Testicular Dysgenesis Syndrome and the Estrogen Hypothesis : A Quantitative Meta-Analysis Background Male reproductive tract abnormalities such as hypospadias and cryptorchidism , and testicular cancer have been proposed to comprise a common syndrome together with impaired spermatogenesis with a common etiology resulting from the disruption of gonadal development during fetal life , the testicular dysgenesis syndrome -LRB- TDS -RRB- . The hypothesis that in utero exposure to estrogenic agents could induce these disorders was first proposed in 1993 . The only quantitative summary estimate of the association between prenatal exposure to estrogenic agents and testicular cancer was published over 10 years ago , and other systematic reviews of the association between estrogenic compounds , other than the potent pharmaceutical estrogen diethylstilbestrol -LRB- DES -RRB- , and TDS end points have remained inconclusive . Impaired spermatogenesis , male reproductive tract abnormalities such as hypospadias and cryptorchidism , and testicular cancer have been proposed to comprise a common underlying syndrome with a common aetiology resulting from the disruption of embryonic programming and gonadal development during fetal life , termed the testicular dysgenesis syndrome -LRB- TDS -RRB- -LRB- Sharpe and Skakkebaek 2003 ; Skakkebaek et al. 2001 -RRB- . A hormonal etiology most likely underlies this syndrome , although it is believed to have more than one cause , possibly including other than endocrine disruption . Some common causes of endocrine disruption include infection , diet and body weight , lifestyle , genetics , and environmental exposure , but endocrine-disrupting chemicals -LRB- EDCs -RRB- , particularly those with estrogen-like properties , have received the most scientific attention . The synthetic estrogenic drug diethylstilbestrol -LRB- DES -RRB- was prescribed to more than 5 million pregnant women from the late 1940s to the early 1970s to prevent abortions and pregnancy-related complications -LRB- Palmlund et al. 1993 -RRB- . Evidence later showed that maternal ingestion of DES during early pregnancy increased the risk of vaginal clear cell adenocarcinoma in female offspring -LRB- Herbst et al. 1971 -RRB- and resulted in an increased incidence of malformations of the testes , the development of epididymal cysts , and impaired sperm quality in male offspring -LRB- Bibbo et al. 1977 -RRB- . During pregnancy , maternal estrogen levels are significantly elevated . However , more than 90 % of maternal endogenous estrogens are effectively sequestered via binding to sex hormone binding globulin -LRB- SHBG -RRB- , and thus the fetus is relatively protected -LRB- Joffe 2001 ; Vidaeff and Sever 2005 -RRB- . On the other hand , DES and ethinylestradiol do not bind well to SHBG , having a higher biopotency if ingested -LRB- Sharpe and Skakkebaek 2003 ; Vidaeff and Sever 2005 -RRB- . Additionally , transgenerational exposure is also possible when lipophilic xenoestrogens are mobilized during pregnancy and lactation -LRB- Colborn et al. 1993 -RRB- . Previous systematic reviews of studies in which pregnant women were exposed to estrogens other than DES have failed to find evidence of an increased risk of urogenital abnormalities in the male offspring -LRB- Raman-Wilms et al. 1995 ; Storgaard et al. 2006 ; Toppari et al. 1996 ; Vidaeff and Sever 2005 -RRB- , and have raised the possibility that nonestrogenic or atypical estrogenic effects of DES exposure in utero induce male reproductive abnormalities . However , none of the effects of DES exposure on either male or female offspring of exposed wild-type pregnant mice were induced when administered to ERKO -LRB- ER-α knockout -RRB- mice -LRB- Couse et al. 2001 -RRB- , strongly suggesting an ER-α -- mediated mechanism . There is , however , a body of experimental data that is consistent with an effect of antiandrogenic industrial chemicals on male sexual differentiation -LRB- Gray et al. 1999 , 2000 -RRB- . Moreover , mechanisms other than endocrine disruption may be involved in testicular toxicity ; for example , the nematocide dibromochloropropane , an alkylating agent , is one of the most potent known testicular toxins in adults -LRB- Joffe 2001 -RRB- . In this review we focus on the estrogen hypothesis of TDS . Although several systematic reviews of the literature on the association between estrogenic agents and the disorders thought to belong to the TDS have been published , they are predominantly qualitative and the only quantitative summary estimate of the association between prenatal exposure to estrogenic agents and testicular cancer was published over 10 years ago -LRB- Toppari et al. 1996 -RRB- . The primary objective of a quantitative meta-analysis is to combine the results of previous studies examining a specific research question to arrive at a summary conclusion about a body of research . It has been found particularly useful when individual studies are too small to yield a valid conclusion , but it can not , however , correct for bias and confounding . When applied to observational studies , subset analysis can be a useful tool to explore the reasons for discrepancies among the results of different studies . The objectives of this research were therefore to carry out a quantitative meta-analysis of the association between three of the end points related to TDS and prenatal exposure to estrogenic agents that would account for both the size and quality of the studies included and yield updated summary estimates in light of the body of research carried out since the formulation of the estrogen hypothesis . Inclusion in this analysis was based on mechanistic criteria , and the plausibility of an ER-α -- mediated mode of action was specifically explored . Moreover , subset analysis has been applied to categories of compounds with estrogenic potencies differing by several orders of magnitude in an attempt to detect the existence of any potency -- response trend . Most of the studies of sperm quantity or quality have been concerned with time trends rather than etiology , and this end point was not considered further here . Material and Methods Identification and selection of literature A computerized search was conducted using the databases PubMed -LRB- National Center for Biotechnology Information 2007 -RRB- and Web of Science -LRB- ISI Web of Knowledge 2007 -RRB- for the period 1970 to April 2007 . The general search keywords were `` estrogen , '' `` risk , '' `` dose , '' and either `` hypospadias , '' `` cryptorchidism , '' or `` testicular cancer . '' A preliminary identification was performed by screening the titles and , if relevant , the abstracts of retrieved literature . The next stage was to check the citations and references of selected studies . This was an iterative process , repeated until no new study could be identified . A set of both inclusion and exclusion criteria was defined , and all relevant literature was then checked for eligibility . The inclusion criteria considered were a -RRB- study design , namely , either a case -- control , cohort , or clinical trial ; b -RRB- written in English ; c -RRB- exposure to one or a mixture of known estrogenic compounds ; and d -RRB- sufficient data reported to be used in meta-analysis . The following exclusion criteria were used : Exposure to a group of compounds -LRB- suspected endocrine disruptors -RRB- for which mode of action was unspecified , for example , pesticides . Studies of exposure to phytoestrogens . Some phytoestrogens have been found to have a greater binding affinity for ER-β than for ER-α and can result in agonistic or antagonistic effects -LRB- Mueller et al. 2004 -RRB- . Studies of maternal endogenous hormones . Studies of the same cohort as this would bias the results towards the particular studies . Incomplete data . Data extraction and quality rating In addition to the number of exposed and nonexposed cases and controls , and risk ratios -LRB- RRs -RRB- with their confidence intervals -LRB- CIs -RRB- , information regarding the study design , estrogenic agent , geographic location of the study , and year of publication were extracted from the selected literature to allow subset