accuracies of all models were similar for all of proportions between training and testing. For DT in maize, the highest prediction accuracy models were BayesB and RKHS at the 90:10 proportion. At the 70:30 and 50:50 proportions, the accuracies of all these models were mostly similar. For EH in maize, BayesB had the highest prediction accuracy at all training-to-testing proportions.Because the highest prediction accuracy was when using the SNP_05 subset, this subset was used to compare the different genomic prediction models Fig. . For soyP < 0.05 increased prediction accuracy in all selected traits of all crops, this subset was used to report the effect of the different training-to-testing population proportions . These trends were similar for all the different sets of marker subsets  in soybean, seeds per plant (SPP) in rice, and ear height (EH) in maize at the 90:10 training-to-testing proportion Fig. . We usedh2 for all traits using all the different combinations of markers sets and three training-to-testing proportions . , maize (Zea mays L.), and rice (Oryza sativa L.). For each crop, two traits were used that varied in broad-sense heritability (H2). For soybean, these traits included canopy wilting  and ear height  [2 = 80%) and seeds per plant  [Data sets for three plant species that vary in LD decay rates were selected for our experiments: soybean  and car2 = 60%, ). For ma2 = 65%) . Lastly 2 = 55%) .δ13C [https://www.panzea.org/data) and rice data (http://ricediversity.org/data/) were publicly available and soybean data are included herein . In soybean, SNP data were obtained using the application of Illumina Infinium SoySNP50K iSelect SNP BeadChip that provided 42,509 SNPs for all 346 accessions  than soybean (150 kb in euchromatic and 5 kb in heterochromatic regions) or rice (123 kb).Additional file  project . Qualitygenotype . The finEleven different statistical models were compared for genomic predictions. These models differ with respect to assumptions about the markers as described in Additional File r ≥ 0.90, 0.80 ≤ r < 0.90, 0.70 ≤ r < 0.80, 0.60 ≤ r < 0.70, and 0.50 ≤ r < 0.60 between alleles. For simplicity, these subsets are subsequently referred to as 0.90, 0.80, 0.70, 0.60, and 0.50, respectively. For example, if a haplotype block consisted of five SNPs that were linked with each other at r ≥ 0.90, then only one SNP out of five was kept in the subset of markers.Ten different marker subsets were compared to evaluate the effects of marker distribution across the genome on the prediction accuracy. Subsetting markers was done based on two approaches: 1) linkage disequilibrium between markers and 2) markers that met a significance threshold. Linkage disequilibrium between markers, which defines a haplotype block, was evaluated using the correlation coefficient between alleles at a pair of genetic loci. Five LD based subsets of markers were selected from the haplotype block that was made using a correlation coefficient (r) of P-value of the significant association of markers with a trait at probability levels of P < 0.5 (SNP_5), P < 0.1 (SNP_1), and P < 0.05 (SNP_05). The significant association between markers and traits was conducted using the Fixed and random model Circulating Probability Unification (FarmCPU) model [P > 0.05 (SNP_NS). The F-test for two-samples of variance was conducted to compare the significant effect between the different sets of markers.In addition to selecting marker subsets based upon haplotype blocks, we also selected subsets of markers based on the U) model . One subThree different sizes of training populations among these species were evaluated to determine the effect of the training population proportion on the genomic prediction accuracy. These three training population proportions consisted of 90 training to 10 testing set, 70 training to 30 testing set, and 50 training to 50 testing set. Random assignments of individuals to training and testing sets were repeated 10 times and the average value of the prediction accuracy are reported in this work. The correlation coefficient (r) between the GEBVs and observed phenotypic values was used to determines predictive ability (r), which was used as the indicator of prediction accuracy in this paper. This approach has been used previously by several groups , 46.h2) was estimated to understand the variation and trend of predictive ability across traits [Y = Xβ + Zu + e, where Y is the vector of observed phenotypes; β is an unknown vector containing fixed effects, including the genetic marker, population structure (Q), and the intercept; u is an unknown vector of random additive genetic effects from multiple background QTLs for individuals/lines; X and Z are the known design matrices; and e is the unobserved vector of residuals. The u and e vectors are assumed to be normally distributed with a null mean and a variance of: 2aK with σ2 a as the additive genetic variance and K as the kinship matrix. Homogeneous variance was assumed for the residual effect . The proportion of the total variance explained by the genetic variance is defined as marker-based narrow sense heritability, which was calculated for all traits using different subsets of markers and different training-to-testing population proportions.Marker-based narrow sense heritability  between markers and significant markers, in soybean for two traits including canopy wilting (CW) and carbon isotope ratio (δ13C). Table S3. Markers distribution in the different subsets of markers, which were selected based on the two methods, linkage disequilibrium (LD) between markers and significant markers, in maize for two traits including days to tasseling (DT) and ear height (EH). Table S4. Markers distribution in the different subsets of markers, which were selected based on the two methods, linkage disequilibrium (LD) between markers and significant markers, in rice for two traits including panicles per plant (PPP) and seeds per plant (SPP). Table S5. Correlations of narrow sense heritabilities and prediction accuracies of 11 different genomic prediction models for canopy wilting (CW) carbon isotope ratio (δ13C), seeds per plant (SPP), panicles per plant (PPP), and days to tasseling (DT) and ear height (EH) at different training-to-testing proportions . Table S6. Prediction accuracy of genomic models for canopy wilting (CW) and carbon isotope ratio (δ13C) in soybean, and seeds per plant (SPP) and panicles per plant (PPP) in rice, and days to tasseling (DT) and ear height (EH) in maize using different subsets of markers at the 90/10% training-to-testing proportion. Table S7. Prediction accuracy of genomic models for canopy wilting (CW) and carbon isotope ratio (δ13C) in soybean, and seeds per plant (SPP) and panicles per plant (PPP) in rice, and days to tasseling (DT) and ear height (EH) in maize using different subsets of markers at the 70/30% training-to-testing proportion. Table S8. Prediction accuracy of genomic models for canopy wilting (CW) and carbon isotope ratio (δ13C) in soybean, and seeds per plant (SPP) and panicles per plant (PPP) in rice, and days to tasseling (DT) and ear height (EH) in maize using different subsets of markers at the 50/50% training-to-testing proportion.Additional file 2: Fig. S1. Prediction accuracies for carbon isotope ratio (δ13) in soybean using BayesB model and a training-to-testing cross-validation proportion of 90:10%. Thirteen sets of marker subsets were selected based on the different significant levels including P < 0.001, P < 0.0025, P < 0.005, P < 0.0075, P < 0.01, P < 0.025, P < 0.05, P < 0.075, P < 0.1, P < 0.25, P < 0.5, P < 0.75, and complete set.Additional file 3. Zip file containing genotypic and phenotypic data files for three datasets: soybean, two traits (δ13C and CW) and 42,509 SNPs for 346 accessions; maize, two traits (DT and EH) and 50,896 SNPs for 273 accessions; rice, two traits (PPP and SPP) and 44,100 markers from two projects: OryzaSNP project and OMAP project.Additional file 4. Zip file containing R scripts used in this study.


