Potential xeroderma pigmentosum group D (XPD), also called excision repair cross-complimentary group two (ERCC2), Lys751Gln and Asp312Asn polymorphisms have been implicated in gastric cancer risk among different ethnicities.We aimed to explore the effect of XPD Lys751Gln and Asp312Asn polymorphisms on the susceptibility to gastric cancer among different ethnicities through a systematic review and meta-analysis. Each initially included article was scored for quality appraisal. Desirable data were extracted and registered into databases. 13 studies were ultimately eligible for the meta-analysis of Lys751Gln polymorphism and 9 studies for the meta-analysis of Asp312Asn polymorphism. We adopted the most probably appropriate genetic model (recessive model) for both Lys751Gln and Asp312Asn polymorphisms. Potential sources of heterogeneity were sought out via subgroup and sensitivity analyses, and publication biases were estimated.Statistically significant findings were apparently noted in Asians but not in Caucasians for both XPD Lys751Gln and XPD Asp312Asn polymorphisms. A statistically significant finding could be seen in noncardia-type gastric cancer for XPD Lys751Gln polymorphism. A statistically significant finding could also be seen in high quality subgroup, small-and-moderate sample size subgroup, articles published after 2007, or PCR-RFLP genotyping subgroup for XPD Asp312Asn polymorphism.Our meta-analysis indicates that XPD Gln751Gln (CC) genotype and Asn312Asn (AA) genotype may seem to be more susceptible to gastric cancer in Asian populations but not in Caucasian populations, suggesting that the two genotypes may be important biomarkers of gastric cancer susceptibility for Asian populations, the assumption that needs to be further confirmed in well-designed studies among different ethnicities. Gln751Gln (CC) genotype may also be associated with noncardia-type gastric cancer risk, which should also be confirmed among different ethnicities in the future. Although worldwide gastric cancer incidence has decreased, its mortality still ranks second It is widely acknowledged that DNA must remain stable to undertake its crucial physiological functions, but it is persistently vulnerable to various endogenous and/or exogenous damages and thus its probable mutations could accumulate and carcinogenesis may occur due to the damaged DNA. DNA repair system, however, plays a vital role in maintaining the functions of normal cells and genome integrity through the reversal of the damaged DNA Nucleotide excision repair (NER), one of the major DNA repair pathways in humans, is capable of removing helix-distorting base lesions produced by ultraviolet light (UV) and an array of chemical agents et al. published the first study involved in XPD Lys751Gln polymorphism in relation to gastric cancer risk In 2005, Huang WY Accordingly, the aim of our meta-analysis was to explore, using the most appropriate genetic model, the effect of XPD polymorphisms on gastric cancer risk among different ethnicities and to identify possible sources of heterogeneity among the eligible studies.A systematic literature search was performed for articles regarding XPD/ERCC2 SNPs associated with gastric cancer risk. The MEDLINE, EMBASE databases, Chinese National Knowledge Infrastructure (CNKI), Web of Science, and BIOSIS databases were used simultaneously with the combination of terms “XPD”, “ERCC2”, “DNA repair”, “NER”, “Lys751Gln”, “Asp312Asn”, or “Arg156Arg”; “gene”; “polymorphism”, “variant”, or “SNP”; and “gastric cancer”, “gastric carcinoma”, or “stomach cancer” up to December 2011. The search was performed without any restriction on language. The scope of computerized literature search was expanded according to the reference lists of retrieved articles. The relevant original articles were also sought manually.Studies concerning the association of XPD/ERCC2 SNPs  with gastric cancer risk were included if the following conditions were met: (i) any study described the association of at least one of XPD/ERCC2 SNPs with gastric cancer; (ii) any study reported the numbers of both controls and gastric cancer cases; (iii) results were expressed as odds ratio (OR) with 95% confidence intervals (CI); and (iv) studies were case-control or nested case-control ones.et al.et al.et al.To identify high-quality studies, we mainly adopted predefined criteria for Quality Appraisal initially proposed by Thakkinstian Helicobacter Pylori infection status.The following data from each article were extracted: authors, year of publication, country, ethnicity of participants , study design, source of controls, number of controls and of cases, genotyping method, distribution of age and gender, Lauren's classification , anatomical classification (cardia or non-cardia cancer), smoking habit, drinking habit and The data were extracted and registered into two databases independently by two investigators (Lin B and Lu Y) who were blind to journal names, institutions or fund grants. Any discrepancy between these two investigators was resolved by the investigator (Xue H), who participated in the discussion with them and made an ultimate decision.All statistical analyses were performed using STATA statistical software . Two-sided Ps<0.05 were considered statistically significant. Hardy-Weinberg equilibrium (HWE) in controls was calculated again in our meta-analysis. The chi-square goodness of fit was used to test deviation from HWE (significant at the 0.05 level).1, OR2, and OR3 were calculated for genotypes CC versus AA, CA versus AA, and CC versus CA for XPD Lys751Gln polymorphism; AA versus GG, GA versus GG, and AA versus GA for XPD Asp312Asn polymorphism; AA versus CC, CA versus CC, and AA versus CA for XPD Arg156Arg polymorphism, respectively. The pairwise differences were used to determine the most appropriate genetic model. If OR1 = OR3≠1 and OR2 = 1, a recessive model is suggested. If OR1 = OR2≠1 and OR3 = 1, a dominant model is implied. If OR2 = 1/OR3 ≠1 and OR1 = 1, a complete overdominant model is suggested. If OR1>OR2>1 and OR1>OR3>1, or OR1<OR2<1 and OR1<OR3<1, a codominant model is indicated Odds ratios (OR) and 95% confidence intervals (95% CI) were used to assess the strength of associations between XPD/ERCC2 SNPs and gastric cancer risk. ORI2) value, representing variation in OR attributable to heterogeneity was then used to quantify the degree of such between-study heterogeneity I2<25% represents no heterogeneity, I2 = 25–50% represents moderate heterogeneity, I2 = 50–75% represents large heterogeneity, and I2>75% represents extreme heterogeneity”. Between-study variance Tau-squared (τ2) value was also used to evaluate between-study variance. A fixed-effects model, using Mantel–Haenszel (M–H) method, was employed to calculate the pooled ORs when homogeneity existed on the basis of Q-test p value no less than 0.1.By contrast, a random-effects model, using DerSimonian and Laird method (D+L), was utilized if there was heterogeneity based on Q-test p value less than 0.1. Even for the homogeneity among-studies, D+L method was also used. Meta-regression analyses and subgroup analyses were utilized to explore and control potential sources of heterogeneity across studies. The significance of pooled ORs was tested by Z test (P<0.05 was considered significant).The Q statistic was used to assess heterogeneity across studies included in the meta-analysis. I-squared , OR2 , and OR3  were 1.22 (p = 0.266), 1.11 (p = 0.369), and 1.02 (p = 0.811), respectively, hardly insinuating a particular model effect of putative susceptible C allele. Heterogeneity chi-squared was 29.83 (d.f. = 12), p value was 0.003, and I2 was 59.8%. After meta-regression analysis using single covariate , p values of coefficient t value for Asians, Caucasians, or Turkish population were 0.001, 0.139, and 0.585, respectively; strongly indicating that Asians single covariate could mostly constitute the source of heterogeneity across studies. τ2 for Asians, Caucasians, or Turkish population single covariate were 0, 0.09178, and 0.1347, respectively. For Asians single covariate, τ2 decreased from 0.1233 to 0, indicating Asians single covariate could account for 100% of the source of heterogeneity across studies. When stratified by ethnicity subgroup analysis, OR1 , OR2 , and OR3  among Asian population were 2.63 (p = 0.002), 1.14 (p = 0.653), and 1.51 (p = 0.034), respectively, highly indicating a recessive model effect of putative susceptible C allele (OR1 = OR3≠1 and OR2  = 1).For XPD Lys751Gln polymorphism, OR1 , OR2 , and OR3  were 1.75 (p = 0.015), 1.15 (p = 0.549), and 1.47 (p = 0.003), respectively, highly indicating a recessive model effect of putative susceptible A allele (OR1 = OR3≠1 and OR2 = 1). Heterogeneity chi-squared was 9.91 (d.f. = 8), p value was 0.272, and I2 was 19.2%, indicating no heterogeneity across studies. After meta-regression analysis using single covariate (ethnicity composed of Caucasians or Asians), p value of coefficient t value for ethnicity single covariate was 0.277, indicating that ethnicity could constitute one of the sources of little heterogeneity across studies. τ2 decreased from 0.0354 to 0.02075, indicating ethnicity could account for 41.4% of the source of little heterogeneity across studies. Similarly, when stratified by ethnicity subgroup analysis, OR1 , OR2 , and OR3  among Asian population were 2.10 (p = 0.026), 1.22 (p = 0.574), and 1.80 (p = 0.008), respectively, further indicating a recessive model effect of putative susceptible A allele (OR1 = OR3≠1 and OR2 = 1).For XPD Asp312Asn polymorphism, ORTaken together, a recessive genetic model was ultimately chosen for both XPD Lys751Gln and XPD Asp312Asn polymorphisms in our meta-analysis.I2 were 0.0%, 0.0%, and 75.1%; and τ2 were 0.0000, 0.0000, and 0.4489, respectively, demonstrating no heterogeneity within Caucasians or Asians, but extreme heterogeneity within Turkish population  and low-and-moderate quality (scores less than 6.5) subgroups. As for XPD Lys751Gln polymorphism, no statistically significant finding was witnessed in either high quality subgroup or low-and-moderate quality counterpart, given that the pooled ORs  were 1.20  for the former and 0.61  for the latter. When ethnicity sub-stratification was performed for high quality subgroup, a statistically significant finding was much apparently witnessed among Asians but no statistically significant finding was noted among Caucasians because the pooled ORs  were 2.54  for the former and 0.98  for the latter, respectively. As for Asp312Asn polymorphism, a statistically significant finding was witnessed in high quality subgroup but not in low-and-moderate quality counterpart, given that the pooled ORs  were 1.56  for the former and 1.42  for the latter. Likewise, when ethnicity sub-stratification was performed for high quality subgroup, a statistically significant finding was much apparently witnessed among Asians but no statistically significant finding was noted among Caucasians because the pooled ORs  were 2.37  for the former and 1.31  for the latter, respectively.The data were additionally stratified, in line with publication time, into the earlier publication subgroup (articles published before or in 2007) and the later publication subgroup (articles published after 2007). As for XPD Lys751Gln polymorphism, no statistically significant findings were observed on the grounds that the pooled ORs  were 0.89  in the former and 1.23  in the latter, respectively. As for Asp312Asn polymorphism, a statistically significant finding was observed in the later publication subgroup but not in the earlier publication subgroup on the grounds that the pooled ORs  were 1.46  in the former and 1.61 in the latter, respectively.When gastric cancer was classified into non-cardia  and cardia subtypes, a statistically significant finding was found among non-cardia type but not among cardia type for XPD Lys751Gln polymorphism on the grounds that the pooled ORs  were 2.03  among non-cardia type and 0.88  among cardia type. As for Asp312Asn polymorphism, no statistically significant finding was observed among cardia type on the grounds that the pooled OR  was 0.89 , but OR  regarding non-cardia type could not be calculated because only one study In terms of pathology, gastric cancer could be classified into intestinal, diffuse, or mixed subtypes, but only 1 study Helicobacter pylori infection or smoking status could not be analyzed in our meta-analysis because necessary relevant data of at least two studies could not be accessible in our meta-analysis.Confounding factors such as And when genotyping techniques were considered, no statistically significant finding was noted in each genotyping technique subgroup for XPD Lys751Gln polymorphism because