analysis to be carried out . When more than one RR was reported , the following priorities were set for choice : Adjusted RRs were used , except when the study provided only unadjusted estimates . When multiple estimates were given , the RR estimator on which the authors had relied for their assessment of causal association was used . Overall RRs were chosen instead of those derived from further stratifications . If an overall estimate was not provided , the RRs of the maximum duration of exposure or the maximum exposure concentration were chosen . Several aspects of the quality of each study were also recorded according to a rating scheme adapted from those previously described -LRB- Altman 1991 ; Rushton 2000 -RRB- . Every criterion was assessed on a scale of 0 to 2 , 0 suggesting that it was not present , 1 when it was unclear , and 2 when that criterion was satisfied . A maximum score of 50 and 52 could be assigned for retrospective -LRB- case -- control -RRB- and prospective -LRB- cohort and clinical trials -RRB- studies , respectively . This enabled a quality sensitivity analysis to be performed to check the influence of studies with low quality on the pooled estimate . Data analysis Graphical representation The RRs and CIs were plotted against the year of publication to determine whether any positive or negative trends in reporting RRs had occurred over time . Similarly , quality scores were plotted against the year of publication to investigate whether the quality of studies improved over time . To assess publication bias , a funnel plot -LRB- SE vs. RR -RRB- was produced based on the assumption that smaller studies are less precise in their RRs and thus have less weight and larger SE and should scatter more widely at the lower end of the graph , whereas larger studies will tend to be closer together -LRB- Sterne et al. 2001 -RRB- . Forest plots present the RRs against the reference of the study and help check homogeneity visually . Statistical pooling Pooled estimates and 95 % CIs were calculated using both a fixed-effects model -LRB- Mantel -- Haenszel method -RRB- and a random-effects model -LRB- DerSimonian -- Laird method -RRB- , allowing evaluation of the dependence of the conclusions of the analysis on the model assumptions . A summary estimate is considered statistically significant at the 0.05 level if its CI does not include unity . The Mantel -- Haenszel pooled effect estimate was used in a chi-square statistical test of homogeneity to assess the between-study variance . The magnitude of the test statistics depends on the weight of each study . When the number of studies is low or the studies themselves are small , the test statistic Q tends to be small . Tests of heterogeneity in meta-analyses are generally low in their power to reject the null hypothesis of homogeneity . For this reason , the chi-square statistical test of homogeneity was carried out at both 0.05 and 0.1 significance levels . Additionally , pooled estimates calculated using fixed effect and random effect models differ only if there is lack of homogeneity between studies . The estimates obtained by both methods were therefore compared to better assess potential heterogeneity between studies , in which case a single summary estimate of effect may be considered inappropriate . Subset and sensitivity analyses To investigate potential sources of heterogeneity between studies , we performed subset analyses for the study design , estrogenic agent , and geographic location . Some studies exploring the influence of hormonal treatment during pregnancy did not specify the type of hormone . From what is known of the hormonal treatment of common conditions occurring during pregnancy , it was deemed reasonable to assume that they would have been likely to include estrogens , and these studies were included in the analysis . The validity of this assumption was tested by applying stricter criteria and calculating a summary estimate of effect excluding any study in which the hormone used had not been specified . Further sensitivity analysis was performed by excluding low-quality studies and extremes -LRB- exclusion of the studies with the largest and smallest RR estimators and exclusion of the studies with the largest and smallest weights -RRB- to verify that either the quality of the studies or one particular study did not have an excessive influence on the pooled estimate . Results A total of 50 studies were identified for the association between in utero exposure to estrogenic agents and hypospadias and/or cryptorchidism , including 16 that had not been included in previous systematic reviews . Sixteen studies , of which 8 were new studies , were included in the calculation of a summary estimate of effect for either or both end points -LRB- Table 1 -RRB- . Studies predating the formulation of the TDS hypothesis often were designed to explore the association of in utero exposure to a range of pharmaceuticals with birth malformations . Other than 2 recent studies for which pesticide exposure was determined by chemical analysis of specific compounds , assessment of exposure to pesticides is generally derived from the occupation of the mother and specific agents are not identified . Of the 12 studies identified for the association with testicular cancer , only 3 were excluded from the calculation of a summary estimate of effect -LRB- Table 2 -RRB- . Hypospadias The data from studies included in the meta-analysis for hypospadias are summarized in Table 3 . Three extreme values , two greater than and one lower than unity , can be identified visually from the forest plot of the RRs and their CIs -LRB- Figure 1 -RRB- . These extremes correspond to studies with larger SEs , and the shape given to the funnel plot -LRB- Figure 2 -RRB- by those smaller positive studies would be consistent with publication bias . These two extreme positive risk ratios were , however , reported after what is commonly referred to as `` third-generation exposure '' to DES , when the mother herself had been exposed to DES prenatally . It was recognized that the inclusion of such studies in the meta-analysis could have introduced heterogeneity , and the influence of this choice was investigated in the subset analysis . Plots of the quality score and RRs versus year of publication did not suggest any significant trends in quality of the studies or estimates of effect over time -LRB- not shown -RRB- . The pooled estimates of effect by both the Mantel -- Haenszel and DerSimonian -- Laird methods are very close to unity , and no relationship between in utero exposure to estrogenic agents and hypospadias could be detected -LRB- Table 4 -RRB- . None of the chi-square tests allowed the rejection of the null hypothesis of homogeneity between the studies at the 0.05 or 0.1 level of statistical significance . The subsets of studies in which exposure to DES and pharmaceutical estrogens were investigated yielded statistically significant risk ratios with both models , although the modest discrepancy between the fixed-effects and random-effects estimates suggests heterogeneity . Summary estimates for the latter subset were no longer significant when studies that included DES exposure were excluded . Although these results were based on four studies that all addressed in utero exposure to oral contraceptives , some heterogeneity between studies remained . Excluding the studies of third-generation exposure to DES , values for the summary estimate of effect were found to be 1.33 -LRB- 95 % CI , 0.63 -- 2.83 -RRB- by the Mantel -- Haenszel method and 1.31 -LRB- 95 % CI , 0.52 -- 3.26 -RRB- by the DerSimonian -- Laird method , a very modest and nonsignificant increase in risk . Excluding third-generation exposure from the DES subset yielded estimates of 2.02 -LRB- 95 % CI , 1.12 -- 3.65 -RRB- by the Mantel -- Haenszel method and 2.00 -LRB- 95 % CI , 0.97 -- 4.15 -RRB- by the DerSimonian -- Laird method , on the basis of two studies investigating exposure to any estrogenic drug during the first trimester of pregnancy . The difference between the results obtained by the two models for studies of third-generation exposure to DES was reduced only slightly by excluding the study by Klip