Greater benefit was seen in younger participants and those with the highest risk. The small number of participants and the nascent status of plasma amyloid testing will rightfully temper embracement of these results. However, both the favorable direction of change after BCG as well as the utility of the APS—a valuable surrogate AD biomarker—may prompt a definitive large-scale multicenter investigation of BCG and AD risk as determined by plasma amyloid peptide ratios and APS.BCG vaccine has been used for 100 years to prevent tuberculosis. Not all countries, including the United States, adopted the initial World Health Organization recommendation to use BCG. Moreover, many Western countries that had routinely used BCG have discontinued its use. Recent population studies demonstrate lower prevalence of Alzheimer’s disease (AD) in countries with high BCG coverage. Intravesicular instillation of BCG is also used to treat bladder cancer that has not invaded the bladder muscle wall and has been shown to reduce recurrence. Several retrospective studies of bladder cancer patients demonstrated that BCG treatment was associated with a significantly reduced risk of developing AD. Plasma amyloid β assessment has become a fertile area of study for an AD biomarker that is predictive of a positive amyloid PET scan. Mass spectrometry-based plasma amyloid 42/40 ratio has proven to be accurate and robust, and when combined with age and ApoE, is shown to accurately predict current and future brain amyloid status. These parameters, amyloid 42/40 ratio, age and ApoE genotype are incorporated into an Amyloid Probability Score (APS)–a score that identifies low, intermediate or high risk of having a PET scan positive for cerebral amyloid. Community recruitment was used for this open-label pilot study. Forty-nine BCG-naïve, immunocompetent individuals completed our study: prior to BCG prime and boost, as determined by the APS, 34 had low risk (APS 0–35), 5 had intermediate risk (APS 36–57) and 10 had high risk (APS 58–100). The APS range for the participant group was 0 to 94. Follow-up plasma amyloid testing 9 months after vaccination revealed a reduction in the APS in all the risk groups: low risk group ( The Bacillus Calmette–Guérin (BCG) vaccine has not only been viewed as an important intervention in tuberculosis control but as an adjuvant for other antigens and as a “pro-inflammatory” agent in cancer therapy. Very shortly after its introduction in humans, Calmette noted its effect, not just on the incidence of tuberculosis, but also on the general mortality of infants . During Increasingly understood is that the pathology of AD predates clinical symptoms by decades ,7. AccurWith the LC-MS method, receiver operating characteristic analysis show that prediction of amyloid PET status by plasma Aβ42/Aβ40 ratio resulted in an area under the curve (AUC) of 0.88. When age and APOE e4 status was added to the model, the AUC went up to 0.94. Moreover, in a small subset of individuals who were negative for amyloid on PET imaging at baseline, the plasma assay predicted future amyloid positivity on PET imaging .Clinicaltrials.gov identifier: NCT04449926). Participant demographics are found in 2N Diagnostics, and analyzed in a blinded manner. The samples were tested utilizing liquid chromatography-mass spectrometry as previously described [8 CFU of lyophilized BCG was reconstituted in 50 mL of saline. A single dose consisting of 0.1 mL (2 × 105 CFU) was administered by slow intradermal injection using a 25 gauge/0.5 mm syringe in the deltoid area. A follow up booster dose was given 1 month after the initial dose. All participants had an anticipated local inflammatory reaction at the vaccination site. None experienced axillary lymphadenopathy nor fever. Participants returned 9 months after the initial vaccination for follow up testing of lymphocyte profile, Il-6 and plasma Aβ42 and Aβ40.Community recruitment for individuals with a family history of “dementia” and 50–80 years of age drew 49 BCG naïve, immunocompetent individuals who participated and completed the study : A Language and Environment for Statistical Computing  using RStudio 2021.09.1 as an Integrated Development Environment,  and GraphPad Prism v. 9.3 . p-values < 0.05 were considered significant.Numerical data were expressed as mean ± standard deviation (SD) and median with interquartile range (IQR). Categorical data were expressed as frequency and percentages. The significance of difference of numerical data between two independent groups was assessed using parametric independent p = 0.52) from that of female participants (64.3 years). Similarly, the length of education of participants was not statistically significant between males and females (p = 0.47). The proportion of patients who tested positive for CMV, defined as IgG titer greater than 0.6 U/mL, was not significantly different between females and males . Although the proportion of male participants who had at least one ApoE4 allele was higher than that of female participants, the difference was not statistically significant . The SAGE scores were not significantly different between females and males either before or after BCG vaccine administration.The study cohort consisted of 49 participants, of which 28 (57.1%) were female and 21 (42.9%) were males . The aveThe Aβ42/40 ratio for each participant was stratified by ApoE genotype . As can p < 0.0001) (p = 0.49).When the Aβ42/40 ratio before and after BCG administration was stratified by age, participants ≤ 65 years old had a significant increase in the Aβ42/40 ratio post BCG vaccine compared to the baseline ratio before vaccine administration  . On the p = 0.016) (The change in APS following BCG vaccination in the participants was stratified by the risk of positive PET scan as assessed by the baseline APS. The APS was reduced after BCG administration in the three risk categories . However= 0.016) .CMV is part of the herpes family of viruses, its designation HHV-5. CMV is mostly known as an opportunist causing life-threatening infection in HIV infected persons or in immunosuppressed organ transplant recipients. In immune competent individuals, infection with CMV is usually asymptomatic; once established, its containment becomes a priority for the immune system, which is unable completely to eliminate it and its continued presence promotes age-like immune changes ,12. The p = 0.03), whereas there was no significant change in the APS following BCG vaccination in participants with positive CMV IgG (p = 0.13).Thus, the change in APS following BCG vaccination was stratified by the CMV IgG status. There was no significant difference in APS before BCG administration between participants having negative or positive CMV IgG . Howeverp = 0.52). On average, the APS was significantly lower after BCG administration in participants having a higher CD4/CD8 ratio (p = 0.003), whereas it was not significantly different in participants with a lower CD4/CD8 ratio (p = 0.75)  .p < 0.0001). The mean CD4/CD8 ratio was not significantly altered in participants following BCG vaccine administration compared to the baseline value .In elderly individuals positive for CMV there is paucity of naïve T-cells and an accumulation of terminal T-cells with no proliferative capacity . This “iIn this study, the reduction of risk of cerebral amyloid as determined by increase in plasma Aβ42/40 ratio and decrease in APS after BCG vaccination is reported. The favorable changes are statistically significant in the younger participants, in those with the highest risk category, those not latently infected with CMV and those participants with a favorable lymphocyte immune risk profile (CD4:CD8 > 1.5).Integral to the study, determination of the amyloid beta peptides by the methods of Schindler et al. of the Bateman laboratory is based on isolation of Aβ from plasma via immunoprecipitation, purification by liquid chromatography and measurement via mass spectrometry. Other groups are developing similar diagnostic testing, the Quanterix SIMOA system which, by reaction with monoclonal antibodies, determines phosphorylated Tau with a 90% sensitivity in AD . A laterIn the past decade, there have been several clinical trials that re-introduce non-pathogenic BCG to stimulate immune remodeling against infectious, autoimmune and allergic diseases ,22,23. TM. tuberculosis [M. avium ss. paratuberculosis (MAP) in AD [While the primary use of BCG is for the prevention of tuberculosis , there irculosis . This noP) in AD . It is nP) in AD .(i) a heterologous TH1, TH17, and CD8+ lymphocytic response and (ii) a non-specific memory in innate immune cells  through epigenetic effects and metabolic rewiring in a paradigm known as “trained immunity” [There is increasing evidence that BCG immunization lowers overall mortality that is beyond its limited protection against TB. In part, this protection is related to increased protection by BCG against other infections including viruses such as Yellow Fever virus, human papillomavirus, respiratory syncytial virus, and SARS-CoV-2. With varying degrees of evidence, such protection has been shown in both experimental animals and humans. Such non-specific protection by BCG against non-mycobacterial infections and perhaps other non-infectious disorders include induction of: mmunity” . Of contmmunity” ,35,36. Epidemiologic studies of intradermal BCG (as a TB vaccine) and intravesicular BCG (for bladder cancer immunotherapy) administration showing an inverse relationship with the prevalence of AD implicate only an association and not necessarily a causal effect. However, our prospective finding that intradermal BCG induces a favorable profile of a peripheral biomarker for AD is a highly promising signal. What could be the mechanism(s) by which BCG induces a favorable impact? Regardless of the inciting event for AD, it is becoming increasingly clear that damaging neuro-inflammation contributes to the neurodegeneration seen with AD ,38. An iIn the APP/PSI AD mouse model, BCG immunization reversed their cognitive decline but did not reduce the burden of Aβ in the brain . Further+CD4+ and IFNγ+CD4+ T cell responses [BCG may also afford protection against demyelinating disease such as multiple sclerosis  and expeesponses .Our finding of an AD benefit from vaccination adds to a growing list of studies that have found an association between vaccination and decreased AD risk. A relationship between Tetanus, Diptheria and Pertussis (Tdap) vaccine and AD was investigated in a retrospective review of 100,000+ medical claims from Veteran Health Administration and private sector cohorts . After cClincaltrials.gov site for BCG/ COVID-19 lists 29 international studies assessing BCG as a preventative against COVID-19. This includes a multicenter US study: ClinicalTrials.gov Identifier: NCT04348370. The large data sets associated with COVID-19 vaccines will undoubtedly be mined for years to come including the role that BCG may have had with COVID-19 [With the preceding discussion of the potential AD benefit from various vaccines, the question can rightly be posed: what about the COVID-19 vaccines. Both epidemiological and clinical studies suggest BCG vaccine has protective effects against COVID-19 ,46. All COVID-19 .The results of this study show the utility of the plasma amyloid-based APS particularly as it is able to identify higher risk individuals at an age when intervention, in this case BCG, can be more successfully introduced. The long history of BCG use, its strong safety profile and success in this pilot trial would make it a strong candidate for further study. Taken as a whole, the reduction of risk of cerebral amyloid as determined by an increase in plasma amyloid 42/40 ratio and decrease in APS after BCG vaccination is reported in this study. The favorable changes are statistically significant in younger participants, in those with the highest risk category, those not latently infected with CMV and those participants with a favorable lymphocyte immune risk profile (CD4:CD8 > 1.5).