pooled ORs  were 0.94  and 1.67  in RFLP and TagMan subgroups, respectively; however a statistically significant finding was noted in RFLP genotyping subgroup but not in Direct sequencing subgroup for Asp312Asn polymorphism because pooled ORs  were 1.90  and 1.24 , respectively.Engin AB et al., also deviated from HWE, the OR  became 1.19  for the all ethnicity, indicating high stability of the results. If all the three deviated-HWE studies Meta-analyses were conducted repeatedly when each particular study had been removed. The results indicated that random-effects estimates before and after the deletion of each study were similar at large, suggesting moderately high stability of the meta-analysis results. For XPD Lys751Gln polymorphism, as shown in For Asp312Asn polymorphism, as shown in Cumulative meta-analyses of XPD Lys751Gln polymorphism association were also conducted among Asians and Caucasians via the assortment of publication time and total number of sample size. As shown in Publication bias was preliminarily examined by funnel plots qualitatively and estimated by Begg's and Egger's tests quantitatively. For XPD Lys751Gln polymorphism, its funnel plot  showed tIn our meta-analysis statistically significant findings were apparently noted in Asians but not in Caucasians for both XPD Lys751Gln and XPD Asp312Asn polymorphisms. Also based on the findings of cumulative meta-analyses, the inclinations toward significant associations in Asians for both XPD Lys751Gln and XPD Asp312Asn polymorphisms could be obviously seen when sorted by publication time and total sample size. XPD Gln751Gln (CC) and Asn312Asn (AA) genotypes may seem to be more susceptible to gastric cancer in Asians. Our meta-analyses suggest that Gln751Gln (CC) and Asn312Asn (AA) genotypes may be important biomarkers of gastric cancer susceptibility for Asians, the assumption that needs to be further confirmed in future well-designed studies in Asian populations. Also the different or even conflicting risk associations, if so, among different ethnicities should be further meticulously investigated and reconfirmed in the future.I2 (%) was 19.2 and τ2 was 0.0354, as shown in I2 (%) and τ2 became 0.0 and 0.0000 for Asians and 41.6 and 0.0587 for Caucasians, respectively, further indicating that, actually, no heterogeneity existed among Asians studies rather than among Caucasians counterparts. Given this, we think that stratification or subgroup analyses are still needed in our meta-analysis. For XPD Asp312Asn polymorphism, our subgroup analyses also indicate that significant associations could be found in the small-and-moderate sample subgroup but not in the large sample counterpart. In large sample subgroup the ORs in the studies conducted by Capellá G et al. For XPD Asp312Asn SNP, although For XPD Asp312Asn polymorphism, a statistically significant finding was witnessed in high quality subgroup but not in low-and-moderate quality counterpart. When ethnicity sub-stratification was performed for such a high quality subgroup, a statistically significant finding was even more apparently witnessed among Asians but still no statistically significant finding was noted among Caucasians. For XPD Lys751Gln polymorphism, no statistically significant finding originally noted in high quality subgroup interestingly changed into a statistically significant finding among Asians but still not among Caucasians when ethnicity sub-stratification was performed for such a high quality subgroup. Those findings observed in different ethnic high quality subgroups further indicate the point that XPD Gln751Gln (CC) and Asn312Asn (AA) genotypes may be more susceptible to gastric cancer in Asians rather than in Caucasians. Certainly, high-quality studies should be more advocated to be designed in the future so as to accurately explore the real associations between XPD polymorphisms and gastric cancer risk among different ethnicities. Besides Asians and Caucasians, other ethnic participants, if possible, should be more appraised in the future.Moreover, as for XPD Lys751Gln polymorphism, 4 Helicobacter pylori infection, alcoholic drinking, and smoking habits may be associated with increased damage to DNA repair Furthermore, a variety of confounding factors such as With the advent of novel genotyping technologies like seminested polymerase chain reaction, TaqMan allelic discrimination test, or real-time PCR, we may witness an upsurge of genetic association studies in the future. As for XPD Asp312Asn polymorphism in our meta-analysis, a statistically significant finding could be noted in PCR-RFLP subgroup but not in Direct sequencing subgroup. As for XPD Lys751Gln polymorphism, however, no statistically significant finding was witnessed in either PCR-RFLP subgroup or TagMan PCR genotyping subgroup. Certainly, the difference should be concerned with extreme caution. Unfortunately, no direct sequencing was used among XPD Lys751Gln polymorphism studies. For a novel genotyping technique to be employed for the study of a particular genetic polymorphism, this technology, to our knowledge, should better be confirmed using direct sequencing. In that case, this new technology can be seen as valid as direct sequencing Finally, the strength of our meta-analysis could be summarized as follows. We sought to find as many publications as we could by means of various searching approaches. The study that appeared to deviate from HWE was not excluded mechanically in our meta-analysis unless there are other convincing grounds for doubting the quality of the study To be sure, there are some unavoidable limitations in our meta-analysis. Firstly, the offered information from the included studies is inconsistent. Put it another way, the information about overall gastric cancer susceptibility is predominantly provided, while more important information about pathologic subtypes or anatomic subtypes of gastric cancer is less provided. Thus, the specific subtype results should be considered with caution. Secondly, with the merely published studies included in our meta-analysis, publication bias is very likely to occur, though no or a little statistically significant publication bias is indicated in our meta-analysis. Thirdly, moderate to severe heterogeneity could be witnessed among the included studies. So as to minimize the potential bias, we designed a rigorous protocol before conducting meta-analysis, and performed a scrupulous search for published studies using explicit methods for study selection, data extraction, statistical analysis, adoption of the most appropriate genetic model and sensitivity analysis.In conclusion, XPD Gln751Gln (CC) and Asn312Asn (AA) genotypes may seem to be more susceptible to gastric cancer in Asian population but not in Caucasian population, suggesting that Gln751Gln (CC) and Asn312Asn (AA) genotypes may be important biomarkers of gastric cancer susceptibility for Asian population, the assumption that needs to be further confirmed in future well-designed studies among different ethnicities. Gln751Gln (CC) genotype may also be associated with the noncardia-type gastric cancer risk, the finding that also needs to be further confirmed.Table S1Scales for Quality Assessment.(DOC)Click here for additional data file.


Objective. This study was performed to determine the prevalence of and associated risk factors for cardiovascular disease (CVD) in Latin American (LA) patients with systemic lupus erythematosus (SLE). Methods. First, a cross-sectional analytical study was conducted in 310 Colombian patients with SLE in whom CVD was assessed. Associated factors were examined by multivariate regression analyses. Second, a systematic review of the literature on CVD in SLE in LA was performed. Results. There were 133 (36.5%) Colombian SLE patients with CVD. Dyslipidemia, smoking, coffee consumption, and pleural effusion were positively associated with CVD. An independent effect of coffee consumption and cigarette on CVD was found regardless of gender and duration of disease. In the systematic review, 60 articles fulfilling the eligibility criteria were included. A wide range of CVD prevalence was found (4%–79.5%). Several studies reported ancestry, genetic factors, and polyautoimmunity as novel risk factors for such a condition. Conclusions. A high rate of CVD is observed in LA patients with SLE. Awareness of the observed risk factors should encourage preventive population strategies for CVD in patients with SLE aimed at facilitating the suppression of cigarette smoking and coffee consumption as well as at the tight control of dyslipidemia and other modifiable risk factors. Systemic lupus erythematosus (SLE) is a complex systemic autoimmune disease (AD), characterized by the production of numerous pathogenic autoantibodies . Diverseper se seems to be an independent risk factor for the development of accelerated atherosclerosis [CVD is the leading cause of mortality, responsible for about 30% of deaths worldwide. Globally, 80% of total CVD deaths occur in developing countries . In addiclerosis . mestizos) that is mainly derived from a European and Amerindian inheritance [SLE is not uncommon in Latin America (LA), the geographical area defined by Mexico, Central America, South America, and the islands of the Caribbean, a rapidly growing region with almost 600 million inhabitants . Latin Aeritance . The inceritance . Despiteeritance . Enormoueritance . So far,eritance –19. ThusFirst, a cross-sectional analytical study was conducted in 310 Colombian patients with SLE in whom CVD was assessed. The subjects were seen at the Center for Autoimmune Diseases Research (CREA) in Bogota, Colombia. All of them fulfilled the 1997 update American College of Rheumatology classification criteria for SLE . This stInformation on patient socio-demographic and cumulative clinical and laboratory data, as well as household description, were obtained by interview, standardized report form, physical examination and chart review. All data were collected in an electronic and secure database. Socio-demographic variables included age at SLE onset, disease duration, educational and socioeconomic status, current occupation, smoking habits, coffee consumption, expositional factors and physical activity. Age at onset of the disease was defined as the first subjective experience of the symptom(s) and/or sign(s) described in any of the items of the classification criteria . DuratioClinical and laboratory variables were registered as present or absent at any time during the course of the disease. Clinical features of the disease were included taking into account the revised American College of Rheumatology criteria  and otheFive subphenotypes were defined and assessed: first, hypertension, defined as having a blood pressure ≥140/90 mm Hg or using any antihypertensive medication : systoliRelevant laboratory variables associated with SLE were recorded. Antinuclear antibodies, antidouble strand DNA antibodies, precipitating antibodies to extractable nuclear antigens , anticardiolipin IgG and IgM, antibeta 2-glycoprotein 1 IgG and IgM antibodies, and lupus anticoagulant were extracted from the patient's clinical record. Other autoantibodies including rheumatoid factor, anticyclic citrullinated peptide, -thyroperoxidase enzyme, -thyroglobulin, -Scl 70, -centromere, -mitochondrial, and -smooth muscle antibodies were also recorded. Inflammatory biomarkers, including erythrocyte sedimentation rate (ESR), and serum high sensitive C-reactive protein (CRP) levels, as well as white blood cell and platelet count, hemoglobin levels, mean corpuscular volume, coombs test, complement , TSH, tetraiodothyronine T4, venereal disease research laboratory, and creatinine were extracted from patient's clinical record. Likewise, serum levels of total cholesterol (TC), triglycerides (TGL), high-density lipoprotein cholesterol (HDL), low-density lipoprotein cholesterol (LDL), and glycemia were determined by blood tests provided by every patient. Patients were assessed for traditional CVD risk factors including current age  –34. Typet student test. Nonparametric values were analyzed by Mann-Whitney U test. Clinical variables with a P ≤ 0.25 were included in a multivariable model. Finally, a multivariate binomial logistic regression model having CVD as the dependent variable was fit. As independent factors, the model included the traditional and nontraditional associations that were statistically significant in bivariate analyses and those variables that were biologically plausible. Moreover, other logistic regression models were made, including interactions between independent factors. In both circumstances, the models were adjusted by gender and duration of the disease. Adequacies of logistic models were assessed using