et al. -LRB- 2002 -RRB- ; the Mantel -- Haenszel method yielded an estimate of 2.46 -LRB- 95 % CI , 0.91 -- 6.67 -RRB- and the DerSimonian -- Laird method of 2.18 -LRB- 95 % CI , 0.64 -- 7.46 -RRB- . The latter study 's cohort had been recruited in a fertility clinic , and whether results obtained with subfertile women are generalizable to all women exposed to DES in utero has been questioned -LRB- Hernandez-Diaz 2002 -RRB- . Although the equality of the results obtained by both methods for the environmental estrogens subset suggests those results are robust , the influence of the weight of the study by Vrijheid et al. -LRB- 2003 -RRB- can not be underestimated , as shown by the sensitivity analysis . Exclusion of this study from the analysis yielded a statistically significant Mantel -- Haenszel estimate but a lower and not statistically significant DerSimonian -- Laird estimate , revealing heterogeneity . A statistically significant estimate was obtained for prospective studies by the Mantel -- Haenszel method , but the wide difference with the estimate using the random effect model was suggestive of heterogeneity . Geographic subsets point to a higher risk in Latin America , although the pooled estimates for this location were based on only two studies and did not reach statistical significance . In addition to the results of the sensitivity analysis presented in Table 4 , a pooled estimate of effect was calculated when a stricter inclusion criterion was applied , namely , excluding results from the study by Monteleone-Neto et al. -LRB- 1981 -RRB- . This had little influence on the overall result , generating summary estimates of 0.97 -LRB- 95 % CI , 0.83 -- 1.13 -RRB- for the fixed effect model or 0.93 -LRB- 95 % CI , 0.80 -- 1.09 -RRB- for the random effect model . Cryptorchidism Data for the six studies included in the meta-analysis for cryptorchidism can be found in Table 5 . The results of only two studies significantly differ from unity , as illustrated by the forest plot -LRB- Figure 3 -RRB- . The small number of eligible studies renders analysis of the funnel plot and potential for publication bias difficult -LRB- Figure 4 -RRB- . The SEs do , however , illustrate well that the studies were all relatively small . No time trends for the estimate of effect or the quality of studies could be detected -LRB- not shown -RRB- . As presented in Table 6 , the pooled estimates of effect by both the Mantel -- Haenszel and DerSimonian -- Laird methods are marginally superior to unity , and their relative divergence implies there may be sources of heterogeneity . Chi-square tests did not , however , detect that any of the subsets were significantly heterogeneous . Excluding studies in which DES exposure was examined , either exclusively or along with hormonal therapeutics , yielded summary estimates consistent with no relationship . Statistical pooling of the studies including DES exposure generated a statistically significant estimate by the Mantel -- Haenszel method , suggesting a doubling of the risk of cryptorchidism after in utero exposure to DES . The same estimate by the DerSimonian -- Laird method did not , however , reach statistical significance and the difference relative to the fixed effect model is indicative of heterogeneity . The heterogeneity introduced by the DES subset of studies can again be observed by comparing the results obtained for all pharmaceutical estrogens with those obtained by pooling the two studies of accidental use of oral contraceptives during pregnancy . Study design also appeared to be a source of heterogeneity . If case -- control studies are prone to recall bias , this subset also included the study with the highest estimate , itself a source of heterogeneity , as shown by the sensitivity analysis . Excluding the study by Depue -LRB- 1988 -RRB- reduced the difference between estimates by both models , the Mantel -- Haenszel estimate then calculated as 1.29 -LRB- 95 % CI , 0.87 -- 1.91 -RRB- and that by the DerSimonian -- Laird method as 1.23 -LRB- 95 % CI , 0.81 -- 1.86 -RRB- . This was also observed for the American subset of studies . When the Depue -LRB- 1988 -RRB- study is omitted , the Mantel -- Haenszel method yielded a no longer statistically significant estimate of 1.34 -LRB- 95 % CI , 0.84 -- 2.14 -RRB- and the DerSimonian -- Laird method an estimate of 1.27 -LRB- 95 % CI , 0.72 -- 2.23 -RRB- . Applying a stricter exclusion criterion to studies examining hormonal treatment did not affect which studies were included in the meta-analysis of cryptorchidism . The study with the highest weight appears to lower the overall estimates , whereas increasing quality seems to reduce heterogeneity and lower the estimate of effect toward unity . These variations did not , however , influence the overall conclusion that aside from the DES studies subset , summary estimates did not detect any association between in utero exposure to estrogenic substances and cryptorchidism . Testicular cancer Nine studies were included in the meta-analysis of testicular cancer and the data used are summarized in Table 7 . Of these , 4 had not been included in the summary estimate previously calculated by Toppari et al. -LRB- 1996 -RRB- . The lack of homogeneity between studies is evident from the forest plot -LRB- Figure 5 -RRB- . Further , the funnel plot -LRB- Figure 6 -RRB- also illustrates the relatively small size of the included studies . Although a positive trend over time was found for the quality of the included studies -LRB- Figure 7 -RRB- , no significant time trend could be detected for the effect size -LRB- not shown -RRB- . Both the fixed and random effect models yield a statistically significant estimate ; however , the discrepancy between the two results is suggestive of heterogeneity despite the result from the chi-square test -LRB- Table 8 -RRB- . Conversely , the subset analysis was limited by the similarity of the question addressed by the studies included . Eight of the nine studies were interested in hormonal exposure and were conducted in the United States . Despite this , statistically significant heterogeneity between the studies was detected at the 0.1 level . Pooling the two studies examining DES exposure specifically produced a raised but statistically nonsignificant result . Despite the unexplained heterogeneity , all estimates that were calculated point to a doubling of the risk of developing testicular cancer after exposure to estrogenic agents in utero . The work on chlorinated biphenyls -LRB- PCBs -RRB- by Hardell et al. -LRB- 2004 -RRB- was the only study examining environmental estrogens . Its size was relatively small , and it did not detect such an effect . Applying a stricter exclusion criterion to studies examining hormonal treatment excluded four studies from the meta-analysis ; namely , Brown et al. -LRB- 1986 -RRB- , Gershman and Stolley -LRB- 1988 -RRB- , Henderson et al. -LRB- 1979 -RRB- , and Weir et al. -LRB- 2000 -RRB- . This resulted in a slightly lower Mantel -- Haenszel estimate of 1.98 -LRB- 95 % CI , 1.23 -- 3.18 -RRB- and if the DerSimonian -- Laird estimate remained equal to 1.59 , because of the wider confidence interval -LRB- 95 % CI , 0.93 -- 2.69 -RRB- , statistical significance was no longer achieved . The sensitivity analysis is consistent with some heterogeneity between the studies , the estimates obtained being relatively sensitive to the exclusion of particular studies varying above and below a risk estimate of 2 . The quality of the studies seemed to explain at least some of this heterogeneity . Discussion While it is clear that hypospadias , cryptorchidism , and testicular cancer are all positively associated with prenatal exposure to DES , this meta-analysis was unable to produce evidence that such effects were associated with environmental estrogens or even accidental use of oral contraceptives during pregnancy . This is consistent with the results obtained in earlier meta-analyses -LRB- Raman-Wilms et al. 1995 ; Toppari et al. 1996 -RRB- . The main limitations of meta-analysis are a -RRB- the susceptibility of its summary results to publication bias , b -RRB- the influence of the quality of studies , c -RRB- the possibility