Undertreatment rates were not statistically significant between men and women in the overall cohort , and male sex was not found to be a significant factor in undertreatment . The result persisted even when patients were divided into four age groups and two groups based on the study time period. The undertreatment rates in both sex groups remained consistent following the introduction of novel oral anticoagulants. In conclusion, there was no evidence of sex disparity with respect to OAC treatment, even after stratifying the cohort by age and ischemic stroke year.Various studies on oral anticoagulants (OAC) use among atrial fibrillation (AF) patients have shown high rates of undertreatment and the presence of sex disparity. This study used the ‘Geisinger Neuroscience Ischemic Stroke’ (GNSIS) database to examine sex differences in OAC treatment among ischemic stroke patients with the pre-event diagnosis of AF in rural Pennsylvania between 2004 and 2019. We examined sex disparities in OAC undertreatment and associated risks based on age group and ischemic stroke year. A total of 1062 patients were included in the study and 1015 patients (96%) had CHA It isSc score ,4,5,6. Clatelets .2DS2-VASc scores [Despite the AHA/ACC/HRS guidelines for the management of patients with atrial fibrillation , some stc scores , while ac scores . Furtherc scores . While tc scores , there iIn a previous study, we investigated the prevalence and factors associated with AF undertreatment in patients with stroke outcomes . The curThe study used a retrospective cohort analyzing data from the “Geisinger Neuroscience Ischemic Stroke (GNSIS)” registry, a database of ischemic stroke patients at Geisinger which includes demographics, family history, and clinical and past medical history. Geisinger is an integrated system delivering healthcare in rural Pennsylvania to approximately 2.6 million people throughout 43 counties. Patients were included in GNSIS if they had a primary diagnosis of ischemic stroke based on the International Classification of Diseases, Ninth/Tenth Revision, Clinical Modification (ICD-9-CM or ICD-10-CM) during a hospital encounter of at least 24 h, and a magnetic resonance imaging of the brain in the same encounter. Further details on the data extraction and pre-processing for the GNSIS database are provided in previously published study articles ,15,16. T2DS2-VASc score. Sex was studied as a risk factor for undertreatment in patients with CHA2DS2-VASc ≥ 2 stratified based on age group or NOACs use. Alternatively, CHADS2 was employed for risk stratification with a score of ≥2 denoting patients at high risk of stroke.The study included ischemic stroke patients ≥ 18 years old with an AF diagnosis ICD (ICD-9-CM and ICD-10-CM) code at any time before the stroke index date, and it excluded patients with an AF diagnosis on the index stroke date. The study analyzed point prevalence among men and women, and patients’ distribution according to CHATo study the impact of NOACs on sex disparity in undertreatment, patients were stratified based on their index stroke date. The first group contained patients with an index stroke date between 2004 and 2010, while the second group included patients with an index stroke date between 2011 and 2019. To examine the sex disparity in OAC treatment in different age groups, the patients were also divided into four subgroups based on age, and undertreatment was examined in each subgroup.The HAS-BLED score is used by clinicians to evaluate the risk of bleeding in AF patients and assesses the history of uncontrolled hypertension, renal or liver disease, stroke, bleeding, labile INR, age ≥ 65 years, medications, and alcohol use . For thip-values was set to 0.05. R version 4.0.3  was used for all statistical analyses.In the study, demographic and comorbidity characteristics for the population were summarized using descriptive statistics. Continuous variables were presented as mean ± standard deviation or median with interquartile range (IQR), and categorical variables were presented as counts and percentages. Statistical analysis among groups included Pearson’s chi-squared test or Fisher’s exact test for the categorical variables, and analysis of variance (ANOVA) or the Kruskal–Wallis test for continuous variables. Multiple logistic regression was performed to examine the association of gender with undertreatment in AF patients, adjusting for age and comorbidities, while the goodness of fit was evaluated using the Hosmer–Lemeshow test. Variables with missingness, such as the National Institutes of Health Stroke Scale (NIHSS), were not included in regression analysis. The alpha value for all p < 0.001). The median age at the index date of stroke was 76.9 years (IQR 68.1–82.9) for men and 82.6 years (IQR 74.9–88.3) for women (p < 0.001). Additional patient characteristics are summarized in p = 0.418), anticoagulants only (129 (25.5%) vs. 146 (26.3%); p = 0.830), or anticoagulants and antiplatelets (120 (23.7%) vs. 106 (19.1%); p = 0.076). There was a significant difference between the CHA2DS2-VASc median score . Further comparison of CHA2DS2-VASc scores distribution based on sex can be seen in Evaluation of ischemic stroke patients from the GNSIS database yielded 1062 patients with an AF diagnosis before the index stroke date . Of thes2-VASc into three categories, 0, 1, and 2+ (p = 0.18), as seen in p = 0.17)  . To examine sex differences in atrial fibrillation, patients were first stratified into four groups based on age quartiles . Group 1p = 0.95). Additionally, sex was not associated with undertreatment rate  in p = 0.11) in p = 0.06) in When grouping the patients based on the index stroke year, the first group (2004–2010) contained 221 patients with 112 (50.7%) identified as undertreated with anticoagulants, 58 (51.3%) men, and 54 (50.0%) women . Undertr2DS2-VASc ≥ 2; however, sex was not shown to be a statistically significant risk factor for OAC undertreatment. When dividing the population based on age and index stroke year, both analyses revealed no apparent sex disparity and no risk association between sex and undertreatment. Following the introduction of NOACs in 2010, the undertreatment rates remained consistent between the 2004–2010 and 2010–2019 groups, without significant sex disparities or associated risk. Similarly, risk stratification using CHADS2 scores did not show a sex disparity or risk association with undertreatment  confirmation. Furthermore, ICD codes do not provide detailed clinical information and sex-related preferences. Previous studies have found that women tend to present with more symptoms, older age, and receive more conservative treatments ,13. AddiThis study found that OAC treatment for AF in ischemic stroke patients remains low according to guidelines in both men and women; however, there does not appear to be a sex disparity, even when stratifying patients by age and index ischemic stroke date. Sex was not associated with OAC undertreatment in our rural population.


ARFIP2 gene and identified with the HyperLasso method, revealed predictive potential and was included in the best-performing regression models. Advanced machine learning approaches showed a significant increase in sensitivity of intermediate eye colour prediction (up to 39%) compared to 0% obtained for the original IrisPlex model. We identified a new potential predictor of eye colour and evaluated several widely used advanced machine learning algorithms in predictive analysis of this trait. Our results provide useful hints for developing future predictive models for eye colour in forensic and anthropological studies.Increasing understanding of human genome variability allows for better use of the predictive potential of DNA. An obvious direct application is the prediction of the physical phenotypes. Significant success has been achieved, especially in predicting pigmentation characteristics, but the inference of some phenotypes is still challenging. In search of further improvements in predicting human eye colour, we conducted whole-exome (enriched in regulome) sequencing of 150 Polish samples to discover new markers. For this, we adopted quantitative characterization of eye colour phenotypes using high-resolution photographic images of the iris in combination with DIAT software analysis. An independent set of 849 samples was used for subsequent predictive modelling. Newly identified candidates and 114 additional literature-based selected SNPs, previously associated with pigmentation, and advanced machine learning algorithms were used. Whole-exome sequencing analysis found 27 previously unreported candidate SNP markers for eye colour. The highest overall prediction accuracies were achieved with LASSO-regularized and BIC-based selected regression models. A new candidate variant, rs2253104, located in the The online version contains supplementary material available at 10.1007/s00414-021-02645-5. Increasing understanding of human genome variability is enabling better use of DNA’s predictive potential . BesidesIn this study, we explored the possibility of increasing the accuracy in predicting eye colour. To this end, we adopted the following strategies: (1) quantitative characterization of samples using high-quality images of the iris analysed with Digital Iris Analysis Tool (DIAT) software; (2) whole-exome sequencing (WES)-based identification of new potential predictors in a group of 150 phenotypically diverse Polish samples using the HyperLasso method and regression-based single-SNP association testing; (3) predictive modelling conducted based on the literature and WES-identified markers, using various machine learning algorithms and independent sets of samples in order to find the most accurate method for eye colour in a moderate dimensional dataset.The study cohort consisted of 999 unrelated individuals , collected together within the NEXT project, funded by the National Centre for Research and Development, grant number DOB-BIO7/17/01/2015. The study was approved by the Ethics Committee of the Jagiellonian University in Kraków (decision no. KBET/122/6120/11/2016), and all volunteers gave written informed consent prior to their inclusion in the study. Recruitment of the participants was carried out in the Police Academy in Szczytno.Whole blood was collected from the volunteers and subjected to DNA extraction using the PrepFiler Express™ Forensic DNA Extraction Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. Quantification of the extracted samples was performed using the Quantifiler™ Human DNA Quantification Kit or the Plexor® HY System.f/22, ISO 200, flash A = 1/8, B = 1/4. Eye colour was classified into 3 categories: blue, intermediate  and brown. Classification was performed based on photographic documentation of both irises, by one assessor. The assignment to a specific category was carried out in two independent rounds of classification, or three, when there was an inconsistency between the first and the second round of assessment. The second approach consisted in an objective, quantitative characterization of eye colouration. Eye colour quantitative evaluation was conducted based on high-quality images and with DIAT software [Phenotyping of the investigated samples for eye colour was conducted in two independent ways: quantitative measurements, used for identification of new SNP candidates from WES analysis, and categorization, used at the predictive modelling stage. The evaluation was performed based on collected high-resolution photographic documentation. Photos of both eyes were taken in identical conditions for all volunteers using a Nikon D5300 camera with an R1C1 Wireless Close-up Speedlight System . Images of the iris were taken from a distance of about 20–30 cm, with the following settings: shutter speed 1/125, aperture software . Blue anExonic sequences (66 Mbp) enriched in regulatory regions of > 160 loci with a known association with human appearance traits (1.5 Mbp) extracted from Nencki Genomics Database and FANTOM , 23 wereP-value < 1 × 10−4 for candidate SNP selection. Because of the character of the data, linear regression for quantitatively described eye colour was used. Results were adjusted for age and sex. In addition, the HyperLasso method (https://www.ebi.ac.uk/projects/BARGEN) was applied as an alternative approach for feature selection. It is a highly attractive method that addresses the computational challenge of simultaneous SNP analysis from large-scale experiments. It is a model selection method that utilizes a Bayesian-based penalized maximum likelihood approach, which can handle high-dimensional inputs [p), i.e. 0 < p < 100. All newly selected candidate SNP markers were subjected to linkage disequilibrium (LD) pruning and one SNP from each LD block (r2 > 0.7) was kept for further analyses. LD analysis was conducted using PLINK 1.9, while remaining analyses were conducted with R v3.5.2 using ‘ordinal’ package and ‘HyperLasso’ code. Since many variants associated with human pigmentation traits have already been identified, an