the Hosmer-Lemeshow goodness-of-fit test. The Nagelkerke R2  was used to estimate the percentage of variance explained by the models. Adjusted odds ratios (AOR) were calculated with 95% confidence intervals (CI). The Wald statistic test was used to evaluate the significance of individual logistic regression coefficients for each independent variable. Statistical analyses were done by the Statistical Package for the Social Sciences .First, univariate analyses were done. Categorical variables were analyzed by frequencies. Kolmogorov-Smirnov normality test was done to evaluate normality for quantitative continuous variables. Parametric data are expressed as the mean and standard deviation, and nonparametric data are described as the median and interquartile range. Second, associations between traditional and nontraditional CVD risk factors were assessed by bivariate analyses. The presence of CVD was assessed through chi square tests or Fisher's exact tests when the variable was dichotomous. Parametric values were analyzed by The Preferred reporting items for Systematic Reviews and Meta-analyses guidelines were followed  to systeThe search was done in PubMed, using the following Medical Subject Headings (MeSH terms) groups: “Lupus Erythematosus, Systemic,” “Cardiovascular Diseases;” “Latin America,” “Ethnic Groups,” “Brazil,” “Mexico,” “Colombia,” “Chile,” “Cuba,” “Panama,” “Venezuela,” “Bolivia,” “Peru,” “Argentina,” “Uruguay,” “Paraguay,” “Ecuador,” “Nicaragua,” “Surinam,” “French Guiana,” “Guatemala,” “Honduras,” “Belize,” “Costa Rica,” “El Salvador,” “Puerto Rico,” “Dominican Republic,” and “Haiti.” Each one of them was cross-referenced with the following MeSH terms/keywords: “risk factors,” “traditional risk factors,” “classic risk factors,” “nontraditional risk factors,” “novel risk factors,” “hypertension,” “metabolic syndrome,“ “obesity,” “smoking,” “tobacco,” “dyslipidemia,” “advanced age,” “menopausal status,” “family history of CVD,” “hyperhomocysteinemia,” “sedentary lifestyle,” “renal impairment,” “male gender,” “type 2 diabetes mellitus,” “insulin resistance,” “hormone replacement therapy,” “coffee,” “ancestry,” “polymorphism, genetic,” “poliautoimmunity,” “autoantibodies,” “antibodies, antiphospholipid,” “lupus coagulation inhibitor,” “circulation anticoagulants,” “antibodies, anticardiolipins,” "beta 2-glycoprotein I," “endothelial cells antibodies,” “systemic inflammation,” “c-reactive protein,” “blood sedimentation,” “tumor necrosis factor,” “cytokines,” “immune complex,” “disease activity,” “SLEDAI,” “organ damage,” “SDI,” “duration of illness,” “immune cells aberrations,” “glucocorticoids,” “steroids,” “DMARD,” “antirheumatic agents,” “methotrexate,” “biological therapy,” “rituximab,” “anti-Inflammatory agents, non-steroidal,” “azathioprine,” “vasculopathy,” “lupus nephritis,” “premature menopause,” “endogenous dyslipidemia,” “sociodemographic factors,” “hypovitaminosis D,” “vitamin D deficiency,” “low vitamin D,” “osteoporosis,” “biomarkers,” “antiphospholipid syndrome,” “thyroiditis, autoimmune,” “Graves Disease,” “Hashimoto Disease,” “scleroderma,” “Sjögren's syndrome,” “rheumatoid arthritis,” "hypertension,” “ischemic heart disease,” “coronary artery disease,” “acute coronary syndrome,” “congestive heart failure,” “myocardial infarction,” “stroke,” “angina,” “thrombosis,” “deep vein thromboses,” “pulmonary embolism,” “periphery arterial disease,” and “atherosclerosis.” Each term was cross-referenced for the greatest number of results. No limits regarding language, period of publication, or publication type were used. The same terms were used for searching in EMBASE and Scopus databases. Each MeSH term and keyword was translated into DeCS  in order to explore sources of information in Spanish, Portuguese, and English through SciELO and Virtual Health Library databases. Reviewers screened all titles and abstracts and applied the eligibility criteria in order to identify studies that were appropriate for inclusion . A studyhttp://dx.doi.org/10.1155/2013/794383. Each record was classified based on the quality score of the studies that was assigned by applying the levels established by the Oxford Centre for Evidence-based Medicine 2011 in order to evaluate the risk of bias [Articles were excluded from the analysis if they dealt with juvenile SLE or were done on animal models  instead of SLE patients. Studies were also excluded if they were reviews or case reports, or if they discussed topics not related to CVD, and/or were not done on LA population. Blinded reviewers  organized selected articles on the basis of publication source, country, author, year, type of study, sample size, traditional and nontraditional risk factors, cardiovascular outcomes evaluated, and main results. Only novel risk factors with statistical significance were included. For details, see Supplementary Table S1 in the supplementary material available online at  of bias . The seaOut of a total of 310 patients, 91.3% 283/310) were women. The median (interquartile range) of age and duration of the disease was 37 (22) and 5 (9) years, respectively. CVD was observed in 36.5% (113/310). The most frequent condition was hypertension (25.2%) , dyslipidemia, use of cytotoxic drugs, serositis, renal involvement , and thrombocytopenia were all risk factors significantly associated with CVD. Instead, ethnicity and leukopenia were factors negatively associated with CVD .Dyslipidemia, pleural effusion, polyautoimmunity, and renal compromise were variables significantly associated with CVD, regardless of gender and duration of the disease by logistic regression analysis . The ass There were 21,161 articles identified in PubMed, EMBASE, and Scopus databases search. Additional records identified through other sources included 814 articles . Therefore, the database searches provided a total of 21,975 publications. Of these, 19,729 were identified as duplicates. A total of 2,246 full text articles were assessed for eligibility. Only 115 articles were included for methodological analysis. Finally, 60 articles that had interpretable data and fulfilled the eligibility criteria were included. In 3 papers, data extraction was made from abstract –43. For classic CV risk factors such as metabolic syndrome (MetS), obesity, dyslipidemia, hypertension, T2DM, sedentary lifestyle, male gender, smoking, advanced age, hyperhomocysteinemia, renal impairment, family history of CVD, and menopausal status were described. Several studies reported nontraditional risk factors such as ancestry, certain single-nucleotide polymorphisms, SLE per se, polyautoimmunity, autoantibodies , markers of systemic inflammation , SLE disease activity, SLE duration, organ damage, immune cells aberrations, medication , vasculopathy, lupus nephritis, endogenous dyslipidemia, bone mineral density, education level, and monthly income. A broad spectrum of CV subphenotypes including hypertension, IHD, CAD, ACS, MI, angina, CHF, stroke, thrombosis, peripheral arterial disease, subclinical atherosclerosis, and mortality due to CVD were described in LA individuals with SLE. For details, see  Out of total of 60 articles that fulfilled eligibility criteria, 46 had interpretable data regarding CVD frequency, which corresponds to a prevalence range of 4%–79.5%. Several The analysis of Colombian patients with SLE discloses a high prevalence of CVD (36.5%). In contrast, prospective North Americans cohort studies showed a prevalence and annual incidence of CVD between 6 and 10%, and 1.5%, respectively , 98, 99.Carotid plaque is prevalent in 21% of SLE patients under age of 35 and in up to 100% of those over age 65 . The mosn = 229) [n = 498) [n = 546) [n = 561) [n = 637) [Independent predictive risk factors for CV events had been assessed in five large prospective cohorts of patients with SLE, including Baltimore  , Pittsbun = 546) , Toronton = 561)  and Systn = 637) . These cn = 637) , 106. In CVD has been assessed in LUMINA multiethnic cohort and GLADEL's longitudinal inception cohort, which demonstrated differences in sociodemographic, clinical , immunologic, and therapeutic characteristics, in SLE patients with CV events , 86, 93.Several studies have assessed smoking as an independent risk factor for CV atherosclerotic disease , 107–111P = 0.03). Relative risk  of hypertension associated with drinking 5 or more cups a day was 1.35 (0.87–2.08) for baseline intake and 1.60 (1.06–2.40) for intake over followup. Other effects attributed to coffee drinking are the increase in systemic vascular resistance, increased serum cholesterol levels, arterial stiffness, plasma rennin activity, epinephrine and norepinephrine, driving an unfavorable effect on endothelial function in healthy population [Several studies have evaluated the association between coffee consumption and CVD in the general population with controversial results. Two Dutch studies , 113 foupulation . On the pulation  detectedpulation , 119.In order to isolate the interaction of smoking and coffee consumption, two regression models were made in which both the independent effect of coffee and smoking consumption on CVD as well as their interaction remains significant, demonstrating synergism between them . Otherwise, coffee consumption has not been evaluated systematically in SLE patients with CVD. However, since there is not a universal accepted tool for assessing coffee consumption, a bias concerning this variable is not precluded. Furthermore, membership bias could also exist because coffee consumption in Colombia is a well-defined tradition.  Diverse lupus cohorts had shown the influence of advanced age, dyslipidemia, obesity, hypertension, and hyperhomocysteinemia, as classical risk factors for CVD , 121. YoLópez-Jaramillo et al.  showed tIt is well known that while traditional CVD risk factors are undoubtedly important in increasing the CVD risk among SLE patients, these do not fully account for the elevated risk of CVD in this population . TherebyFamily and twin studies have repeatedly supported a role for heredity in CAD, particularly in young individuals. Several genetic markers have been proposed as predisposing factors for CVD in SLE patients , 138. ThPhysicians often face the question of how to personalize treatment and prevention of CV events. Framingham risk score (FRS) is widely used to stratify asymptomatic patients into different CV risk categories in order to target the intensity of primary medical intervention. This score is strongly influenced by age and therefore has limited usefulness in young patients with SLE . TherefoRecently, Petri and Magder  proposedSLE entails a CVD equivalent to T2DM; thus, lower lipid goals, aspirin use, and an aggressive monitoring are required , 146. StThere are several new mechanisms of action described for antimalarials, many of them with beneficial effects in the management of CV risk in patients with SLE , 150. ThThe presence of low vitamin D levels has been associated with disrupting self-tolerance. Therefore, it is tempting to speculate that vitamin D might prove useful as a preventive agent by reducing the risk of developing an autoimmune response . In addi As a cross-sectional study, it is placed at level 4 of evidence according to Oxford Evidence Based Medicine . AlthougThe diversity of parameters defining CVD in SLE precluded homogenization of analysis and assessment in the systematic review. Often, one or two CVD subphenotypes were assessed as primary outcomes but many did not consider general CVD compromise. Therefore, it was not possible to perform a meta-analysis and to establish true measures of association such as odds ratios. Finally, we acknowledge that publication bias may exist.CVD is a major cause of morbidity and mortality in SLE patients. SLE and CVD share common pathophysiology mechanisms  with secondary accelerated atherosclerosis. Since traditional risk factors do not completely explain the high rates of CVD in patients with SLE, novel risk factors related to autoimmunity are now recognized. In the current study classical risk factors such as dyslipidemia, coffee consumption, and smoking habit are highlighted, and SLE-related factors  are confirmed. In addition, several factors associated with CVD in LA patients with SLE were reviewed. Altogether, our results should encourage preventive population strategies for CVD in patients with SLE , 159, aiThe Supplementary Material enclose the total description of the systematic literature review, organized by country, author, and year. In this table are all data regarding to type of study, evidence level and contains the prevalence found and their associations with traditional and non–traditional risk factors for CVD in patients with SLE in Latin America.Click here for additional data file.