of including multiple results from the same study , and finally , d -RRB- heterogeneity between studies that could lead to invalid conclusions . The methodology employed in this present review attempts to address these issues . Additionally , the importance of carrying out and reporting a sensitivity analysis was illustrated by the case of hypospadias where the weight attributed to one particularly large study had a nonnegligible influence on the results . In this particular case , the study by Vrijheid et al. -LRB- 2003 -RRB- inferred exposure to phthalates from registry data about occupation , and although such an approach can allow the analysis of a great number of cases , assessment of exposure is much more likely to be prone to confounding . The number of studies included in meta-analyses lies typically between 5 and 15 , and the results presented here also fall within this range . The size of the homogeneity test statistic depends on both the number and size of individual studies . The funnel plots offer a good visual representation of the precision and size of individual studies , and it is clear that most studies published on the association between estrogenic agents and the probable end points of a TDS were found to be relatively small . The chi-square tests had , therefore , a relatively low power to detect heterogeneity . However , in the absence of statistical heterogeneity , the results of the fixed effect and random effect models should be virtually identical , and the comparison of results obtained by applying both the Mantel -- Haenszel and DerSimonian -- Laird models enabled the exploration of sources of heterogeneity despite this low statistical power . If the quality of the studies was found to explain some of the heterogeneity observed , particularly in the case of testicular cancer , the remaining heterogeneity could not be explained solely by the fact that environmental , and therefore generally much weaker , estrogens were included in the analysis . The systematic review of published literature yielded relatively few studies examining the association of male urogenital abnormalities or testicular cancer with environmental estrogens specifically ; a number of studies concerned with an association with broad categories of putative endocrine disruptor , most often pesticides , were excluded from the meta-analyses . This illustrates the difficulties associated with assessment of exposure , pesticide exposure often being inferred from parental occupation rather than direct measurement . Furthermore , there is increasing evidence that , in accordance with pharmacokinetic theory , the effects of xenobiotics acting via the same mechanism can be predicted fairly accurately by concentration addition -LRB- Zhu et al. 2006 -RRB- . Accurately accounting for combined exposure or adjusting for the confounding introduced by environmental exposures will probably require the development of mechanism-specific biomarkers of exposure . When DES is excluded , there is no conclusive evidence of an effect of pharmaceutical estrogens . Exposure to such estrogens is related mainly to the accidental use of oral contraceptives during pregnancy or hormonal pregnancy tests . Such estrogenic pharmaceuticals often are given in combination with progestagens , and it is legitimate to question whether unopposed estrogens would have the same effects as opposed estrogens . This also highlights another difficulty associated with exposure assessment , that of critically sensitive periods of development and the ascertainment of whether exposure took place during a `` window '' of susceptibility to hormone disruption . Nonetheless , studies in which maternal levels of hormones were measured in the first and third trimester of pregnancy do not support an association with elevated estrogen levels but rather indicate that a lower estrogen/androgen ratio and/or higher levels of α-fetoproteins may be beneficial -LRB- McGlynn et al. 2005 ; Zhang et al. 2005 -RRB- . If in animals both estrogenic and antiandrogenic compounds have been associated with end points consistent with those of human TDS -LRB- Fisher 2004 ; Veeramachaneni 2000 -RRB- , epidemiologic evidence remains elusive . Alternatively , the doubling of the risk estimates of all three effects associated with DES exposure would be consistent with a shared etiology and the TDS hypothesis . It does not constitute conclusive evidence of an estrogenic mode of action , however , as common etiologic factors could be related to the underlying condition for which DES was prescribed . Furthermore hypospadias , cryptorchidism , and testicular cancer have all been found to be associated with low birth weight , suggesting a potential association with an underlying placental defect . The understanding of the importance of endogenous estrogens in normal adult testicular function is becoming clearer . Their roles during fetal life , however , remain relatively unclear , but those mediated by the ER-αor ER-β have been shown to differ -LRB- Habert et al. 2006 -RRB- . Interestingly , DES has been found to have similar affinity for both receptors , whereas estradiol has only a slightly stronger affinity for ER-αcompared with ER-β -LRB- Mueller et al. 2004 -RRB- . ER-α has been detected in undifferentiated gonads as early as 10 days postconception in the mouse and found to be localized in the Leydig cells of fetal testis in rodents -LRB- Habert et al. 2006 -RRB- . Studies of the expression of ER-α and ER-β in human and nonhuman primates have so far yielded inconsistent results . Gaskell et al. -LRB- 2003 -RRB- reported that ER-α could not be detected in human fetal testes between weeks 12 -- 19 of gestation , whereas Shapiro et al. -LRB- 2005 -RRB- found that ER-α was apparent by week 12 , its levels peaked at 16 weeks before diminishing , and it was localized in Leydig cells . Current research focus has shifted to the role played by testosterone , anti-Müllerian hormone and insulin-like factor 3 produced by the fetal testes during masculinization . In the male rat , exposure to high levels of estrogens has been shown not only to suppress testosterone production but also to downregulate the expression of the androgen receptor protein in reproductive target tissues including the testes , Wolffian duct , and prostate -LRB- Sharpe 2006 -RRB- . Further research in this area may help shed light on possible mechanisms of injury or relevance of the rodent model . The subset analyses did not generate many clues to explain the heterogeneity of the collected data . This is , however , consistent with the wide geographic variability in the incidence of the conditions of interest -LRB- Boisen et al. 2004 ; Richiardi et al. 2004 -RRB- . Interactions between genetic susceptibility and the environment have been the focus of research in this area -LRB- Martin et al. 2007 -RRB- , and advances in genomics have allowed the identification of polymorphisms associated with hypospadias , cryptorchidism , and testicular cancer -LRB- Beleza-Meireles et al. 2006 ; Kurahashi et al. 2005 ; Starr et al. 2005 ; Yoshida et al. 2005 -RRB- . Such discoveries may , however , give rise to as many questions as they offer to answer . This is well illustrated by the recent identification of the association of a variant of the gene for the ER-α with hypospadias and cryptorchidism in Japanese cohorts -LRB- Watanabe et al. 2007 ; Yoshida et al. 2005 -RRB- that has now been found to be associated with a decreased incidence of hypospadias in a European cohort -LRB- Galan et al. 2007 -RRB- . Conclusion The modest increase in risk for all three end points associated with DES exposure is consistent with a shared etiology and the TDS hypothesis , whereas the results of the subset analyses suggest the existence of yet unidentified sources of heterogeneity between studies or within the study populations . Although 10 years of further research on the potential effects of endocrine disruptors on male reproductive health have provided some clues regarding the etiology and mechanism of conditions such as hypospadias , cryptorchidism , and testicular cancer , there is still no conclusive evidence of the role played by environmental estrogens .