intensive literature review was conducted and 114 SNP markers, previously correlated with pigmentation in general, were selected for further statistical analyses  with Thermo Fisher Scientific support, and covered DNA markers for various human appearance traits, including pigmentation, hair morphology, hair greying, earlobe, monobrow and other traits investigated within the NEXT project. Because of technical problems, four SNPs were replaced by SNPs in LD . DNA libraries were prepared manually and sequenced as described previously [Genetic data for 141 DNA variants in a population of 849 individuals were collected using targeted high-throughput DNA sequencing with Ion AmpliSeq™ technology and an Ion S5™ or Ion Proton™ platform. Two independent Ion AmpliSeq™ custom panels were designed using Ion AmpliSeq™ Designer tool , which were monomorphic in our dataset and were excluded from statistical analyses. The final list of variables also included age and sex. Various machine learning algorithms were evaluated for the most accurate model development. Models were developed to predict eye colour categorized in three classes.All candidate SNP variants selected from the literature and WES analysis were used in prediction modelling except four variants . In order to determine their robustness, two additional regression models, i.e. (1) developed using only one marker—the most important one—chosen in the first round of SNP selection (LOG 1-STEP), and (2) using all the analysed in this study SNPs (LOG FULL), were added for comparison purposes. In addition, we used LASSO, in which the penalized log-likelihood function is considered [glmnet package.Marker selection for regression models was performed by the forward selection method with two classical statistical approaches, the Akaike Information Criterion (AIC)  and the nsidered . This reinfotheo, missForest, cvTools, rpart, randomForest, xgboost, glmnet, class, ROCR, earth, nnet and caret. All tested algorithms with their abbreviations used in the text are listed in Table The performance of seven additional machine learning algorithms was also tested. They included Classification and Regression Trees (TREE), Random Forests (RF), Extreme Gradient Boosting (XGB), Multivariate Adaptive Regression Splines (MARS), Neural Network (NN), Support Vector Machine (SVM) and Naïve Bayes (NB). A random naive classifier, which assigned observations randomly to classes according to apriori probabilities (Naive), was used as a benchmark. Default settings were applied to tested algorithms. Feature selection for TREE, RF, XGB and MARS algorithms is embedded in the learning algorithm, i.e. relevant features were selected during fitting the models. Analyses were carried out using R (v 4.0.3) and the following packages: AUC — area under the ROC curve — expressing the overall prediction accuracy and ranges between 0.5, which corresponds to random classification, and 1.0, which corresponds to perfect classificationAccuracy (acc.) — the percentage of individuals correctly classified into a specific category over the total number of individuals in the analysis, expressed by the equation:Sensitivity (sens.) — true positive rate, expressed by the equation:Specificity (spec.) — true negative rate, expressed by the equation:In order to assess the performance of the fitted models, the predictive modelling cohort was divided into training (70%) and testing (30%) datasets, which is a commonly used strategy, close to optimal for reasonable sized datasets (n ≥ 100) with strong signals (≥ 85% accuracy) . AdditioWe calculated the above measures for each class (eye colour), i.e. the observations corresponding to the considered class are treated as positive examples, whereas observations corresponding to the two remaining classes are treated as negative examples.P-value = 0.349 and P-value = 0.582, respectively). Quantitative measurements of eye colour revealed a whole range of possible phenotypes, from PIE score =  − 1 to 1. Mean PIE score was 0.2 (SD = 0.8). The borderline statistical significance was noted for correlation between quantitatively described eye colour and age  but not sex . Nevertheless, although statistically significant, correlation between PIE score and age was negligible in the studied sample set (r = 0.065). Information about eye colour of the whole studied cohort, and divided into discovery and predictive modelling cohorts, is provided in Table The study cohort consisted of 673 (67.4%) males and 326 (32.6%) females in the age range 19–77 years . Considering the categorized pigmentation phenotype, most individuals had blue eyes (62.8%) followed by brown (18.1%) and intermediate (15.0%). Due to difficulties in unambiguous eye colour categorization, forty-one individuals’ eye colour was not determined (4.1%) mainly due to varying degrees of heterochromia. There was no statistically significant correlation between categorized eye colour and age or sex   candidate DNA markers for eye colour. A literature search revealed a further 114 DNA markers previously associated with pigmentation  overlapped with WES-based selected variants. Overall, WES analysis discovered twenty-seven novel candidates for eye colour. As the four SNPs were monomorphic in the studied dataset, prediction modelling finally involved analysis of 137 SNPs, age and sex  selected in > 50 out of 100 data splits and 2) selected by at least two selection methods. Among them, besides well-known literature predictors, was the novel marker rs2253104 in  selectedHERC2 gene, selected as the best predictor) achieved slightly better AUC values than both AIC-based and FULL models and only marginally lower compared to BIC- and LASSO-based developed models for all eye colour categories. The differences were mostly observed for the intermediate category , while for blue and brown eye colours, the differences were less pronounced. Most importantly, the sensitivity of intermediate eye colour prediction reached high values, especially in the case of LOG AIC: sens. = 0.40 and LOG FULL: sens. = 0.41. LOG BIC and LOG REG also showed relatively good values of 0.29 and 0.17, respectively, but not LOG 1-STEP: 0.00. At the same time, the specificity of the LOG BIC and LOG REG models was very high, reaching values of 0.96 and 0.97, respectively, while LOG FULL and LOG AIC remained reasonably high: 0.85–0.88. Detailed results of prediction performance analysis are shown in Table Five different approaches were analysed in order to find the most accurate methods for regression model development. The highest prediction accuracies were achieved using the BIC and LASSO regularization methods. These values reached acc. = 0.84 and 0.85, respectively, which means that 84–85% of individuals were classified into the correct eye colour category. Slightly lower accuracies were achieved with the AIC method and for the 1-STEP model (for both acc. = 0.79), and the lowest for the LOG FULL model (acc. = 0.74). Moreover, high AUC values were noted for all categories, including intermediate eye colour (AUC = 0.85), equal for models developed with the help of the BIC and LASSO approaches. Using these methods, blue and brown eye colours were predicted with an AUC of 0.96 and 0.93–0.94, respectively. Interestingly, a 1-STEP model . The poorest accuracies were achieved by NB and NN (acc. = 0.77). When it comes to AUC, the highest values were estimated for RF for all eye colour categories. Nevertheless, all these methods  gave similar AUC and accuracy outcomes.However, sensitivity and specificity values varied substantially among the tested methods. The lowest sensitivity values were estimated for RF  and SVM, followed by NB and NN. The greatest differences were found for intermediate eye colour, which was particularly interesting. The highest sensitivity values were estimated for MARS (sens. = 0.39), TREE (sens. = 0.35) and XGB (sens. = 0.34), while they were significantly lower for NN (sens. = 0.18), NB (sens. = 0.16), RF (sens. = 0.11) and SVM (sens. = 0.10). On the other hand, specificity values were the highest for RF and SVM , followed by NB and NN (spec. = 0.95 and 0.94) then TREE, XGB and MARS (spec. = 0.91–0.92). Details of prediction modelling analysis conducted using advanced machine learning algorithms are shown in Table P-value = 5.46 × 10−233). Using the single-SNP association testing of the WES/regulome data under P-value < 1 × 10−4, and the HyperLasso algorithm, which aimed to select the subset of SNPs that best predicted the trait under study simultaneously controlling the type I error of the selected variants [Accuracy of phenotype prediction from genetic data is essential for the successful application of predictive methods in biomedical studies including anthropology, paleogenetics and forensics . Severalvariants , we idenARFIP2, newly identified by HyperLasso (Table ARFIP2 is located on 11p15.4 and encodes for ADP-ribosylation factor-interacting protein 2 (ARFIP2), which is highly expressed in various tissues. This protein has been shown to be involved in several cellular processes and signalling pathways. They include Rac1-mediated signalling, triggering actin polymerization [ARFIP2 was among the downregulated genes in human melanoma cells treated with arbutin [ARFIP2 has been associated with lung cancer [ARFIP2 was selected in 65% of LOG AIC models and in 51% of LOG REG models, therefore more frequently than, e.g. rs12203592 in IRF4 or rs1408799 in TYRP1 (LOG REG), the other well-established eye colour predictors  methods, also deserves more attention. The research by Yuasa et al. showed a north–south geographic gradient of the rarer T allele, which was interpreted as a possible case of adaptive evolution [OCAC2 variant is responsible for reduced efficiency of melanogenesis [HERC2), rs1800407 (OCA2) and rs1689182 (SLC45A2) [OLFM3; [MC1R, rs8049897, DEF8; [A large number of algorithms have been developed to deal with a variety of increasingly demanding and computationally challenging data analyses. Analysis of AIC, BIC and LASSO methods of marker selection conducted in this study revealed that all of them are robust, since they produced models with better performance compared to models without any selection method applied (i.e. LOG FULL). We confirmed that BIC, which more heavily penalizes the introduction of additional variables, produced the most parsimonious models. Together with BIC, LASSO yielded models with the best predictive performance. Interestingly, focusing on SNPs selected by at least two out of three feature selection methods and in at least 50% of data splits, we found the well-known pigmentation markers and the intronic variant rs2253104 in rization , which irization , inducinrization . Interes arbutin , which i arbutin . Therefo arbutin . So far,g cancer . Rs22531volution . Indeed,ogenesis  and thusogenesis . The incSLC45A2)  as well  [OLFM3; ), or oth7, DEF8; ) , higher than reported for IrisPlex  and Snip colours . A small colours .interm. = 0.34–0.39); however, a slightly reduced sensitivity of brown eye colour prediction was observed for these models when compared to the regression model. It is well known that more advanced machine learning methods may better cope with recognition of complex phenotypes, including intermediate eye colour, due to the ability to identify possible nonlinear dependencies between variables, such as interactions. Nevertheless, while some advanced methods were found to demonstrate increased sensitivity or specificity in predicting certain categories, none of these approaches outperformed the regression method developed following prior features selection using BIC or LASSO, when AUC or accuracy metrics were compared. Moreover, differences between the tested methods were modest. These results suggest that more sophisticated learning algorithms may need larger datasets to demonstrate their superiority and do not reveal their potential in low- and medium-dimensional data. Also, a systematic review [Besides classical regression, several more advanced machine learning algorithms were evaluated. The study demonstrated that advanced machine learning methods showed even higher sensitivity values of intermediate eye colour prediction  and ranc review  were sucARFIP2 was selected by at least two different feature selection methods for regression predictive models, which turned out to be the most accurate. None of the sophisticated machine learning algorithms outperformed the overall prediction accuracy of regression models developed following prior features selection using BIC or LASSO regularization, indicating that medium-dimensional data does not use the whole potential of these more advanced algorithms.In summary, whole-exome sequencing of 150 individuals has allowed identification of 27 DNA variants that are relevant for eye colour prediction which have not been reported before in pigmentation predictive studies. Besides well-known pigmentation-associated variants, rs2253104 in Supplementary file1 (DOCX 54.2 KB)Below is the link to the electronic supplementary material.