Recent animal and human research indicates that stress around the time of encoding enhances long-term memory for emotionally arousing events but neural evidence remains unclear. In the present study we used the ERP old/new effect to investigate brain dynamics underlying the long-term effects of acute pre-encoding stress on memory for emotional and neutral scenes. Participants were exposed either to the Socially Evaluated Cold Pressure Test (SECPT) or a warm water control procedure before viewing 30 unpleasant, 30 neutral and 30 pleasant pictures. Two weeks after encoding, recognition memory was tested using 90 old and 90 new pictures. Emotional pictures were better recognized than neutral pictures in both groups and related to an enhanced centro-parietal ERP old/new difference (400–800 ms) during recognition, which suggests better recollection. Most interestingly, pre-encoding stress exposure specifically increased the ERP old/new-effect for emotional (unpleasant) pictures, but not for neutral pictures. These enhanced ERP/old new differences for emotional (unpleasant) scenes were particularly pronounced for those participants who reported high levels of stress during the SECPT. The results suggest that acute pre-encoding stress specifically strengthens brain signals of emotional memories, substantiating a facilitating role of stress on memory for emotional scenes. Acute stress initiates various bodily adaptation processes to establish physiological homoeostasis and affects cognitive processes such as attention, learning and memory. The link between stress and cognitive functioning has been the focus of intensive basic and clinical research. Experiencing episodes of extreme stress can lead to mental disorders such as post-traumatic stress disorder (PTSD), which is characterized by severe impairment in cognitive functioning and memory  However, studies that investigated the effects of stress and elevated stress hormone levels on learning and memory in healthy populations have reported mixed findings In contrast, acute stress and stress level glucocorticoid doses around the time of encoding have been shown to improve later memory performance for emotionally arousing events in humans and animals A key advantage of ERPs compared to other brain imaging techniques is that they provide measures of neural activity with extraordinary time resolution in real time and thus makes them ideally suited to examine neural events responsible for human memory Recent picture memory studies using longer retention intervals from 24 hours up to one year In the present study, we examined the influence of acute pre-encoding stress on brain dynamics associated with the long-term memory  for emotionally arousing and neutral scenes. In accordance with our previous ERP long-term recognition memory studies Participants provided informed written consent for the protocol approved by the Review Board of the University of Greifswald and received financial compensation for participation. The study conforms with The Code of Ethics of the World Medical Association (Declaration of Helsinki) printed in the British Medical Journal (18 July 1964).2) participated in the study. Exclusion criteria were checked in a standardized telephone interview and included smoking, current or lifetime diagnosis of mental disorders, medical conditions and medication intake within the prior three weeks, and during study participation. Participants were instructed to refrain from physical exercise, meals and caffeine intake within 3 h prior to the experimental sessions. Female participants reported a regular cycle with six subjects in follicular and 17 in luteal phase. All participants had normal or corrected-to-normal vision.Fifty-two healthy students  from the University of Greifswald : 22.2, range 19–27 kg/m = 1.1, p = .19), sex, handedness, BMI and menstrual cycle for females <1; see Participants were randomly assigned to either the stress or control condition. Groups did not differ for age  were exposed to the Socially Evaluated Cold Pressure Test (SECPT) as described by Schwabe et al. Stimuli consisted of 180 pictures  taken from the International Affective Picture Series (IAPS) Each picture set consisted of 90 pictures . 21 pictures were added before  and after  picture presentation to avoid serial position effects on subsequent memory performance. These pictures were not included in the analyses. = 475.08, p<.001) and pleasant  = 883.74, p<.001) pictures. Additionally, emotional pictures  were rated as more arousing than neutral pictures  = 349, p<.001). As in our previous study  = 2.6, p = .118) and arousal <1) ratings. No differences were observed between both picture sets.In addition, individual hedonic valence and arousal ratings for all pictures in our sample were obtained to check for correspondence with normative ratings of the IAPS and EmoPicS. As expected, unpleasant pictures were rated as more unpleasant  than neutral  of 2000, 2500 or 3000 ms. A 500 ms fixation cross preceded the onset of each picture to ensure that participants fixated the center of the screen. The pictures were presented in pseudorandom order for each participant with the restriction that no picture from the same valence category was presented in two consecutive trials. Participants were instructed to attentively watch the pictures and to avoid eye blinks and body movements during ERP measurement. No mention of a memory test was made .Two weeks after the encoding session participants returned to the lab for memory testing. After attaching the EEG electrodes, a recognition test was conducted during which 90 previously seen pictures  were presented randomly intermixed with 90 new pictures that were matched for content, valence and arousal. Each picture was displayed for 3000 ms and preceded by a 500 ms fixation cross. Participants were instructed to attentively watch the pictures and to avoid eye blinks and body movements during ERP measurement. Following each picture, participants had to indicate whether they had seen the picture before or not, by pressing either a “yes” or “no” button. The assignment of left and right button presses to yes/no responses was counterbalanced across participants. After recognition, participants were asked to rate all previously seen pictures for their subjective hedonic valence and arousal using the SAM rating procedure EEG signals were recorded continuously from 257 electrodes using an Electrical Geodesics (EGI) HydroCel high-density EEG system with NetStation software on a Macintosh computer. The EEG recording was digitized at a rate of 250 Hz, using the vertex sensor Cz as recording reference. Scalp impedance for each sensor was kept below 30 kΩ. All channels were bandpass filtered online from 0.1 to 100 Hz. Offline reduction was performed using EMEGS ERPs were computed for the three emotional picture categories  in each experimental group (stress vs. control). Only trials with correct responses  were included in ERP averages.To identify sensor clusters representative for the old/new effect, visual inspection and single-sensor waveform analyses were used in concert. On the basis of this inspection, ANOVAs, including the factors Emotion , Memory (old vs. new) and Group (stress vs. control), were calculated for each time point and each individual sensor Mean ERP amplitudes of both scalp clusters in the corresponding time windows were analyzed separately using an ANOVA involving the within-subject factors Emotion  and Memory (old vs. new) as well as the between-group factor Group (stress vs. control).In addition, group differences were observed for old and new neutral and unpleasant pictures in the time window between 250 and 300 ms over the frontal electrodes see . Mean N2For behavioral performance, hit rates, false alarm rates and the discrimination index Pr for recognition were analyzed using an ANOVA involving the factors Emotion  and Group (stress vs. control). All analyses were conducted with SPSS 19.0 .For effects involving repeated measures, the Greenhouse-Geisser procedure was used to correct violations of sphericity.Autonomic and subjective measurements indicated a successful stress induction by the SECPT see . = 46.29, p<.001), painful  = 104.94, p<.001) and unpleasant  = 67.63, p<.001) than the participants in the warm water control condition. In addition, hand immersion was harder to tolerate in the stress group  = 59.25, p<.001) compared to the warm water control group.As expected, participants rated the hand immersion in the SECPT condition as significantly more stressful  = 4.14, p<.05), systolic  = 69.85, p<.001) and diastolic  = 8.69, p<.01) blood pressure compared to the control group. Moreover, during hand immersion, participants in the SECPT group showed significantly elevated autonomic reactions  = 6.80, p<.05; systolic blood pressure: F = 15.48, p<.001; diastolic blood pressure: F = 50.63, p<.001) compared to the control group  = 42.44, p<.001) indicated that pictures with emotional contents were better remembered than neutral pictures. Unpleasant pictures (Mean (hr) = .81) were better recognized than pleasant (Mean (hr) = .74) pictures  = 24.36, p<.001). Consistent with hit rate, correct discrimination (Pr) between old and new pictures was better for emotional compared to neutral pictures  = 47.66, p<.001). False alarm rates differed between emotional contents  = 4.21, p<.05) and were slightly higher for pleasant compared to unpleasant pictures. No group effects were observed for false alarms <1, p = .434) or hit rates  = 3.21, p = .079). In contrast, a main effect for group  = 6.94, p<.05) indicated that picture discrimination (Pr) was overall better in the warm water control compared to the stress group. Post hoc tests showed that discrimination was better for neutral pictures in the controls compared to stress group  = 7.01, p<.05); however these group differences did not occur for emotionally arousing pictures  = 2.83, p = .10).As expected for hit rate (hr), a main effect of Emotion  = 9.18, p<.01) over frontal sensor sites. A significant interaction  = 3.228, p<.05) indicated that novel neutral pictures prompted a larger N200 than correctly recognized old neutral pictures in the control group  = 4.50, p<.05). This effect did not occur in the SECPT group <1, p = .731). In contrast, new unpleasant pictures prompted a larger negativity than correctly recognized old unpleasant pictures in the SEPCT group but not in the controls  = 4.32, p<.05; Control group: F<1, p = .718). For pleasant scenes, the N200 in response to novel pictures, compared to old, did not differ between stress and control group  = 1.20, p = .278).In the time window from 250 to 300 ms, new pictures evoked a larger ERP negativity than old pictures  = 39.30, p<.001), but not over centro-parietal sensor sites <1, p = .770). No main effects of group or any interactions were significant over frontal <1, p = .542) and centro-parietal sensors <1, p = .450) in the early time window.In the early time window from 300 to 500 ms, correctly recognized old pictures prompted more positivity, relative to correctly classified new pictures over frontal  = 19.45, p<.001) over frontal electrodes but no interaction with emotion <1, p = .50). No group differences were observed between SECPT and control group <1, p = .943).In the late time window between 400 and 800 ms, there was a prominent old/new effect  = 5.46, p<.01) with emotional pictures showing larger old/new differences than neutral pictures  = 9.06, p<.01)  = 4.45, p<.05). These group differences were not observed during correct recognition of neutral or pleasant pictures. The mean amplitude changes during correct recognition of old and new pictures as a function of emotion and group are listed in For centro-parietal sensors, correctly recognized old pictures showed greater ERP positivity than new pictures  = 13.45, p<.01). Notably, this old/new difference was modulated by emotion (Emotion×Memory: F.01) see . The oldTo further examine the relationship between acute pre-encoding stress and memory, correlational and median split analyses were conducted. As a measure of overall experienced stress, the mean average of the four subjective stress ratings  was calculated. Significant correlations were observed between the reported stress level and the centro-parietal old/new effect (difference score: old minus new) for emotionally arousing pictures , in which participants were divided into a high stress (Mean: 75.0) and low stress group (Mean: 37.11). In the present study, we examined the influence of acute pre-encoding stress on brain potentials of long-term memory for emotional and neutral scenes. Correct recognition of previously seen pictures evoked enhanced positivity over centro-parietal sensor sites in the late time window (400–800 ms) compared to viewing of new pictures. This late old/new difference was more pronounced for emotional picture contents. Interestingly, although memory performance was not generally facilitated by stress exposure, we found that acute pre-encoding stress specifically enhanced the late centro-parietal old/new effect for emotional (unpleasant) pictures. Furthermore, participants experiencing high levels of stress showed higher ERP old/new differences for emotionally arousing (unpleasant and pleasant) pictures, but not for neutral pictures. Taken together, pre-encoding