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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O B O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B I O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O B I O O O O O O O O O O O O O O O

cryptorchidism
diethylstilbestrol
endocrine disruption
environment
estrogen
hypospadias
oral contraceptives
testicular cancer
testicular dysgenesis


Prediction of outcome in individuals with diabetic foot ulcers : focus on the differences between individuals with and without peripheral arterial disease . The EURODIALE Study Aims/hypothesis Outcome data on individuals with diabetic foot ulcers are scarce , especially in those with peripheral arterial disease -LRB- PAD -RRB- . We therefore examined the clinical characteristics that best predict poor outcome in a large population of diabetic foot ulcer patients and examined whether such predictors differ between patients with and without PAD . Introduction Diabetic foot ulcers are a common and much feared complication of diabetes , with recent studies suggesting that the lifetime risk of developing a foot ulcer in diabetic patients may be as high as 25 % -LSB- 1 -RSB- . Foot ulceration requires long and intensive treatment , has important effects on quality of life of both patients and care-givers -LSB- 2 -RSB- and is associated with major healthcare costs -LSB- 3 -- 5 -RSB- . Although in recent years much effort has been put into the development of international guidelines in order to stimulate the delivery of uniform and structured care -LSB- 6 -RSB- , prospective data on outcomes and predictors of outcome in patients with diabetic foot ulcers are limited . The population of diabetic patients who present with foot ulceration is heterogeneous : although most patients have peripheral polyneuropathy , there are several other characteristics that may vary among patients , such as the presence of peripheral arterial disease -LRB- PAD -RRB- , infection and co-morbidities . PAD is present in approximately one-half of all patients with foot ulcers -LSB- 7 -RSB- and is considered an important predictor of outcome -LSB- 8 , 9 -RSB- . Therefore , outcome data on this important subgroup of patients with diabetic foot disease are needed . Such a requirement is underlined by the fact that although diabetic foot ulcers are usually reported and analysed as one clinical entity , marked differences in patient , foot and ulcer characteristics can exist between patients with and without PAD -LSB- 7 -RSB- . These observations raise the question of whether predictors of outcome in patients with and without PAD may differ . The aim of the present study was therefore : -LRB- 1 -RRB- to obtain prospective data on outcome of individuals presenting with a new diabetic foot ulcer , including patients both with and without PAD ; -LRB- 2 -RRB- to assess clinical characteristics that best predict poor outcome -LRB- i.e. non-healing of the foot ulcer -RRB- from this large set of patients ; and -LRB- 3 -RRB- to examine whether such predictors differ between patients with and without PAD . Methods Study design and population The EURODIALE consortium is an international collaborative network that was created to stimulate further research in the field of diabetic foot disease . Its main objective was to assess outcome and the major predictors of clinical outcome in a large sample of European patients with diabetic foot ulcers . The design and rationale of this study have been described in detail elsewhere -LSB- 10 -RSB- . Briefly , between 1 September 2003 and 1 October 2004 , 1,232 patients with a new foot ulcer were included in 14 diabetic foot centres in ten European countries . The mean -LRB- range -RRB- number of included patients per centre was 88 -LRB- 40 -- 125 -RRB- . All participating centres have a longstanding expertise in the field of diabetic foot disease . Patients included were those presenting for the first time with a new foot ulcer within a period of 12 months , either at the outpatient or inpatient clinics of participating centres . Excluded patients were those who had been treated at the participating centres for an ulcer on the ipsilateral foot during the previous 12 months and those with a life expectancy of less than 1 year . Participants attended follow-up visits on a monthly basis . At baseline and during all follow-up visits , data were collected and recorded on standardised case record forms . This was done by dedicated investigators in each centre who were trained during plenary meetings and on-site visits . Recorded data included demographics , data on co-morbidities and foot and ulcer characteristics , as well as management . The local ethics committees of the 14 hospitals approved the study protocol and all patients gave written informed consent . Management of diabetic foot ulcer All patients were treated according to protocols based on the International Consensus on the Diabetic Foot -LSB- 11 -RSB- , which include off-loading , diagnosis and treatment of infection , assessment of vascular status , treatment of PAD and regular wound debridement . Potential predictive factors Potential determinants of healing were chosen on the basis of -LRB- 1 -RRB- current literature ; -LRB- 2 -RRB- expert opinion after extensive discussions during EURODIALE meetings ; and -LRB- 3 -RRB- suitability for use in daily clinical practice . In addition to sex , age at baseline and duration of diabetes , several disease-specific characteristics and co-morbidities were investigated -LSB- 10 -RSB- . Ulcer characteristics All patients underwent a standardised examination according to the PEDIS system . This was developed by the International Consensus on the Diabetic Foot to enable classification of patients for clinical research purposes -LSB- 11 , 12 -RSB- and classifies foot ulcers according to five categories : perfusion , extent , depth , infection and sensation.Perfusion assessment included evaluation of the presence of pedal pulses and measurement of the ankle -- brachial pressure index -LRB- ABPI -RRB- using a handheld Doppler device ; PAD was considered to be present if ABPI was < 0.9 and/or two foot pulses were absent.Extent -LRB- i.e. size -RRB- was determined by multiplying the largest by the second largest diameter perpendicular to the first and divided into three categories : < 1 cm2 , 1 -- 5 cm2 and > 5 cm2.Depth was described as either deep or superficial if a full thickness lesion of the skin was or was not extending through the subcutis , respectively.Infection was diagnosed if two or more of the following signs were present : frank purulence , local warmth , erythema , lymphangitis , oedema , pain , fever and foul smell . The term infection covers both soft tissue infection and bone infection.Evaluation of sensation -LRB- peripheral neuropathy -LSB- PNP -RSB- -RRB- included pressure sensation -LRB- 10 g monofilament on plantar aspect of hallux , metatarsophalangeal joints 1 and 5 -RRB- , tactile sensation -LRB- cotton wisp on dorsum of foot -RRB- , vibration sensation -LRB- 128 Hz tuning fork on dorsum of the hallux -RRB- and blunt/sharp discrimination -LRB- dorsum of foot -RRB- . PNP was diagnosed if the results of two or more of the aforementioned tests were abnormal.In addition , the location of the ulcer was divided into plantar -LRB- on the plantar toes , plantar mid - or forefoot and plantar hind foot -RRB- and non-plantar -LRB- on the dorsal or interdigital part of the toes , on the dorsal or lateral aspect of the foot and heel ulcers -RRB- . Ulcer duration was divided into three categories : < 1 week , between 1 week and 3 months , and > 3 months . Co-morbidities The following disabling co-morbidities were assessed : presence of severe