The data are nationally representative and are based on analysis of cross-sectional data for the non-institutionalized, U.S. household population. The survey consisted of interviews conducted in participants’ homes and standardized health examinations conducted in mobile examination centers. Distributions of total hair mercury levels expressed as micrograms per gram hair Hg and the association of hair Hg levels with sociodemographic characteristics and fish consumption are reported. Geometric mean (standard error of the geometric mean) hair mercury was 0.12 μg/g (0.01 μg/g) in children, and 0.20 μg/g (0.02 μg/g) in women. Among frequent fish consumers, geometric mean hair mercury levels were 3-fold higher for women (0.38 vs. 0.11 μg/g) and 2-fold higher for children (0.16 vs. 0.08 μg/g) compared with nonconsumers. The NHANES 1999–2000 data provide population-based data on hair mercury concentrations for women and children in the United States. Hair mercury levels were associated with age and fish consumption frequency.Exposure to methyl mercury, a risk factor for neurodevelopmental toxicity, was assessed in U.S. children 1–5 years of age ( Mercury is a naturally occurring heavy metal whose presence in the environment is widespread and persistent . Hg occuin utero.The mammalian nervous system is highly vulnerable to MeHg . ExposurMore recent prospective epidemiologic data from New Zealand, the Faroe Islands, and the Seychelles assessed developmental effects of lower level MeHg exposure in fish-consuming populations resulting from maternal and fetal exposures to MeHg . The U.STotal blood and hair Hg are indicators of MeHg exposure in fish consumers and others who are not exposed to inorganic and elemental Hg occupationally or incidentally . ExposurHair Hg concentration is the preferred biomarker for evaluating Hg exposure for extended periods of time such as periods of weeks or months . Hair inThe Centers for Disease Control and Prevention (CDC), the U.S. EPA, U.S. Food and Drug Administration, Department of Energy, and the National Oceanographic and Atmospheric Administration funded a comprehensive Hg assessment component as part of the 1999–2001 National Health and Nutrition Examination Survey (NHANES). The NHANES Hg assessment component targeted children 1–5 years of age and women 16–49 years of age. Preliminary blood and hair Hg results from NHANES 1999 were reported in 2001 . A detaiThe NHANES survey is conducted to provide continuous health and nutrition data on the U.S. population for all ages . StandarThe NHANES survey design is a stratified, multistage, national probability sample of the civilian, noninstitutionalized U.S. population; regional estimates are not produced (NCHS 2002). One or more participants within the eligible households sampled are included in the survey. Annual samples are nationally representative and include approximately 6,000 interviewed and 5,000 examined persons. NHANES 1999–2001 included expanded samples of Mexican Americans, non-Hispanic blacks, pregnant females, adolescents 12–19 years of age, and adults ≥ 60 years of age.The NHANES 1999–2000 Hg assessment component included a dietary interview and blood, hair, and urinary Hg assessments. Venipuncture blood collection was conducted on persons ≥ 1 year of age, hair collection on children 1–5 years of age and women 16–49 years of age, and urine collection on persons ≥ 6 years of age. The NHANES dietary assessment component included a 24-hr dietary recall interview on all examined persons and fish and shellfish consumption frequency questions on persons ≥ 1 year of age. All survey examination protocols, questionnaires, and reporting criteria were approved by the NCHS institutional review board; total hair Hg values of ≥ 15 μg/g or total blood Hg values of ≥200 μg/L were reported to participants. Signed informed consent was obtained for all survey participants. Participants or their guardians provided consent for participants < 18 years of age. Examined persons received remuneration for their participation in the survey ranging from $30 to $100, depending on their age and examination content; transportation and child care expenses were also compensated.A minimum of 5–10 mg of hair was required for the hair analysis assay. Approximately 100 strands of hair (~ 50 mg) were gathered and cut from the occipital region of the scalp (approximately the diameter of a pencil eraser). A 1.5 × 2 in. Post-it  was placed over the end of the hair strands closest to the scalp; the paper was marked with an arrow indicating the end of hair closest to the scalp. A plastic clip was placed over the paper to secure the hair sample. The samples were placed in a resealable plastic bag and shipped to the Research Triangle Institute for analysis. Respondents or proxies for children were asked if the respondent’s hair had been given a permanent or been treated with a hair dye or a hair relaxer product within the last month. The analysis compared total hair Hg in persons with treated hair (any of the products listed) to those with untreated hair.Hair segments of 0.4 in. (1.0 cm) closest to the scalp, approximately 1 month’s growth, were analyzed for total Hg concentration using a cold vapor atomic fluorescence spectroscopy method . The metPercent recovery of the Hg analyte was monitored by analyzing hair samples spiked with a known Hg reference standard before the digestion process. Mean percent recovery of Hg (± SD) in the spiked samples was 96.2 ± 12.1%]. The precision of analysis was assessed from duplicate extracts  and from duplicate hair sample analyses . The mean precision (± SD) for duplicate extract and sample analyses was 5.4 ± 8.7% and 11.7 ± 14.4%), respectively. The limit of detection (LOD) for total hair Hg varied by analytic batch because of the laboratory’s batch-specific standardization methodology. Method detection limits ranged from 0.0006 to 0.06 μg/g. Whenever the values for hair Hg were below a batch LOD, a fill value equal to the batch-specific LOD divided by the square root of 2 was used .Fish and shellfish consumption frequency during the 30-day period before hair collection was reported. The fish and shellfish frequency questions were administered by bilingual interviewers in English or Spanish. A proxy respondent reported for children < 6 years of age and proxy-assisted interviews were conducted with children 6–11 years of age . No infoRace and ethnicity were categorized as non-Hispanic white, non-Hispanic black, and Mexican American based on self-reported information provided by the survey participants. The sample size for the other race/ethnicity group that includes Asians and American Indians was too small to present separate estimates; the total population estimates include data for the other race/ethnicity group composed of 103 children and 195 women. Age in years at the time of the household interview was used. Pregnancy status was self-reported during private interviews administered to women ≥ 12 years of age at the mobile examination center. Pregnancy status was confirmed with a urinary pregnancy test in females ≥ 18 years of age in NHANES 1999 and in females ≥ 8 years of age in NHANES 2000.Weighted estimates were produced using NHANES mobile examination center–examined sample weight values. These NHANES 1999–2000 sample weights adjust for the differential probabilities of selection and nonresponse in the survey sample . The samDistributions, extreme hair Hg values, influential observations based on an analysis of the survey sample weights and hair Hg data, laboratory QC data, and correlation analyses were examined initially . After tn = 785) and 96% of women  with hair Hg data. Detectable total hair Hg levels were measured in 84 and 89% of hair specimens obtained for children and women, respectively.A total of 12,160 persons were selected to participate in NHANES 1999–2000; 9,282 completed the household interview and health examination components (76.3%). Of the 1,250 children 1–5 years of age selected for the survey, 1,013 (81%) were interviewed and examined. Hair specimens were obtained from 838 examined children (83% of examined children); hair collection response rates were lower among non-Hispanic black and Mexican-American males because of insufficient hair specimen collection. Hair samples were obtained from 44 and 82% of non-Hispanic black and Mexican-American males respectively, compared with 90% of non-Hispanic white males. This response was not random, and sample weight adjustments were applied during final data analysis. A total of 2,314 women 16–49 years of age were selected to participate; of these, 1,819 were interviewed and examined (79%). Hair specimens were obtained from 1,726 women (95% of the examined women). Fish and shellfish consumption data were obtained for 93% of children  hair Hg levels than girls in all race/ethnicity groups (not shown). For women, non-Hispanic white females had significantly higher GM hair Hg levels than did non-Hispanic blacks and Mexican Americans.The GM hair Hg level of children who received hair treatments during the past month did not differ from that of the untreated group. Thirty-seven percent of women reported using a hair treatment; the GMs total hair Hg levels of the treated and untreated hair groups were the same (0.20 μg/g).GM hair Hg increased with increasing frequency of fish consumption for children and adults  and 2. Tn = 292) did not differ from that in nonpregnant women. Analysis by race/ethnicity among the pregnant sub-sample was limited to comparisons of GM and median hair Hg values because of small sample sizes; race/ethnicity differences were not statistically significant among the pregnant women. Non-Hispanic white and Mexican-American women had higher hair Hg levels than did non-Hispanic black women. GM hair Hg levels among the pregnant, frequent fish consumers (n = 79) did not differ from that among nonpregnant frequent fish consumers .GM hair Hg levels of pregnant women (y = log (hair Hg). LS means for non-Hispanic blacks and Mexican Americans were 0.19 and 0.16 μg/g, respectively, compared with 0.09 μg/g for non-Hispanic whites. Children who consumed fish one or more times during the previous month had higher total hair Hg levels than did children who did not consume fish. LS means for children (Separate multiple linear regression models were developed for children and women  and 4. Tchildren  who consA separate regression model was developed for women 16–49 years of age . Pregnanp < .0001). Hair-to-blood Hg ratios were computed using total hair Hg (ng/g hair) and blood Hg (μg/L) data. The SUDAAN Proc Ratio procedure for correlated variables was used to compute the ratios . The mean ± SE hair Hg level reported in a study of 189 New Jersey pregnant women was 0.53 ± 0.07 μg/g . These subgroups may consume more fish than the general U.S. population and have higher MeHg exposure. Seafood consumption among Asian Americans and Pacific Islanders (AAPI) in King County, Washington, averaged 117 g/day , compareThe NHANES data may be useful for assessing the prevalence of health risks in the U.S. population when the associated risks of low-level Hg content are better defined and may be used to support diet and health research, policy, and monitoring activities. Diet remains the primary contributor to MeHg exposure in populations. More than 50% of NHANES participants consumed fish during a 30-day reference period. Annual seafood consumption projections for the U.S. population indicate that 75–93% of adult women and 58–72% of children 2–5 years of age consume seafood .Hair Hg analysis in national samples of U.S. children and women of childbearing age provide a useful biomarker for long-term Hg exposure. Acceptance of the hair collection procedure was high among survey participants and excellent method precision was achieved, allowing for of detection of hair Hg in approximately 84 and 89% of children and women, respectively. Total hair Hg is associated with age, race/ethnicity, and fish consumption frequency. Among women of childbearing age, total hair Hg levels of pregnant and non-pregnant women were the same.