stress prompts an enhanced old/new effect during recognition of emotional pictures that varies with individual stress levels.Correct recognition of previously presented compared to correctly rejected new pictures was associated with enhanced ERP positivity over centro-parietal sensor sites (400–800 ms). This late old/new difference over posterior sensor sites has been reliably described as an electrophysiological correlate reflecting retrieval processes with specific episodic recollection Previous work has indicated that stress influences learning and memory processes depending on the exact timing of the stressor Even though the ERP old/new effect for neutral pictures did not differ between stressed and control participants, we found that new neutral pictures prompted an enhanced negativity (250 to 300 ms) compared to old neutral pictures in control participants. A fronto-central N200 has been discussed as an ERP component reflecting perceptual novelty during information processing, because it is elicited when a perceptual mismatch between the repetitive standard and an infrequent target is detected or when visual stimuli with novel features are presented Although memory performance for emotional picture contents was not enhanced in the stress group on the behavioral level, we observed enhanced late centro-parietal ERP old/new differences for unpleasant pictures following pre-encoding stress exposure, indicating that the neural signature of the memory trace is enhanced for unpleasant stimuli encoded in the context of stressful experiences Another line of research has described enhanced memory based on mood-congruency The amygdala is assumed to be the key brain structure for mediating stress effects on attentional networks and memory formation, interacting with several brain structures  triggered by locus coeruleus-originating noradrenaline innervation From an evolutionary perspective, remembering the emotionally arousing aspects of a stressful experience is important for survival. But, experiencing highly arousing, life-threatening episodes under conditions of extreme stress can result in exceptionally strong, over-consolidated traumatic (fear) memories (re-experienced in flashbacks or nightmares). These memories often lack the integration of specific neutral context information, and thus may lead to the development of Posttraumatic Stress Disorder (PTSD) and an overgeneralization of fear To summarize, acute exposure to stress significantly increased the late centro-parietal old/new effect during retrieval of emotionally arousing pictures. Moreover, this effect was most pronounced in participants reporting high subjective stress experience. These findings suggest that recollection of emotional memories seems to be particularly facilitated when the stressful event around the time of encoding is also evaluated as intense, stressful and unpleasant.


Progranulin and C1q/TNF-related protein-3 (CTRP3) were recently discovered as novel adipokines which may link obesity with altered regulation of glucose metabolism, chronic inflammation and insulin resistance.We examined circulating progranulin and CTRP3 concentrations in 127 subjects with (n = 44) or without metabolic syndrome (n = 83). Furthermore, we evaluated the relationship of progranulin and CTRP3 levels with inflammatory markers and cardiometabolic risk factors, including high-sensitivity C-reactive protein (hsCRP), interleukin-6 (IL-6), estimated glomerular filtration rate (eGFR), and adiponectin serum concentrations, as well as carotid intima-media thickness (CIMT).r = 0.30, P = 0.001) and IL-6 , whereas CTRP3 concentrations exhibit a significant association with cardiometabolic risk factors, including waist circumference (r = −0.21), diastolic blood pressure (r = −0.21), fasting glucose (r = −0.20), triglyceride (r = −0.34), total cholesterol (r = −0.25), eGFR (r = 0.39) and adiponectin (r = 0.26) levels. Serum progranulin concentrations were higher in patients with metabolic syndrome than those of the control group  and the number of metabolic syndrome components had a significant positive correlation with progranulin levels . In multiple regression analysis, IL-6 and triglyceride levels were significant predictors of serum progranulin levels (R2 = 0.251). Furthermore, serum progranulin level was an independent predictor for increased CIMT in subjects without metabolic syndrome after adjusting for other cardiovascular risk factors (R2 = 0.365).Circulating progranulin levels are significantly related with inflammatory markers, hsCRP (Serum progranulin levels are significantly associated with systemic inflammatory markers and were an independent predictor for atherosclerosis in subjects without metabolic syndrome.NCT01668888ClinicalTrials.gov  Inflammation is known as a pivotal pathogenic mechanism of obesity-related disorders such as type 2 diabetes, metabolic syndrome, and atherosclerosis. Adipose tissue functions as a major endocrine organ by adipokine-mediated modulation of a number of signaling cascades in target tissues that exhibit pro-inflammatory or anti-inflammatory activity Progranulin was first purified as a growth factor from conditioned tissue culture media ob/ob mice significantly lowered blood glucose levels by activation of the Akt signaling pathway and suppression of gluconeogenic enzymes in the liver C1q/TNF-related protein-3 (CTRP3) is a novel adipokine that is a structural and functional adiponectin paralog In the present study, we aimed to clarify the clinical significance of progranulin and CTRP-3 in the context of metabolic syndrome and atherosclerosis. We examined their circulating concentrations in subjects with or without metabolic syndrome, especially after excluding individuals with type 2 diabetes or CVD. In addition, we evaluated the relationship of serum progranulin and CTRP3 levels with various cardiometabolic risk factors, such as insulin resistance, high sensitivity C-reactive protein (hsCRP), IL-6, estimated glomerular filtration rate (eGFR), and adiponectin levels, as well as CIMT, which is a reliable indicator of early carotid atherosclerosis.Subjects who visited the Health Promotion Center of Korea University Guro Hospital for a routine health check-up were enrolled between October 2009 and March 2011 using predefined inclusion and exclusion criteria. Inclusion criteria were apparently healthy volunteers with age between 20 and 80 years. We exclude the participants had a history of CVD , type 2 diabetes, stage 2 hypertension , malignancy, or severe renal or hepatic disease. This study excluded subjects with a history of chronic inflammatory conditions that may affect the study results, and subjects that had taken medications that might affect inflammatory status within the last 6 months were also excluded. Participants were free of any lipid-lowering therapies for at least a 6-month period prior to enrollment. Finally, one hundred twenty-seven apparently healthy subjects who agreed to participate in the study were enrolled. Forty four subjects had metabolic syndrome and 83 participants were classified as a normal control group. Metabolic syndrome was defined according to the criteria established by the National Cholesterol Education Program Adult Treatment Panel III using the adjusted waist circumference for Asians 2 (kg/m2) and waist circumference was measured at the midpoint between the lower border of the rib cage and the iliac crest. All blood samples were obtained the morning after a 12-hour overnight fast, and were immediately stored at −80°C for subsequent assays. Serum triglyceride and high density lipoprotein cholesterol (HDL-C) levels were determined enzymatically using a chemistry analyzer . Low density lipoprotein cholesterol (LDL-C) concentrations were estimated using the Friedewald formula 2) = 175×(Scr)−1.154×(Age)−0.203× Body mass index (BMI) was calculated as weight/heightThe IMT of the common carotid artery was determined using high-resolution B-mode ultrasonography  with a 5–12 MHz transducer. Measurements of CIMT were made using IMT measurement software  at 3 levels of the lateral and medial walls, 1–3 cm proximal to the carotid bifurcation. The mean IMT was the average value of 99 computer-based points in the region. The CIMT level in this study was calculated as the average of the left and right mean IMT values. All measurements were recorded by one trained technician who was blinded to the subjects’ anthropometric and laboratory data.t-test or Mann-Whitney U test for continuous variables, and the Chi-square test was used to test for differences in the distribution of categorical variables. Spearman’s correlation test was performed to determine the relationships of serum progranulin and CTRP3 levels with study variables. P-values for the linear trend of serum progranulin and CTRP3 levels according to the tertiles in the number of metabolic syndrome components were calculated by analysis of variance (ANOVA). Multiple linear stepwise regression analysis with progranulin and CTRP3 levels as dependent variables was performed to identify the risk factors that determine serum progranulin and CTRP3 concentrations in the study subjects. The second multiple linear stepwise regression analysis was performed to determine the risk factors for the CIMT values in subjects with or without metabolic syndrome. The significance level for entry and for stay in the model was chosen to be 0.15 . All statistical results were based on two-sided tests. Data were analyzed using SAS 9.2 . We regarded a P-value <0.05 as statistically meaningful.Each variable was assessed for a normal distribution. Data are expressed as mean ± SD or median (inter-quartile range [25%–75%]). Differences between groups were tested using an independent two-sample P = 0.051), whereas there was no significant difference in serum CTRP3 levels.The clinical and biochemical characteristics of the study subjects are presented in r = 0.304, P = 0.001 and r = 0.300, P = 0.001, respectively), but had no significant correlations with various metabolic parameters, including BMI, waist circumference, glucose tolerance, blood pressure, and lipid profiles  and a negative correlation with CTRP3 levels . Moreover, serum progranulin levels increased significantly according to the number of metabolic syndrome components  and triglyceride (P<0.001) levels were significant determining factors for serum progranulin levels (R2 = 0.251), whereas sex (P<0.001), triglyceride levels (P<0.001) and LDL-cholesterol levels (P = 0.02) were significant decisive factors for circulating CTRP3 concentrations (R2 = 0.321) (Serum progranulin levels had significant positive correlations with serum hsCRP and IL-6 levels (profiles . On the = 0.321) .r = 0.236, P = 0.035). Similarly, the results of our multiple stepwise linear regression analysis showed that age, sex, BMI, HDL-cholesterol, and circulating progranulin (P = 0.039) levels were significant predictors for CIMT in subjects without metabolic syndrome (R2 = 0.365)(R2 = 0.433).Spearman correlation analysis also showed that serum progranulin level was significantly positively correlated with CIMT in subjects without metabolic syndrome ( = 0.365). On the The present study showed that circulating progranulin levels mainly have a significant relationship with inflammatory markers, such as hsCRP and IL-6, whereas circulating CTRP3 concentrations exhibit a significant association withcardiometabolic risk factors, including waist circumference, diastolic blood pressure, fasting glucose levels, lipid profiles, eGFR, and adiponectin levels. Furthermore, we found that serum progranulin level is an independent marker for carotid atherosclerosis in subjects without metabolic syndrome, even after adjusting for other cardiovascular risk factors.Progranulin, also known as proepithelin or acrogranin, is a widely-expressed, 593 amino acid secreted glycoprotein Our study demonstrates that serum progranulin is an independent maker for subclinical atherosclerosis, represented as CIMT. Atherosclerosis is a chronic inflammatory process resulting from the interaction of modified lipoprotein, macrophages, and the normal cellular elements of the arterial wall CTRP3 is a newly-discovered adipokine whose structure contains a 246 amino acid sequence protein, and is regarded as an adiponectin paralog 2), were very few in this cohort (n = 2). Therefore, to clarify the relationship of renal dysfunction with CTRP3, further studies including the subjects with renal impairment should be followed. Lastly, the data about smoking, alcohol, and exercise were not available in this cohort, so we could not adjust the effect of these lifestyle factors.There are some limitations to this study. First, because it was a cross-sectional study, no causality could be defined. It is not clear whether circulating progranulin and CTRP3 levels are causative factors or markers of the pathogenesis of inflammatory diseases and atherosclerosis. Secondly, this study enrolled only Asian subjects without diabetes or CVD, so the relationship of serum progranulin and CTRP3 levels to metabolic risk factors should be further evaluated in other ethnic populations and in the context of different interventions for the treatment of diabetes and CVD. Thirdly, the subjects with renal insufficiency, defined as an eGFR <60 Click here for additional data file.