visual impairment -LRB- defined as the inability to read a newspaper after correction -RRB- , end-stage renal disease -LRB- ESRD -RRB- -LRB- defined as dependency on haemodialysis or peritoneal dialysis or a previous renal transplant procedure -RRB- , heart failure -LRB- New York Heart Association -LSB- NYHA -RSB- classification III or IV -RRB- , any neurological disorder -LRB- excluding diabetic polyneuropathy -RRB- resulting in loss of motor or sensory function -LRB- e.g. stroke -RRB- and inability to stand or walk without help . Study main outcome Main outcome was complete healing -LRB- with or without minor amputation -RRB- of the foot , within the maximum follow-up period of 1 year . Healing was defined as healing -LRB- intact skin -RRB- of the whole foot at two consecutive visits . If more than one ulcer was present , the foot was defined as healed once all ulcers were healed . Outcome information was not obtained in 144 patients -LRB- 11.7 % of the patients included -RRB- who dropped out of the study and were therefore excluded from the analyses . Reasons for dropout were non-compliance -LRB- n = 24 -RRB- , inability to follow the patient -LRB- lack of transportation , no social support , too sick to attend ; n = 25 -RRB- or if care had been taken over by other specialists -LRB- n = 29 -RRB- ; in 66 patients the reason for dropout could not be discovered . At baseline these participants were slightly older and had a higher incidence of heart failure , deeper ulcers and ulcers of longer duration than those included in the analyses -LRB- n = 1,088 ; Table 1 -RRB- . Table 1Baseline characteristics of participants included and those excluded -LRB- dropouts -RRB- from the present studyVariableIncluded -LRB- n = 1,088 -RRB- Dropouts -LRB- n = 144 -RRB- p valueAge -LRB- years -RRB- 64.7 ± 12.568.0 ± 11.60.003 Male sex , n -LRB- % -RRB- a703 -LRB- 64.6 -RRB- 85 -LRB- 59.0 -RRB- 0.189 Duration of diabetes , n -LRB- % -RRB- a0 .418 < 5 years148 -LRB- 14.1 -RRB- 19 -LRB- 13.5 -RRB- 5 -- 10 years169 -LRB- 16.1 -RRB- 17 -LRB- 12.1 -RRB- > 10 years731 -LRB- 69.8 -RRB- 105 -LRB- 74.5 -RRB- Deep ulcer , n -LRB- % -RRB- a476 -LRB- 43.8 -RRB- 80 -LRB- 55.6 -RRB- 0.007 Size of ulcer , n -LRB- % -RRB- a0 .843 < 1 cm2403 -LRB- 37.2 -RRB- 50 -LRB- 35.0 -RRB- 1 -- 5 cm2563 -LRB- 52.0 -RRB- 76 -LRB- 53.1 -RRB- > 5 cm2117 -LRB- 10.8 -RRB- 17 -LRB- 11.9 -RRB- Duration of ulcer , n -LRB- % -RRB- a < 0.001 < 1 week184 -LRB- 17.0 -RRB- 10 -LRB- 7.0 -RRB- 1 week -- 3 months627 -LRB- 58.1 -RRB- 68 -LRB- 47.6 -RRB- > 3 months269 -LRB- 24.9 -RRB- 65 -LRB- 45.5 -RRB- Plantar location , n -LRB- % -RRB- a493 -LRB- 48.2 -RRB- 62 -LRB- 46.3 -RRB- 0.675 Pretibial oedema , n -LRB- % -RRB- a197 -LRB- 18.2 -RRB- 29 -LRB- 20.3 -RRB- 0.538 Heart failure NYHA III -- IV , n -LRB- % -RRB- a117 -LRB- 10.9 -RRB- 23 -LRB- 16.1 -RRB- 0.065 Neurological disorder , n -LRB- % -RRB- a70 -LRB- 6.5 -RRB- 9 -LRB- 6.3 -RRB- 0.918 Inability to stand or walk without help , n -LRB- % -RRB- a107 -LRB- 9.9 -RRB- 15 -LRB- 10.4 -RRB- 0.843 Visual impairment , n -LRB- % -RRB- a164 -LRB- 15.3 -RRB- 19 -LRB- 13.2 -RRB- 0.507 ESRD , n -LRB- % -RRB- a63 -LRB- 5.8 -RRB- 7 -LRB- 4.9 -RRB- 0.639 Polyneuropathy , n -LRB- % -RRB- a826 -LRB- 78.5 -RRB- 105 -LRB- 76.1 -RRB- 0.515 Infection , n -LRB- % -RRB- a591 -LRB- 57.2 -RRB- 82 -LRB- 61.2 -RRB- 0.380 PAD , n -LRB- % -RRB- a505 -LRB- 47.5 -RRB- 78 -LRB- 56.1 -RRB- 0.056 Unless otherwise stated , data are mean values ± SD.aPercentages may not sum to 100 due to missing information Statistical analyses All statistical analyses were carried using the STATA software package version 9.2 -LRB- STATA , College Station , TX , USA -RRB- . Comparisons between groups ' characteristics were made with χ2 tests -LRB- frequency data -RRB- or Student 's t test -LRB- continuous data -RRB- . Multiple imputation of missing values of predictor variables Values for one -LRB- n = 188 -RRB- , two -LRB- n = 35 -RRB- or three -LRB- n = 13 -RRB- predictor variables were not available for 236 participants ; the number of missing values per predictor ranged from 0 to 6 % . In order to decrease bias and increase power of the analyses -LSB- 13 -RSB- , we used multiple imputation chained equations -LRB- procedure ` ICE ' in STATA -RRB- to impute those missing values -LRB- 1.7 % of all required values -RRB- rather than performing complete case analyses -LSB- 14 , 15 -RSB- . With ICE the imputation model of a single variable uses all the other variables as predictors by appropriate regression models -LRB- i.e. linear , logistic or multinomial if imputed variable is continuous , dichotomous or categorical -RRB- . We generated five imputed datasets that were used to fit the regression models of interest -LRB- in each dataset and in the final , i.e. the combined dataset -RRB- . Parameter estimates and standard errors were combined across the five replicates according to the procedure described by Rubin -LSB- 16 -RSB- and Carlin et al. -LSB- 17 -RSB- -LRB- procedure ` micombine ' in STATA -RRB- . Development of predictive models First , univariable logistic regression analyses were performed for all potential predictor variables with the outcome of interest -LRB- non-healing -RRB- , with values presented as univariable odds ratios -LRB- ORs -RRB- along with the respective 95 % CI . Second , all potential predictors were entered simultaneously in a multivariable logistic regression model that was reduced to a most parsimonious model using a backward selection method based on Akaike 's Information Criterion . These models yielded a set of variables that best predict -LRB- and can be regarded as independent predictors of -RRB- outcome . Results Clinical outcome Within the 1 year follow-up , 77 % of the 1,088 patients healed , 12 % were still undergoing treatment , 5 % underwent a major -LRB- i.e. above the ankle level -RRB- amputation and 6 % died -LRB- before healing of the foot ulcer -RRB- . Among the patients who healed , 17 % underwent a minor amputation ; this rate was similar to that in those patients who did not heal -LRB- 20 % , p = 0.425 -RRB- . When stratifying patients according to the presence or absence of PAD , significantly -LRB- p < 0.001 -RRB- worse healing rates were observed in patients with than in those without PAD -LRB- 69 % vs 84 % , respectively -RRB- . Major amputation and mortality rates were also higher in patients with -LRB- 8 % and 9 % , respectively -RRB- than in patients without PAD -LRB- 2 % and 3 % respectively ; p < 0.001 -RRB- . Baseline characteristics of patients with PAD compared with those without PAD are provided in Table 2 . Table 2Patients ' baseline characteristics according to their PAD statusVariablePatients with PAD -LRB- n = 505 -RRB- Patients without PAD -LRB- n = 558 -RRB- p valueAge -LRB- years -RRB- 69.1 ± 11.260.5 ± 12.3 < 0.001 Male sex , n -LRB- % -RRB- a321 -LRB- 65.6 -RRB- 366 -LRB- 63.6 -RRB- 0.490 Duration of diabetes , n -LRB- % -RRB- a0 .265 < 5 years63 -LRB- 12.9 -RRB- 80 -LRB- 14.9 -RRB- 5 -- 10 years72 -LRB- 14.7 -RRB- 93 -LRB- 17.4 -RRB- > 10 years354 -LRB- 72.4 -RRB- 363 -LRB- 67.7 -RRB- Deep ulcer , n -LRB- % -RRB- a266 -LRB- 52.7 -RRB- 200 -LRB- 35.8 -RRB- < 0.001 Size of ulcer , n -LRB- % -RRB- a0 .002 < 1 cm2173 -LRB- 34.4 -RRB- 219 -LRB- 39.5 -RRB- 1 -- 5 cm2259 -LRB- 51.5 -RRB- 294 -LRB- 53.0 -RRB- > 5 cm271 -LRB- 14.2 -RRB- 42 -LRB- 7.5 -RRB- Duration of ulcer , n -LRB- % -RRB- a < 0.001 < 1 week58 -LRB- 11.5 -RRB- 120 -LRB- 21.7 -RRB- 1 week -- 3 months296 -LRB- 58.0 -RRB- 318 -LRB- 57.5 -RRB- > 3 months148 -LRB- 29.5 -RRB- 115 -LRB- 20.8 -RRB- Plantar location , n -LRB- % -RRB- a197 -LRB- 40.9 -RRB- 284 -LRB- 55.0 -RRB- < 0.001 Pretibial oedema , n -LRB- % -RRB- a111 -LRB- 22.0 -RRB- 83 -LRB- 14.9 -RRB- 0.002 Heart failure