Antioxidant effects after single ingestion of RW or dealcoholized RW (DRW) have been observed in several studies, but results after regular consumption are contradictory. Thus, we examined if single or repeated consumption of moderate amounts of RW or DRW exert antioxidant activity ex vivo with H2O2 .Total phenolic content and concentration of other antioxidants in plasma/serum, total antioxidant capacity (TEAC) in plasma as well as DNA strand breaks in peripheral leukocytes were measured in healthy non-smokers A) before, 90 and 360 min after ingestion of one glass of RW, DRW or water; B) before and after consumption of one glass of RW or DRW daily for 6 weeks. DNA strand breaks (SB) were determined by single cell gel electrophoresis (Comet Assay) in untreated cells and after induction of oxidative stress 2O2 induced SB were reduced after DRW. In contrast, regular RW consumption reduced SB in untreated cells but did not affect H2O2 induced SB.Both RW and DRW transiently increased total phenolic content in plasma after single consumption, but only RW lead to a sustained increase if consumed regularly. Plasma antioxidant capacity was not affected by single or regular consumption of RW or DRW. Effects of RW and DRW on DNA SB were conflicting. DNA strand breaks in untreated cells increased after a single dose of RW and DRW, whereas HThe results suggest that consumption of both RW and DRW leads to an accumulation of phenolic compounds in plasma without increasing plasma antioxidant capacity. Red wine and DRW seem to affect the occurrence of DNA strand breaks, but this cannot be referred to antioxidant effects. However, a sustained antioxidant response is necessary to achieve protection from oxidative stress related diseases. Hence, the aim of our study was to investigate if regular intake of moderate amounts of red wine could improve antioxidant parameters in healthy subjects, and if dealcoholized red wine could be an alternative source providing polyphenols without increasing the alcohol intake. We conducted two randomized, controlled intervention trials to examine the effects of single and repeated intake of native and dealcoholized red wine, respectively, on total phenolic content and antioxidant parameters in blood as well as on DNA strand breaks in peripheral leukocytes in healthy volunteers.Red wine is rich in polyphenols ,4 and isex vivo , decreastrations  or protetrations  have beetrations -20, but trations -23. Resu [et al.  and van  [et al.  even sugMen and women, aged 18 – 50 y, free from any known diseases were included in the study. Smoking, excessive exercise (> 10 h/wk), pregnancy and lactation, present or former alcohol or drug abuse, medications interfering with antioxidants or intake of alcohol, supplementation of vitamins, minerals or probiotics (2 weeks before or during the study) were exclusion criteria. The study was performed according to the guidelines of the Declaration of Helsinki and was approved by the ethics committee of the University of Bonn. All subjects gave their informed written consent.The study consisted of two parts, a single-dose analysis investigating the effects of a single ingestion of red wine (RW) versus dealcoholized red wine (DRW) and a long-term dietary intervention trial in which RW and DRW were consumed daily for 6 wk. Both trials were prospective, randomized and controlled studies.ex vivo H2O2 treatment to induce oxidative stress) were measured before, 90 and 360 min after ingestion. Subjects were instructed to abstain from polyphenol rich foodstuff and from any alcoholic drinks 24 h before the first blood sampling until completion of the study. For this purpose they received a list of acceptable foods which are considered to be low in polyphenols. Compliance was controlled by a self-estimated 24-h dietary record. One hour after the first blood sampling, participants could choose from a breakfast buffet composed of foods low in polyphenols . All subjects were allowed to leave the study center at 90 min, when the initial blood samples had been drawn, and returned at 360 min for the follow-up blood samples. They were allowed to have lunch in between following the dietary restrictions concerning antioxidants.After an overnight fast, 27 healthy non-smokers  or 175 mL dealcoholized red wine  daily within one hour after dinner for 6 wk. The control group (25 subjects) did not receive any study drink. Subjects were instructed not to drink more than 2 cups (150 mL each) of coffee, black or green tea, and 2 glasses (200 mL each) of fruit juice per day, and to renounce from grape juice, multivitamin juices, and alcoholic beverages starting one week before the intervention throughout the whole study period.Blood samples were drawn before and after intervention after an overnight fast (between 07.30 and 09.00 a.m.), and about 12 h after the last ingestion of RW or DRW. In addition to the laboratory parameters measured in the single-dose analysis, α-tocopherol concentration in serum was determined as changes are expected only in the long term . To contThe red wine used in the present studies was Spätburgunder, 1999, Marienthaler Klostergarten, Ahr, Germany. Dealcoholized red wine was produced by vacuum extraction of alcohol from the same batch. The amounts of flavonoids and phenolic acids ingested from a single serving of RW 200 mL) and DRW (175 mL) are listed in Table 00 mL andet al. [et al. [The subjects received a standardized dietary record which they completed for 24 h  or 7 days , respectively. To determine polyphenol intake as exactly as possible, polyphenol rich foods were listed in detail. Calculation of the intake of flavonoids  and phenolic acids  was based on data of Linseisen et al.  and Radt [et al. , which w [et al.  and cate [et al. , and bla [et al. .® tubes  containing Li-heparin or no anticoagulant. Samples were protected from light and stored on ice until centrifugation . Plasma samples for determination of total phenolic content and antioxidant capacity were stored at -70°C, and for determination of albumin, uric acid and bilirubin at -20°C. For measurement of ascorbic acid, plasma was mixed with 5% trichloro acetic acid and centrifuged . Supernatants were stored at -70°C. Serum was frozen at -20°C until measurement of α-tocopherol. For determination of DNA damage, heparinized blood was kept in the dark at room temperature until processing 60 – 120 min after sampling.Peripheral venous blood was collected in Vacutaineret al. [et al. [Total phenolic content in plasma (TPP) was determined by the Folin-Ciocalteu method modified by Serafini et al.  to avoid [et al. , we centPlasma antioxidant capacity was determined by the Trolox equivalent antioxidant capacity (TEAC) assay as described previously . The antet al. [To control, if antioxidants other than polyphenols could have an impact on TEAC, the following major antioxidants in blood were also measured. Vitamin C concentration in plasma was determined colorimetrically according to Speitling et al. . Experim2O2 (induction of oxidative stress), and lysed. During subsequent electrophoresis, DNA regions that are relaxed due to strand breaks migrate towards the anode forming a typical comet structure.Single Cell Gelelectrophoresis (Comet Assay) was performed to assess DNA strand breaks in peripheral leukocytes. In this assay, cells are embedded in agarose on slides, incubated in PBS (control cells) or Het al. [in vivo situation as far as possible and to avoid artifacts by density centrifugation, whole blood was used instead of isolated mononuclear cells. Ten μL heparinized whole blood were diluted with 90 μL PBS  (Sigma) to obtain a single cell suspension. After preparation of the slides, they were incubated for 20 min at 4°C with either 300 μmol/L H2O2 (Sigma) in PBS to induce DNA strand breaks or in pure PBS (controls). Electrophoresis was performed at 4°C and 1 V/cm for 4 min on a flat-bed electrophoresis apparatus with electrode spacing of 39 cm . Experiments were performed in duplicate.This assay was performed according to the standard protocol of Bauch et al.  adapted aqua bidest.  and DNA was dyed with 150 μL propidium iodide (Sigma) . Measurements were performed with the image analysis software package Comet Assay II  coupled to a fluorescence microscope  with a CCD camera . Fifty cells were measured on each slide and Tail Intensity (TI) and Tail Moment (TM) were calculated to quantify DNA strand breaks.For analysis, gels were rehydrated in in vivo [2O2 ex vivo and were calculated as the difference between treated and untreated cells.DNA strand breaks in untreated cells reflect the steady state between DNA damage and repair in vivo -37 and aThe sample size calculation was based on plasma antioxidant capacity (TEAC), which was considered as main outcome and based on data from a previous study with healthy subjects who ingested polyphenol rich fruit-juices or a fruit-vegetable concentrate . With 6 Mean TEAC difference in the previous long-term study was 0.075 mmol/L. To detect changes ≥ 0.075 mmol/L and differences between the groups with a power of 90%, a sample size of 25 subjects in each group  was calculated. Level of significance was 5% for all tests.p < 0.05 were considered statistically significant. Statistical tests were performed with SPSS 10.0 for Windows .Values given are means ± SD unless indicated otherwise. Differences between the groups were examined using the Mann-Whitney U-test. The effects of the study drinks on the investigated parameters were evaluated by comparing the values obtained at different time intervals with baseline for each group using the Wilcoxon signed rank test. Differences indicated by The day before the study the subjects ingested 22.9 ± 47.4 mg polyphenols . This corresponds to 8 – 16% of the average intake observed in another German cohort ,28 and ip = 0.008) or DRW (p = 0.008). After 360 min values were higher than at baseline in groups RW (p = 0.02) and DRW (p = 0.008), but also in control subjects (p = 0.01). Vitamin C concentration in plasma decreased 360 min after RW consumption (p = 0.008), but increased in group DRW (p = 0.02). Significant changes of endogenous antioxidants, i.e. uric acid, albumin or bilirubin, have been observed after consumption of RW and DRW, and also in the control group, but TEAC was not altered significantly in any group.Results for the antioxidant parameters in plasma are presented in Table p = 0.05). Hydrogen peroxide-induced DNA strand breaks decreased in group DRW at 360 min, whereas no alterations could be observed after RW consumption or in controls. Statistical analysis provides the same results for TI  and DRW , but not in controls (37.7 ± 21.3 vs. 41.0 ± 24.8 mg/d). During intervention, the flavonoid intake in group RW exceeded that in group DRW (p = 0.005), and it was higher in group RW (p < 0.001) and DRW (p = 0.005) than in controls. Intake of phenolic acids increased during supplementation of RW  and DRW , but also in controls . The amounts did not differ between the groups before or during intervention.Flavonoid intake increased significantly in group RW , but did not change in group DRW or controls. A significant increase of plasma uric acid concentration was observed in controls (p = 0.04), and a decrease of bilirubin in group DRW (p = 0.02) , but not in group DRW and controls. Susceptibility of leukocytes towards H2O2-induced DNA damage was not altered in any group. Differences in endogenous or ex vivo induced DNA strand breaks could not be observed between the groups, neither before nor after 6 wk intervention . Hence, the observed effects of RW on DNA strand breaks in peripheral leukocytes can be attributed to the additional intake of flavonoids rather than to phenolic acids.et al. [The major