Cell membranes are composed of many different lipids and protein receptors, which are important for regulating intracellular functions and cell signaling. To orchestrate these activities, the cell membrane is compartmentalized into microdomains that are stably or transiently formed. These compartments are called “lipid rafts”. In gamete cells that lack gene transcription, distribution of lipids and proteins on these lipid rafts is focused during changes in their structure and functions such as starting flagella movement and membrane fusion. In this paper, we describe the role of lipid rafts in gamete maturation, fertilization, and early embryogenesis. Fertilization is the process in which 2 different gamete cells, a sperm and an oocyte, unite to produce a zygote. For fertilization to be successful, these gamete cells must differentiate and activate specific signaling pathways. For example, after sperm has differentiated completely, various extracellular factors such as epididymosomes and albumin alter the structure and function of the plasma membrane of the sperm. In addition, in terminally differentiated gamete cells, various sterols, sphingolipids, glycolipids, and glycosylphosphatidylinositol- (GPI-) anchored proteins are localized on cell membrane microdomains that are called lipid rafts. Lipid raft components are often examined by using detergent-resistant membrane domains (DRMs), which enrich these components so that their distributions and functions can be visualized on the cell surface by using putative raft markers , 2. SincSperm are highly differentiated haploid cells with a head and a tail (flagellum) . The heaβ1; Hsp90β1), 70 kDa heat shock protein 5, chaperones, and other exosomes i) is a key regulator of many cellular functions [2+]i stimulates the continuation of the second meiotic division and formation of the second polar body, followed by formation of male and female pronuclei [2+]i is highly conserved among species [3) triggers the increase in [Ca2+]i, which propagates from the endoplasmic reticulum. In contrast, the binding of sperm to the oocyte plasma membrane is species specific. Currently, there are 2 hypotheses about how a sperm binds and activates an oocyte, namely, a transmembrane receptor mechanism that involves G proteins and a soluble sperm factor mechanism [ Xenopus oocytes have a G protein-coupled sperm receptor that activates PLC on the plasma membrane -GlcNAcβ1 → 3Gal) also is found on glycosphingolipids and glycoproteins [β1 → 3Galα1 → 4R' and NeuAcα2 → 3Galβ1 → 3-GalNAcβ1 → 3Galα1 → 4R', respectively [Many cell-surface antigens that are markers of pluripotency have been identified in the ICM, ES cells, and embryonic carcinoma cells. The most common marker of murine ES cells is stage-specific embryonic antigen (SSEA)-1. The expression of this antigen changes dramatically in preimplantation mouse embryos. Specifically, SSEA-1 is highly expressed in the morula stage, suppressed after compaction, and then expressed only in the ICM of the blastocyst . The Lewproteins –91. Mousproteins , 93. Simproteins . The SSEectively –97. The ectively . In viabectively . A similectively . Becauseectively . These pectively –104 thatM1, cholesterol, and tyrosine-phosphorylated proteins also have been found in the cleavage furrow and plane of sea urchin embryos [γ, which are tyrosine phosphorylated at the site of cytokinesis. Furthermore, activation of these enzymes is required for furrow progression. These studies suggest that caveolae and lipid rafts contribute to cytokinesis in early developmental embryos.In addition to cell adhesion, lipid rafts play an important role in cytokinesis , which i embryos . In thes


To clarify the incidence and predictive risk factors of cervical spine instabilities which may induce compression myelopathy in patients with rheumatoid arthritis (RA).Three types of cervical spine instability were radiographically categorized into “moderate” and “severe” based on atlantoaxial subluxation , vertical subluxation , and subaxial subluxation (SAS: irreducible translation ≥2 mm versus ≥4 mm or at multiple). 228 “definite” or “classical” RA patients (140 without instability and 88 with “moderate” instability) were prospectively followed for >5 years. The endpoint incidence of “severe” instabilities and predictors for “severe” instability were determined.+ [VS− SAS−], VS+ [SAS− AAS±], and SAS+ [AAS± VS±]), developed “severe” instabilities more frequently  than those initially without instability . The incidence of cervical canal stenosis and/or basilar invagination was also higher in patients with initial instability  than in those without instability . Multivariable logistic regression analysis identified corticosteroid administration, Steinbrocker stage III or IV at baseline, mutilating changes at baseline, and the development of mutilans during the follow-up period correlated with the progression to “severe” instability (p<0.05).Patients with baseline “moderate” instability, including all sub-groups (AASThis prospective cohort study demonstrates accelerated development of cervical spine involvement in RA patients with pre-existing instability—especially VS. Advanced peripheral erosiveness and concomitant corticosteroid treatment are indicators for poor prognosis of the cervical spine in RA. Rheumatoid arthritis (RA) is a chronic inflammatory disease, which affects 0.5–1% of the adult population We previously presented a prospective multicenter cohort study for the cervical spine in established RA patients This prospective cohort study was conducted at 21 investigation sites . The study protocol was approved by the institutional review board at each facility. Written informed consent was obtained from each patient. The study was conducted in concordance with the principles of the Declaration of Helsinki and with the laws and regulations of Japan.Between 2001 and 2002, in 21 facilities, 634 outpatients who fulfilled the American Rheumatism Association 1958 criteria for “definite” or “classical” RA Lateral cervical radiographs, obtained in full-flexion, neutral, and full-extension positions with the standardized protocol , were used for evaluating cervical spine instability. Cervical radiographs were measured twice at one-week intervals by each of four rheumatologists who were not associated with clinical follow-up of the patients, and the presence of instabilities was determined using the average length of measured values.Instability was defined as AAS in patients with the anterior atlantodental interval (ADI) >3 mm “Severe” instability, which indicates impending neurological deficit, was defined as AAS in patients with ADI ≥10 mm “Moderate” instability was defined in patients with instability who did not meet the “severe” criteria.Cervical canal stenosis, which is severe enough to induce compression myelopathy, was identified in patients with space available for the spinal cord (SAC)  ≤13 mm due to “severe” AAS or “severe” VS at C1–C2 level Basilar invagination, which may compress the brainstem and result in life-threatening symptoms, was recognized in patients with the tip of the odontoid process above the McRae line due to “severe” VS Bilateral hand radiographs were used for classifying the severity of peripheral joint destruction into five categories: Steinbrocker classification stages I–IV Patient age <55  years, sC-reactive protein (CRP) level was quantified at baseline and assessed whether ≥3.8 mg/dl, a reported average value in RA patients with myelopathic deterioration Rheumatoid factor (RF) was measured at baseline and positivity was assessed At baseline, patients received intensive use of disease modifying anti-rheumatic drugs (DMARDs): methotrexate (MTX) (≤8 mg/week), salazosulfapyridine , D-penicillamine (≤100 mg/day), or intramuscular gold (≤25 mg/2 weeks). Afterward, MTX, approved for RA since 1999 in Japan, rapidly replaced other DMARDs. Oral corticosteroids were allowed to relieve RA symptoms (≤10 mg/day prednisolone). In more aggressive cases, biologic therapies—infliximab (3 mg/kg/8 weeks), etanercept (10–25 mg/twice a week), adalimumab (40 mg/2 weeks), or tocillizumab (8 mg/kg/4 weeks)—were applied. Patients were categorized based on drug administration at baseline and through more than half of the follow-up period. Since no biologic agent was approved for RA until 2003, patients who had biologic administration for more than half of the follow-up period were similarly identified.+), “moderate” VS without SAS but with or without AAS (shown as VS+), and “moderate” SAS with and/or without either AAS and/or VS (shown as SAS+). To elucidate the influence of baseline instability on the development of cervical spine involvement in RA, the >5-year incidence of the progression of prior instabilities and/or the development of additional instabilities including “severe” instabilities, canal stenosis, and basilar invagination for each group was compared. Distributions of parameters for cervical spine involvement and of RA stages and mutilating changes were compared between baseline and >5-year follow-up. The χ2 test, Fisher exact test, paired t-test, or Wilcoxon signed-rank test was used. In addition, intra-class correlation coefficient or κ coefficient was calculated to determine intra- and inter-observer reliabilities for the measurement of radiographic parameters.Patients were divided into four groups based on pre-existing cervical spine involvement: no instability, “moderate” AAS alone . All statistical tests were two-sided. Statistical significance was assessed with p<0.05 and p<0.01. When the incidence of cervical spine lesions was compared between patients with AAS+, 24 with VS+, and 7 with SAS+. Baseline demographics and disease characteristics of the patients are shown in Table S1. There were no significant differences in these variables between 21 investigation sites.Baseline radiographic analysis of the cervical spine identified that 228 patients consisted of 140 without any instability, 57 with AASIn radiographic measurements, intra-observer reliabilities for parametric ADI, Ranawat value, SAC, and subaxial translation were 0.90–0.94, 0.80–0.85, 0.89–0.95, and 0.81–0.84 by intra-class correlation coefficient. Inter-observer reliabilities were 0.95, 0.90, 0.94, and 0.91. Intra-observer reliabilities for non-parametric basilar invagination and RA stages with mutilating changes were 0.79–0.83 and 0.74–0.79 by κ coefficient. Inter-observer reliabilities were 0.87 and 0.86. All the values indicated an acceptable reproducibility.To understand the total incidence of cervical spine subluxations, we investigated the incidence of instabilities which consisted of “moderate” and “severe” categories. Then, to clarify the incidence of compression myelopathy-inducing subluxations, we examined the incidence of “severe” instabilities. Forty-three point six percent of 140 patients without cervical spine instability at baseline developed instabilities at >5-year follow-up: AAS in 32.1%, VS in 11.4%, and SAS in 16.4% with some combinations. Furthermore, 12.9% of patients initially without instability presented with “severe” instabilities—“severe” AAS in 3.6%, “severe” VS in 6.4%, and “severe” SAS in 5.0% Table 1.