NYHA III -- IV , n -LRB- % -RRB- a64 -LRB- 12.7 -RRB- 47 -LRB- 8.5 -RRB- 0.027 Neurological disorder , n -LRB- % -RRB- a40 -LRB- 8.0 -RRB- 27 -LRB- 4.9 -RRB- 0.039 Inability to stand or walk without help , n -LRB- % -RRB- a65 -LRB- 12.9 -RRB- 36 -LRB- 6.5 -RRB- < 0.001 Visual impairment , n -LRB- % -RRB- a89 -LRB- 17.9 -RRB- 66 -LRB- 12.0 -RRB- 0.007 ESRD , n -LRB- % -RRB- a35 -LRB- 7.0 -RRB- 25 -LRB- 4.5 -RRB- 0.082 Polyneuropathy , n -LRB- % -RRB- a383 -LRB- 77.2 -RRB- 424 -LRB- 79.3 -RRB- 0.429 Infection , n -LRB- % -RRB- a293 -LRB- 60.9 -RRB- 282 -LRB- 53.4 -RRB- 0.016 Unless otherwise stated , data are mean values ± SDaPercentages may not sum to 100 due to missing information Predictors of healing Table 3 shows the univariable associations of the potential predictors of non-healing in the overall population and Table 4 presents the variables retained in the predictive models after backward selection in the combined imputed datasets . The estimates were similar to those obtained in the complete cases dataset -LRB- n = 854 -RRB- indicating that missing values were non-selective -LRB- data not shown -RRB- . These include the following eight characteristics , all of which predict lower probabilities of healing : older age , male sex , larger ulcer size , heart failure , inability to stand or walk without help , ESRD , PNP and PAD . These variables were consistently identified in all five imputed datasets . Table 3Association of each potential predictor with non-healing in the overall population -LRB- n = 1,088 -RRB- Predictor variablesOutcome : healingOR95 % CIp valueAge , per 10 year increase1 .321.17 -- 1.49 < 0.001 Sex , men vs women1 .501.07 -- 1.970.018 Duration of diabetes0 .712 5 -- 10 vs < 5 yearsa0 .960.56 -- 1.65 > 10 vs < 5 yearsa1 .050.69 -- 1.60 Depth of ulcer , deep vs superficial1 .661.25 -- 2.20 < 0.001 Size of ulcer < 0.001 1 -- 5 vs < 1 cm2a2 .251.60 -- 3.17 > 5 vs < 1 cm2a4 .222.64 -- 6.72 Duration of ulcer < 0.001 1 week to 3 months vs < 1 weeka1 .811.15 -- 2.85 > 3 months vs < 1 weeka2 .611.60 -- 4.27 Location , plantar vs non-plantar0.730 .55 -- 0.980.035 Pretibial oedema , yes vs no1 .791.27 -- 2.510.001 Heart failure -LRB- NYHA III -- IV -RRB- , yes vs no2 .031.35 -- 3.050.001 Neurological disorder , yes vs no1 .440.85 -- 2.460.176 Inability to stand or walk without help , yes vs no2 .501.62 -- 3.79 < 0.001 Visual impairment , yes vs no1 .360.94 -- 1.980.105 ESRD , yes vs no2 .201.30 -- 3.730.004 Polyneuropathy , yes vs no1 .410.98 -- 2.040.065 Infection , yes vs no1 .471.09 -- 2.000.012 PAD , yes vs no2 .311.72 -- 3.10 < 0.001 aReference categoryTable 4Multivariable models with independent predictors of non-healing in the whole study population and in patients with and without PADVariableAll patientsPatients with PADPatients without PADOR95 % CIp valueOR95 % CIp valueOR95 % CIp valueAge , per 10 year increase1 .281.11 -- 1.470.0011.420.17 -- 1.73 < 0.0011.550.91 -- 2.630.105 Sex , men vs women1 .721.23 -- 2.400.0021.971.25 -- 3.110.003 -- -- -- Size of ulcer < 0.001 < 0.0010.008 1 -- 5 vs < 1 cm2a2 .261.58 -- 3.223.221.95 -- 5.321.250.74 -- 2.12 > 5 vs < 1 cm2a3 .882.37 -- 6.343.841.97 -- 7.483.481.62 -- 7.46 Duration of ulcer -- -- 0.086 1 week to 3 months vs < 1 weeka -- -- -- -- 2.141.05 -- 4.36 > 3 months vs < 1 weeka -- -- -- -- 2.180.98 -- 4.84 Heart failure -LRB- NYHA III -- IV -RRB- , yes vs no1 .550.99 -- 2.430.0541.540.87 -- 2.740.141 -- -- -- Inability to stand or walk without help , yes vs no2 .001.27 -- 3.140.0032.361.34 -- 4.170.0031.910.86 -- 4.240.112 ESRD , yes vs no2 .511.41 -- 4.480.0023.041.38 -- 6.700.0062.000.76 -- 5.250.161 Polyneuropathy , yes vs no1 .420.96 -- 2.080.078 -- -- -- 1.700.89 -- 3.250.108 Infection , yes vs no -- -- -- 1.631.03 -- 2.580.036 -- -- -- PAD , yes vs no1 .711.23 -- 2.370.001 N/AN/AaReference categoryN/A , not applicable Since we hypothesised that , from an aetiological point of view , predictors of non-healing would differ between patients with and those without PAD , predictive models were also fitted for these two groups separately -LRB- Table 4 -RRB- . In patients with PAD almost all of the predictors identified in the whole study population , with the exception of PNP , were again found to be independent predictors of healing . In addition , the presence of infection emerged as an additional independent predictor of non-healing . In patients without PAD , older age , larger ulcer size , inability to stand or walk without help , ESRD , PNP and , in addition , longer ulcer duration were independent predictors of poorer healing . The observed interaction between infection and PAD status partly supports the classification of foot ulcer disease into four stages as suggested by Armstrong et al. -LRB- University of Texas classification system -RRB- -LSB- 9 -RSB- . Accordingly , upon analysis of the odds of non-healing per PAD × infection status , it was only in those patients with both PAD and infection that the odds of non-healing were markedly increased compared with those without PAD or infection : OR 2.82 , CI 1.88 -- 4.22 , p < 0.001 in unadjusted analyses -LRB- Fig. 1 -RRB- vs OR 1.87 , CI 1.20 -- 2.91 , p < 0.001 after adjustments for the other variables included in the predictive model . Fig. 1ORs of healing per PAD and infection -LRB- Inf -RRB- status Discussion The EURODIALE study is one of the few large prospective , international studies on outcome and determinants of outcome in diabetic foot disease . Despite the severity of the underlying disease and the important co-morbidity -LSB- 7 -RSB- , clinical outcome of this population within a 1 year follow-up can be considered favourable . In our cohort , 77 % of the patients healed -LRB- with or without a minor amputation -RRB- , 5 % underwent a major amputation and 6 % died . However , healing rates in patients with PAD were considerably worse . In addition , predictors of healing also differed between the groups with and without PAD . The presence of infection , which is generally regarded as an important predictor of healing , was only predictive in individuals with PAD . With regard to the overall outcome in our cohort , two recent studies found relatively comparable outcomes . Jeffcoate et al. -LSB- 18 -RSB- reported healing -LRB- excluding minor amputations -RRB- rates of 66 % and an amputation rate of 5 % with a similar prevalence of PAD . In a German cohort , Beckert et al. -LSB- 19 -RSB- found healing rates between 57 % and 93 % and major amputation rates of 3 % , although the data as presented in that report can not be easily compared because of their unique classification system . Oyibo et al. -LSB- 20 -RSB- also found similar rates of major amputation in their cohort -LRB- 5 % -RRB- . Our study shows that the combination of PAD and infection has a major impact on healing rates -LRB- Fig. 1 -RRB- ; this significant interaction between PAD and infection is , in our opinion , one of the major findings of this study . In the patients without PAD we did not observe an association between infection and non-healing , which suggests that in these patients current antibiotic regimens and surgical techniques seem adequate to save a limb with adequate perfusion . However , within the total population of individuals with diabetic foot disease in developed countries , the group of infected and ischaemic ulcers accounted for almost one-third of all patients in our earlier report -LSB- 7 -RSB- . In a recent study , a significant relation between PAD , infection and poor outcome was also observed : in that study 's large cohort of outpatients with type 2 diabetes , PAD was an independent predictor of infection-related mortality -LSB- 21 -RSB- . Unfortunately , there is very little insight into the pathophysiology and