advantage of our study compared to others is the investigation of both single and regular consumption of RW and DRW which has only been done so far by Cartron et al. . In bothHowever, our study has some constraints, regarding the different polyphenol intake with RW and DRW, which does not allow a direct comparison of both study drinks. In future studies, drinks with equal polyphenol composition should be applied. Moreover, in the single dose analysis TPP increased 360 min after intervention in groups RW and DRW, but also in controls. This could be due to 1) unknown amounts of polyphenols in "permitted" foods (e.g. white bread or pasta). These foods are generally considered to be low in polyphenols and thus, are not listed in the polyphenol database ,28. NeveIt is unlikely that random inhomogeneities of the groups Table  would haOne glass of RW or DRW is sufficient to transiently increase concentration of phenolic compounds in plasma, but accumulation after regular consumption could only be shown for RW. Polyphenols from RW and DRW seem to affect DNA damage in leukocytes, but antioxidant effects probably play only a minor role. Endogenous DNA strand breaks increased after a single dose of RW or DRW but could be reduced after 6 wk daily consumption of RW. As DNA damage is involved in cancer initiation, the mechanisms and the physiological relevance of these findings are worth being considered in future studies.Red wine (RW); dealcoholized red wine (DRW); total antioxidant capacity (TEAC); total phenolic content in plasma (TPP).The author(s) declare that they have no competing interests.BMA participated in study design and coordination, carried out the trolox equivalent antioxidant capacity assay and the single cell gel electrophoresis, did the statistical analysis and drafted the article. SE was involved in study design and realization and helped to draft the manuscript. KK measured total phenolic content and vitamin C concentration in plasma, and analysed the dietary records together with LG. RF did the sample size calculation and contributed to the statistical analysis. US supervised the clinical part of the study. W-UM supported the implementation, data analysis and interpretation of the single cell gel electrophoresis. RG had the initial idea and supervised the study. All authors read and approved the final manuscript.


This study was performed to compare changes in urinary incontinence (UI) symptoms and pelvic floor structure during pregnancy between nulliparous and multiparous women.A cross-sectional survey was performed among pregnant women from July 2016 to January 2017. In total, 358 pregnant women from two hospitals underwent an interview and pelvic floor transperineal ultrasound assessment. A questionnaire regarding sociodemographic, gynecological, obstetric features and the International Consultation on Incontinence Questionnaire-Short Form (ICIQ-SF) were used for the interview. Imaging data sets were analyzed offline to assess the bladder neck vertical position (BNVP), urethral angles , and hiatal area (HA) at rest and at maximal Valsalva maneuver (VM).χ2 = 9.15, P = 0.002), frequency , usual amount of UI  and scores of interference with daily life  during pregnancy were higher in multiparous than nulliparous women. A larger bladder neck descent (BND) , HA , α angle , β angle , and γ angle  at VM were discovered in pregnant women with UI than without UI. Multiparous women had a significantly higher BND  and a larger α angle , β angle , and γ angle  at VM than did nulliparous women.After excluding 16 women with invalid data, 342 women were included. The prevalence (Multiparous women experienced more obvious UI symptoms and pelvic floor structure changes during pregnancy than did nulliparous women. In 2002, the International Continence Society (ICS) defined urinary incontinence (UI) as “the involuntary passage of urine for any reason” . UI is aHowever, studies of UI during pregnancy have mainly focused on nulliparous women; a limited number of epidemiological studies have targeted multiparous women . In a syThe pathophysiology of UI during pregnancy involves pregnancy-associated pelvic floor changes . PreviouEthics approval for the study was granted by the Ethics Committee of Shenzhen Materity & Child Health Care Hospital (2016–30). All participants provided written informed consent.Three hundred fifty-eight women with a singleton pregnancy who were undergoing routine antenatal assessment in the obstetric clinics of various hospitals of Shenzhen from July 2016 to January 2017 were recruited. The exclusion criteria were (1) a history of UI before pregnancy; (2) a history of abdominal and vaginal surgery; (3) diabetes and hypertension; and (4) placenta previa, threatened abortion, amniotic fluid abnormalities, fetal growth restriction, or vaginal bleeding. The elimination criteria were (1) a failure to complete all investigation content and (2) unreliable pelvic ultrasound data.An interview of the patients was conducted by XJ Y to investigate the patients’ age, gestational weeks, body mass index (BMI), constipation during pregnancy, number of pregnancies, prior abortions/miscarriages, and delivery history. UI was diagnosed using the International Consultation of Incontinence Questionnaire-Short Form (ICIQ-SF). This questionnaire can also be used to evaluate the severity of UI. It comprises three scored items (Questions 1–3), frequency of UI , usual amount of UI , interference with everyday life , and a self-diagnostic item  . This inAfter completing the interview covering the patients’ general information and symptoms of UI, transperineal ultrasound examination was performed using a Voluson E8 system  with a 5–9 MHz three-dimensional autosweep transducer. The pregnant women should empty urine before examination and then lay in the supine position. The probe was smeared with a coupling agent and then covered with a condom. A vaginal probe was placed between the two labia, directly below the urethral orifice. The symphysis pubis was set as the axis point indication, and the median sagittal plane of the vagina, urethra, and anal canal was then obtained. Volume scanning was performed, the selection box was properly adjusted, and the BN, urethra, vagina, proximal anal canal, distal anal canal, and pubic symphysis were scanned. Two- and three-dimensional cross-sectional sonograms of the pelvic floor in the resting position and at maximal Valsalva maneuver (VM) were collected. The VM process lasted for approximately 5 s until satisfactory images were obtained. Ultrasound assessment was performed by three experienced investigators  who were blinded to all other information. During the examination, the probe remained closely attached to the perineum to prevent air interference and ensure that the quality of the picture was not affected.2) bordered by the pubovisceral muscle, pubic symphysis and the inferior pubic ramus . All data sets were analyzed by one of two investigators (DL or LC) who were blinded to the UI symptoms. The reference line was a horizontal line placed at the inferoposterior margin of the symphysis pubis. The following parameters were measured at rest and VM: (1) bladder neck vertical position (BNVP): distance (in cm) between the BN and the inferior-posterior margin of the symphysis pubis. A positive value indicated that the BN was positioned cranial of the symphysis pubis and a negative value that is was positioned caudal of the symphysis pubis. Bladder neck descent (BND) is the difference of BN between VM and rest ; (2) α aic ramus , Fig. 12 borderet test was used to analyze quantitative data. Chi-square test was used for categorical data. Covariance analyses  were conducted to compare the ultrasound parameters between pregnant women with and without UI and between nulliparous and multiparous women. All statistical tests were two-tailed. The level of significance was set at 0.05.SPSS 13.0  and GraphPad Prism 5.0  were used for all statistical analyses. Descriptive statistics were presented as mean ± standard deviation for continuous variables and as frequency (percentage) for categorical variables. Student’s Ten pregnant women failed to perform the VM effectively , and six did not finish the interview. Therefore, 342 pregnant women were included in our final data analysis. Among those who completed the study, 179 were nulliparous women and 163 were multiparous women. χ2 = 9.15, P = 0.002). The scores for the frequency of UI ((1.55 ± 0.56) vs. (1.21 ± 0.63)), usual amount of UI((2.53 ± 0.85) vs. (2.14 ± 0.52)), and interference with daily life ((3.58 ± 1.86) vs. (2.67 ± 2.09)) were significantly higher in multiparous than nulliparous women .The results of the comparison of the prevalence and severity of UI between the nulliparous and multiparous women are shown in The results of the covariance analysis of pelvic floor ultrasound parameters  are shown in Multiparous women experienced more severe symptoms of UI than did nulliparous women. The rate of UI in nulliparous women was 31.8%, which was significantly lower than that in multiparous women (47.9%). Our results are basically consistent with those of previous studies . UI, desMost previous studies of the relationship between UI during pregnancy and pelvic structure merely took nulliparous women into consideration. In addition to the higher prevalence of UI and more severe UI symptoms among multiparous than nulliparous women, our study also revealed that multiparous women experienced more obvious changes in their pelvic structure than did nulliparous women. Urethral support plays a critical role in the pathogenesis of UI. The BN position, BN mobility, and HA can be regarded as objective indicators of the supportive capacity of the pelvic floor, which can be measured effectively by transperineal ultrasound . The α aMultiparous women experienced more obvious pelvic changes than did nulliparous women in the present study. Multiparous women had larger α, β, and γ angles and a larger BNVP at VM as well as a larger BND than did nulliparous women during pregnancy after adjustment for the influence of gestational weeks. As mentioned above, the α angle, β angle, γ angle, and BNVP at VM and the BND have been found to differ between pregnant women with and without UI; thus, we were not surprised to find that multiparous women experienced more significant pelvic floor changes than nulliparous women during pregnancy.Multiparous women also showed higher age, larger numbers of pregnancies, and a higher prevalence of prior miscarriages/abortions than did nulliparous women in our study, which might explain why multiparous women were more susceptible to UI and pelvic floor changes than nulliparous women. The previous studies reported that higher age leads to the loss of nerve function and a decrease in the total number of striated muscle fibers of the urethral sphincter at a rate of 2% per year and a gradual decline in the maximum urethral closure pressure of approximately 15% per decade . A previOne of the limitations of our study is its cross-sectional design. To elucidate the pathophysiology of delivery-induced UI, women should ideally be examined before, during, and after their pregnancy to assess preexisting differences in the pelvic floor anatomy and the absence of UI before, during, and after pregnancy . AdditioOur results indicate that multiparous women had a higher prevalence of UI and more severe UI symptoms than did nulliparous women, which could be associated with weaker pelvic floor support.10.7717/peerj.3615/supp-1Data S1UI, urinary incontinence; R, rest; VM, maximal Valsalva maneuver; VM-R/R-VM, the difference of VM and rest; BNVP, bladder neck vertical position; HA, hiatal area.Click here for additional data file.