+ at baseline developed VS in 22.8% at >5-year follow-up, which was higher than those without instability at baseline (11.4%) with a trend toward significance (p = 0.041). Moreover, 33.3% of patients initially with AAS+ exhibited “severe” instabilities at a significantly higher rate than that of patients initially without instability —especially “severe” AAS in 19.3%  (Fifty-seven patients with AASp<0.003) Table 1+ at baseline, the >5-year incidence of SAS was 41.7%, which was significantly higher than in those without instability at baseline . “Severe” instabilities were developed in 75.0% of patients initially with VS+, also more frequently than those initially without instability —notably “severe” VS in 70.8% . In addition, the incidence of “severe” SAS in 20.8% of patients with baseline VS+ was higher than in those without baseline instability (5.0%), and trended toward significance (p = 0.017) (In 24 patients with VS= 0.017) Table 1.+ at baseline, 42.9% developed “severe” instabilities at >5-year follow-up, which was higher than that of patients without instability at baseline (12.9%) although the comparison did not reach significance (p = 0.061). “Severe” VS occurred in 28.6% of patients initially with SAS+ . “Severe” SAS appeared in 28.6% of patients with baseline SAS+  (Of seven patients with SAS= 0.060) Table 1.+ (p<0.01 for both groups). The Ranawat value decreased in all patient groups   Figure 2+ exhibited stenosis (p = 0.014)—particularly SAC ≤13 mm at C1–C2 in 10.5% (p = 0.036). Patients with baseline VS+ presented with cervical stenosis in 16.7% (p = 0.041) who all showed SAC ≤13 mm at C1–C2 (p = 0.017). Patients with baseline SAS+ developed stenosis in 14.3% (p = 0.295) who demonstrated SAC ≤12 mm at C2–C7 (p = 0.179) , 15.8% of those with baseline AAS= 0.179) Table 2.+ (p<0.003) and 5.3% of those with AAS+ (p = 0.415)  Table 2.+, 37.5% in those with VS+, and 14.3% in those with SAS+) than in patients without instability   Table 2.+, and with VS+ and p = 0.04 for those with SAS+)  Figure 2Radiographic cervical spine analysis at baseline and at >5-year follow-up identified the development of “severe” instabilities in 58 of 228 patients (25.4%), as summarized in In the univariable analysis, previous joint surgery, CRP ≥3.8 mg/dl, corticosteroid administration, Steinbrocker stage III or IV at baseline (without the development of mutilating changes during the follow-up period), mutilating changes at baseline, and the development of stages I–IV into mutilating changes during the follow-up period were statistically significant for the development of “severe” cervical spine instabilities at >5-year follow-up (p<0.05) Table 4.In the multivariable analysis, we established a multivariable logistic regression model including the variables described above Table 4.To reduce the risk for overfitting variables In the current study, we assessed and compared the >5-year incidence of cervical spine instabilities with impending neurological deficit in 228 established RA patients with and without baseline instability, which demonstrated that the development of these serious lesions was accelerated in response to the type of baseline cervical spine instability—especially VS. Multivariable logistic regression analysis identified advanced peripheral erosiveness, concomitant corticosteroid treatment, and marginally previous joint surgery as independent risk factors for the development of severe cervical spine instabilities in RA. This study clarifies these clinically important findings with statistically robust, more conclusive evidence, which provides a comprehensive understanding of the progression of cervical spine involvement in patients with RA.The incidence of cervical spine instabilities depends on the diagnostic criteria for subluxation and the severity of RA. In this study, we used previously validated criteria +, 19.3% progressed to “severe” AAS and 22.8% developed VS. Notably, in patients initially with VS+, 70.8% progressed to “severe” VS, 41.7% newly developed SAS, and 20.8% further developed “severe” SAS. Patients initially with SAS+ also frequently had the progression of instabilities, although no statistical significance was obtained due to the small number of cases. Additionally, distributions of radiographic parameters support this higher progression tendency of instabilities in patients with initial instability. These results indicate accelerated development of cervical spine involvement in RA patients with pre-existing instability—especially VS.“Severe” cervical spine instabilities occurred in 33.3–75.0% of patients with baseline “moderate” instability, while these lesions only appeared in 12.9% of patients initially without instability. In patients initially with AAS+. In our previous study, we found that patients with pre-existing VS and/or SAS had substantially worse cervical spine instabilities +. The development of cervical canal stenosis and/or basilar invagination, severe enough to induce spinal cord and/or brainstem compression, are accelerated in patients with pre-existing instability—especially VS.Cervical canal stenosis and/or basilar invagination occurred in 14.3–37.5% of patients with baseline “moderate” instability, while these complications only appeared in 7.1% of patients initially without instability. The highest incidence of both these lesions was detected in patients initially with VSRecently, advances in RA therapies have drastically changed the clinical course even in patients with active disease Reported evidence indicates that patients with “severe” cervical spine instabilities, narrowed spinal canals, and/or cranial migration of the odontoid process are at risk for irreversible neurological damage Our multivariable analysis identified independent predictive risk factors for the development of “severe” instabilities: corticosteroid administration, Steinbrocker stage III or IV at baseline, mutilating changes at baseline, and the development of stages I–IV into mutilating changes during the follow-up period. These four variables, with significance in all the univariable, multivariable, and stepwise multivariable analyses, are relatively robust predictors for the progression of cervical spine involvement in RA. In addition, the consistent results from these analyses support further validation of the clinically-led multivariable model before applying stepwise approach.Prolonged and high dose administration of corticosteroids is shown to aggravate cervical subluxations in RA patients This study, which began in 2001, detected no obvious therapeutic effect of MTX and biologic therapies. However, their ineffectiveness cannot be concluded because their approval occurred in 1999 and 2003, respectively, and our patients had long RA histories predating these treatments. In addition, the dosages of medications used were lower than those used in Western countries. Reduced effectiveness of RA therapies indicates our results should be carefully interpreted.Mutilating changes are reported to highly correlate with aggressive cervical instabilities in RA Steinbrocker stage III or IV was also statistically significant for the progression to “severe” instability. Previous joint surgery was a marginally significant predictor; a high prevalence of cervical spine instabilities in RA patients undergoing joint surgery is often reported Table S1, followed patients were younger than patients lost to follow-up. In addition, patients with baseline “severe” instability, speculated to have worse conditions, showed a lower follow-up rate. This indicates that many older, advanced RA outpatients potentially drop out due to polyarthralgia, worsening general condition, and/or cervical problems. Furthermore, patients who developed “severe” instabilities during the follow-up period might be lost more frequently, implying that the endpoint incidence of “severe” instabilities is higher than the observed data suggests. Taken together, patients' loss to follow-up is likely not completely random in this study.This study is limited in that the duration of RA ranged widely. The currently used classification system for the severity of RA might be a somewhat obsolete and insensitive method of assessment. The biggest limitation is a low follow-up rate of 45.3%. As shown in Survival analyses such as the Cox proportional hazards analysis should provide more useful information for identifying predictive factors for “severe” instability when cervical radiographs obtained at multiple time points are available. The mixed-effects model may also be more suitable in the case of data missing at random between patients with and without the development of “severe” instabilities. The application of these statistical analysis methods is an issue to be considered in future studies.This prospective cohort study demonstrates accelerated development of cervical spine involvement in RA patients with pre-existing instability—especially VS. Established mutilating changes and progressive development of non-mutilating into mutilating changes strongly indicate poor prognosis of the cervical spine in patients with RA. Steinbrocker stage III or IV, corticosteroid treatment, and possibly previous joint surgery also correlate with the progression of cervical spine instabilities in RA.Table S1Baseline demographics and disease characteristics in 634 enrolled patients.(PDF)Click here for additional data file.