treatment of infection in individuals with PAD . Currently it is not clear why infection is more prevalent and more difficult to treat in individuals with PAD . Remarkably , very few patients with PAD were included in most of the randomised trials on antibiotic therapy in diabetic foot infections -LSB- 22 , 23 -RSB- . It has previously been demonstrated that lower limb tissue levels of antibiotics can be markedly decreased as a result of impaired perfusion in PAD -LSB- 24 -RSB- . It is open to speculation whether aggressive revascularisation will improve control of infection in these patients . Although some earlier studies have examined the impact of co-morbidities on ulcer healing -LSB- 25 -RSB- , no studies , to our knowledge , have systematically assessed in a multivariable analysis the effects on ulcer healing of patient characteristics including co-morbidities , as well as foot and ulcer characteristics at baseline . In our study , older age , ulcer size and several co-morbidities were independent predictors of non-healing in patients with and without PAD . Recently a number of larger studies reported data on determinants of outcome in diabetic foot disease such as the single-centre study by Beckert in Germany and the UK multi-centre study initiated by Jeffcoate -LSB- 19 , 26 -RSB- . The former focused on wound-based characteristics and also found that the presence of PAD -LRB- defined as absence of pedal pulses -RRB- was an independent predictor of outcome -LRB- healing -RRB- , while infection was not . In the recent UK multi-centre study , ulcer area was a strong predictor of outcome , as was the presence of PAD ; co-morbidities were not taken into account in the regression analyses . Surprisingly , depth of the ulcer was not associated with outcome in our multivariable model , a finding also shown by Ince et al. -LSB- 26 -RSB- . In a retrospective study , Miyajima et al. -LSB- 27 -RSB- reported on patient characteristics that determined major lower extremity amputation and found that haemodialysis was an independent predictor of major amputation ; in this study wound characteristics were not part of the regression analyses . The poor prognosis of foot ulcers for individuals in our study with ESRD is in line with earlier reports , in which amputation rates of 57 % in individuals on haemodialysis were observed -LSB- 25 -RSB- . Although in our study ESRD was a predictor of non-healing in patients with and without PAD , it seemed to have a particularly negative effect in the latter patient group . PAD is frequently diagnosed and associated with adverse outcomes in haemodialysis patients -LSB- 28 -RSB- . PAD in ESRD patients is more severe and is accompanied by diffuse vascular calcifications , involvement of both distal infrapopliteal and foot arteries , and by impaired microcirculatory perfusion -LSB- 29 -- 31 -RSB- . The severity of PAD in ESRD may explain the importance of ESRD in our healing models ; additional mechanisms are probably impaired host defences in chronic renal failure and uraemia , or the presence of more resistant micro-organisms -LSB- 32 , 33 -RSB- . The outcome of patients without PAD in our study was relatively favourable : 84 % of the patients healed with or without minor amputation , 2 % underwent a major amputation and 3 % died . In our multivariate models , loss of sensation was associated with a poorer outcome in these patients , suggesting that loss of protective sensation is not only a key factor in the development of an ulcer , but also affects its outcome . This may be related to the preserved mechanism of off-loading the ulcer in individuals with intact protective sensation . However , neuropathy may also have direct effects on wound healing . Although data on the effect of neuropathy on wound healing in humans are scarce , some animal studies suggest that denervation may contribute to impaired wound healing in diabetes -LSB- 34 , 35 -RSB- . A large dataset on individuals with neuropathic ulcers comes from retrospective database analyses in which healing rates of 47 % were observed -LSB- 36 -RSB- . Although this study reported on healing at 20 weeks -LRB- whereas the current one examined healing rates at 1 year -RRB- , the different results compared with our study are striking and may be related to an increased awareness of the importance of adequate off-loading , as a result of publication of international guidelines and reports on casting techniques -LSB- 37 -- 39 -RSB- . There are several limitations to our study . Individuals who were lost to follow-up were excluded from the analyses as healing status could not be obtained ; these individuals were slightly older and had a greater incidence of heart failure , deep ulcers or ulcers with a longer duration and PAD at baseline . In addition , we excluded patients who had had a previous ulcer within 12 months prior to presentation -LRB- i.e. we probably excluded patients with recurrent ulcers -RRB- . Also we excluded patients with a life expectancy shorter than 1 year because of anticipated problems with follow-up . The estimates obtained in our models may therefore have underestimated the probability of non-healing in patients with a recurrent foot ulcer , although in one earlier study healing rate of neuropathic foot ulcers did not decrease in patients with multiple recurrences -LSB- 40 -RSB- . Since our study was embedded in daily clinical practice , limitations had to be set with regard to the number and type of data collected . It was therefore not possible to record more characteristics of these patients such as medication and extensive documentation of all complications . Moreover , to facilitate data collection , some continuous data -LRB- e.g. ulcer size -RRB- had to be transformed into a limited set of categories . Nevertheless , the set of potential predictors used in the present study do cover relevant patient and disease-specific aspects that can be easily assessed and used for patient risk estimation in clinical practice . Finally , our predictive model is based on outcomes that can be obtained in developed countries with access to the necessary resources such as antibiotic treatment and revascularisation ; our results , therefore , are most relevant for diabetic foot ulcer patients in developed countries . In conclusion , the results of this study have several implications . Both ulcer characteristics and several patient-related characteristics affected the outcome of diabetic foot ulcers . Therefore , a holistic approach by healthcare professionals who are familiar with the treatment of complicated diabetic patients is essential in order to identify the high-risk patient and start appropriate treatment . We found that patients with and without PAD differ in clinical characteristics , outcome and predictors of outcome . Taking into account these findings and the different pathophysiology and treatment of PAD and non-PAD ulcers , we feel that that diabetic foot ulcer with and without PAD should be defined as two separate disease states . The prevalent combination of PAD and infection is a unique entity ; an important challenge lies in the development of evidence-based strategies to improve the poor outcome of these patients . Both studies comparing different antibiotic regimens in PAD , and studies evaluating the effects of early revascularisation on control of infection are urgently needed .

O O B O O O O O O O O O O O O O O O O B I I O O O O O B O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O B O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B I O O O O O O O O O O O O O O O O O O O O B O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O

diabetes
foot ulcer
infection
outcome
peripheral arterial disease
predictive model