Probiotics are widely used to improve gastrointestinal (GI) health, but they may also be useful to prevent or treat gynaecological disorders, including bacterial vaginosis (BV) and candidiasis. BV prevalence is high in South Africa and is associated with increased HIV risk and pregnancy complications. We aimed to assess the availability of probiotics for vaginal health in retail stores  in two major cities in South Africa.A two-stage cluster sampling strategy was used in the Durban and Cape Town metropoles. Instructions for use, microbial composition, dose, storage and manufacturers’ details were recorded.Lactobacillus acidophilus (53.5%), L. rhamnosus (37.6%), Bifidobacterium longum ssp. longum (35.6%) and B. animalis ssp. lactis (33.7%). Products for vaginal health contained only common GI probiotic species, with a combination of L. acidophilus/B. longum ssp. longum/B. bifidum, L. rhamnosus/L. reuteri or L. rhamnosus alone, despite L. crispatus, L. gasseri, and L. jensenii being the most common commensals found in the lower female reproductive tract.A total of 104 unique probiotics were identified in Cape Town and Durban . Cape Town had more products than Durban (94 versus 59 probiotics), although 47% were common between cities (49/104). Only four products were explicitly for vaginal health. The remainder were for GI health in adults (51.0%) or infants (17.3%). The predominant species seen overall included This survey highlights the paucity of vaginal probiotics available in South Africa, where vaginal dysbiosis is common. Most vaginal products contained organisms other than female genital tract commensals.The online version of this article (doi:10.1186/s12905-017-0362-6) contains supplementary material, which is available to authorized users. Lactobacillus spp. dominance. Commensal Lactobacillus spp. in the genital tract have been thought to contribute to genital health by their ability to lower vaginal pH, to produce bactericidal compounds, and to competitively outcompete potentially pathogenic bacteria  and the eight districts making up the Cape Town metropole  were collected Fig.  and b. TTM version 12 . Descriptive measures  were used to summarize the data. The shelf life of products was estimated by calculating the time difference between time of survey and expiry date of the product.Product characteristics were electronically captured in a spread sheet. Statistical analysis was conducted using STATAA total of 104 unique probiotic products were surveyed from South Africa (SA), with 94 available in Cape Town and 59 in Durban. Forty-five products were only found in Cape Town, ten were only available in Durban, and 49 products were common to both cities Fig. . ProbiotOf the 104 probiotics identified, more than half (51%) had GIT health claims for adults and 17% were indicated for GIT and skin conditions in infants. Less than 10% of the probiotic products had claims in multiple health categories such as GIT and immunological health. Medicinal claims of probiotics in both cities were similar Fig. . Less thLactobacillus  and Bifidobacterium  , L. acidophilus with B. bifidum (23 products), B. longum ssp. longum with B. bifidum (23 products), L. acidophilus with L. rhamnosus (19 products), and L. rhamnosus with B. longum ssp. longum (18 products) ] Fig. . The mos bifidum 6.7%] (FiL. acidophilus/B. longum ssp. longum/B. bifidum (Bioflora Vagiforte® Plus), two species L. rhamnosus/L. reuteri  or the single bacterial species L. rhamnosus .The vaginal probiotics contained either a combination of the three species 9 viable organisms ranging from 4 × 107 to 9 × 1011 cfu per dose, with daily dosage recommended. The daily dose (capsules per day) of products depended on therapeutic indication: prophylaxis (chronic), treatment (acute) or as an adjunct to antibiotic treatment. However, organisms included in these different probiotic preparations did not differ according to indication. Interestingly, some products formulated with exactly the same bacterial species, strains and cfu were marketed by different manufacturers under both similar and multiple health claims . In some instances the same manufacturer produced a range of probiotic products with identical bacterial strain content and dose while marketing them as distinct products with different health claims  to 12  with a mean of 3. Overall, probiotic products claimed to contain a median of 1.5 × 10® and 5/18 produced by Bioflora CC), but did not appear to be systematically different in formulation, probiotic composition or expiry date. The average shelf life time was 14 months (from time of survey to recorded product expiry date) for both products stored at room temperature and at 4 °C. There was no correlation between shelf life and formulation of the product. Probiotics were predominantly recommended for oral administration . Of the four vaginal products available, two were intended for oral administration, one was for both oral and vaginal administration and one for vaginal dosing only, and all were stored at room temperature.The most common formulation was capsules (59.8%), with some products available as liquids (18.7%), tablets (13.1%) or powder (8.4%). The majority  were stored on the shelf (room temperature), and 17.3% (18/104) were stored in the fridge (4 °C). Probiotics maintained at 4 °C tended to be from the same manufacturer  of the surveyed products were manufactured in SA [88.1% (59/67) of shelf products versus 11.9% (8/67) of refrigerated products]. The total of 104 products were produced by 39 manufacturers, of which 27 (69.2%) were based in SA, mainly in Johannesburg (12/27), Pretoria (6/27) and Cape Town (6/27). Of the four vaginal products, two were manufactured in SA , one in Canada  and one in the US (UltraFlora® Women’s).Bifidobacterium spp. but only a few Lactobacillus spp. and thus, it needs to be ingested , ranging from USD 0.13–3.29 (ZAR 1.80–46.00). Probiotic prices did not differ statistically significantly between Cape Town and Durban (mean of ZAR 6.50 vs. ZAR 6.00), nor when comparing probiotics manufactured locally to those that were imported, or probiotics stored on the shelf to those requiring refrigeration. Instead, prices of products differed according to manufacturer with products from MetagenicsThe commensal microbiota in the female genital tract is important to protect women against STIs, including HIV. BV is associated with increased risk of infection with HIV , other Shttp://www.statssa.gov.za), proxies for social-economic status and general health in both cities. Factors contributing to this discrepancy in probiotic availability are likely to be complex, but may include cultural and ethnic differences in the populations, and differences in traditional, complementary and alternative medicine use [The aim of this survey was to determine the availability of vaginal probiotics in South Africa, a region with high BV and HIV burden in young women. We identified 104 products available in Cape Town and Durban, with more products marketed in Cape Town than in Durban. Although there were fewer stores in Durban selling probiotics, these two major cities do not differ in population size, gross domestic product (GDP), average income, unemployment rates or medical aid coverage  published [In addition to eostasis , interfeeostasis –63, modueostasis –66, stabeostasis . The incublished , 69 and In the USA, the FDA applies a complex framework of regulation to validate manufacturers’ claims for the products they market . In thisThis survey had some limitations. The exact shelf lifetime could not be determined because only the expiry date and date of survey, but not the date of production were captured. We did not look at any other cities in the country but restricted our study to two of the larger cities, and only looked at one centre per district within these cities.This study provides a strong rationale for the development and clinical evaluation of additional probiotics for vaginal health, as these products are underrepresented in the South African probiotic market. Adjunctive therapy with probiotics targeting young women with BV in regions with highest BV and HIV incidence has the potential to provide considerable benefit. A cost analysis of the products in our study confirmed that the prices of these probiotics still represent a considerable barrier in their limited uptake in the segments of the population most in need. This highlights the need for the development and testing of cheaper, alternative products, which will have the added benefit of being tailor-made for the South African population. The increased availability of vaginal probiotics represents an intervention with significant potential that can help decrease the burden of BV in the South African community.