Methylenetetrahydrofolate reductase (MTHFR) converts 5,10-methylene tetrahydrofolate to 5-methyl tetrahydrofolate and affects the activity of cellular cycles participating in nucleotide synthesis, DNA repair, genome stability, maintenance of methyl pool, and gene regulation. Genetically compromised MTHFR activity has been suggested to affect male fertility. The objective of the present study was to find the impact on infertility risk of c.203G>A, c.1298A>C, and c.1793G>A polymorphisms in the MTHFR gene.PCR-RFLP and DNA sequencing were used to genotype the common SNPs in the MTHFR gene in 630 infertile and 250 fertile males. Chi-square test was applied for statistical comparison of genotype data. Linkage disequilibrium between the SNPs and the frequency of common haplotypes were assessed using Haploview software. Biochemical levels of total homocysteine (tHcy) and folic acid were measured. Meta-analysis on c.1298A>C polymorphism was performed using data from ten studies, comprising 2734 cases and 2737 controls.c.203G>A and c.1298A>C were found to be unrelated to infertility risk. c.1793G>A was protective against infertility (P = 0.0008). c.677C>T and c.1793G>A were in significant LD (D’ = 0.9). Folic acid and tHcy level did not correlate with male infertility. Pooled estimate on c.1298A>C data from all published studies including our data showed no association of this polymorphism with male infertility , azoospermia , or oligoasthenoteratozoospermia . Eight haplotypes with more than 1% frequency were detected, of which CCGA was protective against infertility (p = 0.02), but the significance of the latter was not seen after applying Bonferroni correction.Among MTHFR polymorphisms, c.203G>A and c.1298A>C do not affect infertility risk and c.1793G>A is protective against infertility. Haplotype analysis suggested that risk factors on the MTHFR locus do not extend too long on the DNA string. Infertility is the inability of a couple to conceive even after one year of regular, unprotected intercourse. The disorder is multifactorial in nature and affects about 15–20% of the couples trying for pregnancy MTHFR deficient mice exhibit global DNA hypomethylation, hyperhomocysteinemia and increased S-Adenosyl homocysteine (SAH), and most importantly, compromised spermatogenesis Methylenetetrahydrofolate reductase (MTHFR), which catalyzes conversion of 5, 10- methylene tetrahydrofolate to 5-methyl tetrahydrofolate, is an important enzyme of folate pathway. This pathway maintains the methyl pool required for regulatory functions and conversion of homocysteine (hcy) to methionine MTHFR enzyme Compromised activity of the MTHFR enzyme results in increased homocysteine levels MTHFR gene have been suggested to be candidates for male infertility MTHFR gene, ii) undertake meta-analysis on c.1298A>C to have a pooled estimate regarding its impact on infertility risk, and iii) correlate biochemical levels of homocysteine and folic acid with infertility.In a recent study, we analyzed c.677C>T polymorphism in an Indian population and conducted a meta-analysis to conclude significantly increased risk of male infertility in carriers of this substitution The Institutional Human Ethics Committee of the Ajanta Hospitals and IVF Centre and that of the King George Medical University (KGMU), Lucknow, approved this study. We recruited 630 infertile men from the Ajanta Hospitals and IVF Centre Pvt. Ltd., Alambagh and the Department of Urology, KGMU, Lucknow, for a case-control study. Informed written consent of each patient was obtained in response to a fully written and verbal explanation of nature of the study. Patients suffering from varicocele, diabetes, mumps, and those showing chromosomal anomalies etc. were excluded from the study. Upon examination of semen quality, patients were categorized according to the WHO 1999 criteria  [31]. FoMTHFR gene were genotyped using PCR-RFLP and direct DNA sequencing methods. The methodology and primers used in the study are detailed in Taq DNA polymerase enzyme (NEB), and 40 ng of genomic DNA. PCR cycling was carried out using ABI Veriti thermal cycler . PCR amplification conditions consisted of denaturation at 95°C for 10 minutes, followed by 35 cycles of denaturation at 95°C for 30 seconds, annealing at a suitable temperature for 30 seconds  Genomic DNA was isolated from lymphocytes of the peripheral blood of the patient and control samples using the phenol-chloroform-isoamyl method http://ihg.gsf.de/cgi-bin/hw/hwa1.pl. Chi square test was used to compare allele and genotype data between cases and controls using Vassar Stats Online Calculator (http://faculty.vassar.edu/lowry/VassarStats.html), adopting dominant, recessive, co-dominant, and additive models. Fisher exact test was applied wherever Chi square test was not applicable. In addition to comparison of all patients with controls, genotype and allele distribution were compared between case groups based on sperm motility (motility <30% and > = 30%) and sperm count . A P-value of less than 0.05 was considered to be statistically significant. The significance in case of multiple comparisons was assessed against a reference P value obtained after applying Bonferroni correction.Genotype data of control samples for all polymorphisms were analyzed for fitness in the Hardy Weinberg Equilibrium using the online calculator available at http://www.broadinstitute.org) LD was calculated and plotted using the Haploview software (Version 4.2) developed at ‘The Broad Institute’ . Folic acid level was measured using chemiluminescence based kit from Siemens diagnostic (Germany) on Immulite 1000 reader.Data on c.1298A>C polymorphism was available for a few other populations as well. Therefore, we have pooled data on c.1298A>C from our study and other published studies, comprising a total of 2734 cases and 2737 controls in order to perform meta-analysis.Identification of relevant studiesTo identify the case-control studies that analyzed MTHFR c.1298A>C polymorphism in male infertility, we conducted a systematic literature search through “ Pubmed”, “Google Scholar”, and “Scirus” databases using the keywords “MTHFR and male infertility”, “MTHFR 1298A>C and male infertility”, and “folate metabolism and male infertility”. The search was limited to the articles in English language and till September 2012 as the publication date. Retrieved studies were screened to meet the inclusion criteria that: (i) each trial was an independent case-control study, (ii) the purpose of all the studies and statistical methods were similar, (iii) SNP typing was done at high resolution level, and (iv) inclusion of the patients was done according to standard diagnosis parameters. Studies not providing enough information (genotype data), and those not well described were considered for exclusion. The citations of the articles included were screened carefully to identify maximum numbers of relevant studies.Data extractionThe articles satisfying the inclusion criteria were read carefully to extract details regarding first author, year of publication, ethnicity, total number of cases and controls, genotype and allele frequency, and the type of infertility phenotype. Data extraction was performed by NG and independently confirmed by SS.Statistical analysis2 value with the classification given by Higgins and Thompson; viz. 25%, 50% and 75%, which correspond to low, medium, and high heterogeneity, respectively Meta-analysis was performed using the Comprehensive Meta-Analysis (CMA) software (Version 2), which allows data entry in different formats. Odds ratio was chosen as a measure of the effect size. Heterogeneity was assessed using Chi square based ‘Q’ test, considering P-values <0.10 to be statistically significant Publication bias was investigated using the funnel plot of precision by log odds ratio method. Asymmetry of the funnel plot was assessed by Egger’s regression intercept test. Egger’s test estimates bias using precision (inverse of the standard error) to predict standardized effect (effect size divided by the standard error), and significance of the intercept was determined using ‘t’ test, considering P value of <0.05 to be significant.We have analyzed four polymorphisms in the MTHFR gene in 630 infertile men and 200 fertile controls by PCR-RFLP and direct DNA sequencing methods. MTHFR genotypes distribution among control samples fitted well in the Hardy-Weinberg equilibrium for c.1298A>C (0.31), c.1793G>A (1.00), and intronic (0.65) polymorphisms, but not for c.203G>A polymorphism. The frequency of allele ‘A’ or genotype ‘GA+AA’ at c.203G>A locus were 0% and less than 1%, respectively, in both fertile and infertile individuals , and genTo find a correlation of each SNP with semen parameters, we cross-classified genotype data with reference to sperm motility and count  and 4. G2 = 0.04). c.677C>T and c.1298A>C polymorphisms  showed intermediate concentration of LD. However, c.677C>T and c.1793G>A polymorphisms were in significant LD . c.1793G>A and intronic polymorphisms were also in significant LD   of <1%; therefore, only three SNPs in the coding region and one intronic SNP were included in LD and haplotype analysis. The data for c.677C>T site from our previous study = 0.196) . Eight h= 0.196) . The dis= 0.196) . HoweverWe measured homocysteine and folic acid levels in a subset of infertile and fertile individuals. Folic acid level ranged from 6.2 to 17.8µg/L in infertile and from 6.8 to 17.1 µg/L in fertile men. Average folic acid level in infertile and fertile groups was 12.05µg/L and 11.97µg/L, respectively, with no significant difference between the two groups . tHcy leLiterature searchUsing strictly defined criteria, we could retrieve forty two studies. Few studies had used MTHFR in connection with measurement of vitamin B12, folic acid, and homocysteine. A large number of studies had not analyzed c.1298A>C polymorphism. Few others were either review articles or were conducted on animal models. We found only ten case-control studies looking for correlation of c.1298A>C polymorphism with male infertility Quantitative data synthesisheterogeneity = 0.058, Q = 16.47, df(Q) = 9, I2 = 45.35, var = 0.029, τ2 = 0.0310 SE = 0.031, τ = 0.172). Random effects model was used to pool the data; however, the results of both the models were similar (Fixed OR = 1.035 and Random OR = 1.045). Pooled odds ratio did not show significant association of “AC+CC” genotype with male infertility  (Genotype comparison was made adopting dominant model (‘AC+CC’ vs ‘AA’). Significant level of between studies heterogeneity was observed (P = 0.59) .heterogeneity = 0.697; I2 = 0.00), and pooled odds ratio did not show significant association of “AC+CC” genotype with azoospermia . However, in case of OAT, the data were heterogeneous  and no significant association of “AC+CC” genotype with OAT, adopting either fixed  or random  effects model, was observed (Genotype data were stratified according to infertility phenotype (OAT and azoospermia) in order to calculate group-wise effect sizes. The data for azoospermic individuals were homogenous  = 6, I2 = 34.09, var = 0.001, τ2 = 0.02, SE = 0.033, τ = 0.14); however, there was no change in the conclusion .Since the control data for three studies Funnel plot and Egger’s test were used to quantify publication bias. The distribution of the studies on the funnel plot did not reveal any evidence of asymmetry, suggesting the absence of bias in quantitative assessment of the pooled data . The absSimilarly, meta-analysis comparing allele distribution did not show an association of c.1298A>C polymorphism with male infertility (P = 0.495), OAT (P = 0.831) and azoospermia (P = 0.864).The impact of MTHFR gene SNPs on infertility risk has been studied in various ethnic groups A to C transversion at the 1298 locus does not affect infertility risk in Indian men. The results of our study are in concordance with a previous study conducted on Indian population MTHFR risk alleles are isolated and do not stretch too long on the DNA string.To dissect the nature of association between c.1298A>C polymorphism and male infertility, we performed a meta-analysis on data pooled from all published studies. SNP data on 2734 cases and 2737 controls from 10 cases-controls studies were included in the pooled estimate. In overall analysis, 1298 genotype (AC+CC) did not show any association with infertility. Similarly, a stratified analysis on the basis of infertility phenotype did not reveal any significant association between this SNP and azoospermia or OAT. We could not conduct analysis on the basis of ethnicity due to lack of many studies from a particular ethnic group. Exclusion of the studies not following the Hardy-Weinberg equilibrium Folic acid supplementation is known to reduce Hcy level and the incidence of birth defects in women, but evidently it also serves an important complementary function in men In conclusion, the presence of ‘T’ at locus 677 increases the risk of infertility. On the other hand, ‘A’ allele at locus 1793 is protective against infertility in Indian men, but c.203G>A and c.1298A>C do not correlate with fertility status. The exact mechanism underlying the impact of folate pathway on fertility is not yet clear; however, some possible mechanisms have been put forward. Reduced MTHFR activity due to inadequate intake of folate and vitamin B12 or due to genetic alterations may lead to hyperhomocysteinemia, which might cause auto-oxidation resulting in oxidative stress Table S1Categorization of infertile patients: Infertile patients were categorized into groups following the WHO (1999) criteria.(DOCX)Click here for additional data file.Table S2Meta-analysis: Summary of the studies included in the meta-analysis on c.1298A>C polymorphism.(DOCX)Click here for additional data file.