Wei Anyang, Department of Urology, Medical Center for Overseas Patients, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong 510515, China. Email: firstname.lastname@example.org; He Shuhua, Department of Urology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong 510515, China. Email: email@example.com
Erectile dysfunction (ED) is increasingly recognized as a public health problem. Several studies have reported the influence of the insertion/deletion (I/D) polymorphism in the Angiotensin-converting enzyme (ACE) gene on ED susceptibility, but the results remain controversial. To derive a more precise estimation of the relationship, a meta-analysis was conducted using data published previously by other groups. A total of six case-control studies, including 1039 cases and 927 controls, were selected. The pooled odds ratios (ORs) and respective 95% confidence intervals (CIs) were calculated by comparing the carriers of D-allele with the wild homozygotes (ID + DD vs. II). Comparisons of other genetic models were also performed (ID + II vs. DD, DD vs. II, DI vs. II and D vs. I). In the overall analysis, no significant association between the polymorphism and ED risk was observed (OR=1.07, 95% CI = 0.84 − 1.37, p = 0.575 for ID + DD vs. II). In the subgroup analysis by ethnic, no significant association was detected among Asian, Latino and European for the comparison of ID + DD vs. II (Asian: OR=1.27, 95% CI = 0.89 − 1.81; Latino: OR=0.76, 95% CI = 0.46 − 1.27; European: OR=1.06, 95% CI = 0.67 − 1.66). Results from other comparative genetic models also indicated the lack of associations between this polymorphism and ED risk. In conclusion, this meta-analysis indicates that the ACE I/D polymorphism might not contribute to the risk of ED.
Erectile dysfunction (ED), defined as the inability to achieve, or maintain, a sufficient erection for sexual intercourse (Melman & Gingell, 1999), is increasingly recognized as a public health problem. Recent studies reported an overall prevalence of ED up to 44% of men in a population-based sample (Smith et al., 2009) and about half of all men aged 40–70 years suffer some degree of ED (Feldman et al., 1994; Teles et al., 2008). The risk for the development of ED increases owing to a number of factors such as advanced age, smoking, diabetes mellitus, hormonal dysregulation, psychogenic factors, heart disease, hypertension, hypercholesterolaemia, vascular insufficiency, neurological dysfunctions caused by surgery and side effects of drug and radiation therapy (Sullivan et al., 2001; Lue et al., 2004; Burnett, 2006). Indeed, ED or sexual complications are common in men with cardiovascular disease (Jackson, 2009) and both diseases have overlapping risk factors, aetiology and clinical outcomes (Kloner, 2008). The shared aetiological factor is thought to be endothelial dysfunction, with ED serving as a marker preceding the onset of symptoms of other vascular diseases (Kloner, 2008; Salem et al., 2009). Angiotensin-converting enzyme (ACE), a key enzyme in the renin-angiotensin system (RAS), plays the important roles of regulating circulatory homeostasis. It converts the biologically inactive angiotensin I to the active angiotensin II, a potent vasopressor, in addition to degrading the vasodilator bradykinin (Erdos & Skidgel, 1987). Recent evidence has pointed out that angiotensin II might play a role in the initiation of detumescence after erection (Kifor et al., 1997; Becker et al., 2001a,b). Thus, the gene encoding ACE has been proposed as a potential candidate influencing the predisposition to both cardiovascular disease and ED (Eisenhardt et al., 2003; Tsai et al., 2007; Mollsten et al., 2008).
The human ACE gene is located on chromosome 17q23 consists of 26 exons and 25 introns, and many polymorphisms have been identified (Kitsios & Zintzaras, 2009). The most widely studied polymorphism Insertion/Deletion (I/D) is located on intron 16. (Haiman et al., 2003). It is characterized by the presence (insertion, I) or absence (deletion, D) of a 287-bp Alu repetitive sequence, which results in three genotypes: II, ID and DD (Haiman et al., 2003). The I/D polymorphism accounts for 20–50% of the variance in ACE expression or activity in blood and tissues among individuals (Rocken et al., 2005). Interestingly, there is an association between the genotype of the ACE I/D polymorphism and serum ACE level: individuals with homozygote DD have the hightest, individuals with heterozygote ID are an intermediate and individuals with homozygote II have the lowest serum ACE levels (Rigat et al., 1990). Since this publication, a number of studies have been performed in an attempt to characterize the influence of the ACE genotypes in various diseases (for review, see ref. Sayed-Tabatabaei et al., 2006), and, although a significant association between the DD genotype of the ACE gene polymorphism and ED in a sample of Koreans was observed (Park et al., 1999), results of association studies regarding the relationship between ACE I/D polymorphism and ED risk have remained inconsistent. More specifically, Mazo et al. reported that in patients with metabolic syndrome, the prevalence of the DD genotype was found to be significantly higher compared with the non-ED group, reinforcing the idea that the I/D polymorphism may indeed play an important role in the development of ED (Mazo et al., 2008). More recently, However, Andersen and colleagues did not detect a significant association between the ACE gene polymorphism and ED risk in Brazilian population (Andersen et al., 2010). This is in agreement with two other studies demonstrating no association between the gene I/D polymorphism and ED in a Mexican (Rosas-Vargas et al., 2004) and German population (Eisenhardt et al., 2010).
Data from many small and individually underpowered association studies produced inconclusive results, therefore, our goal was to evaluate the effect of the I/D polymorphism in ACE on ED risk and to provide more information on these controversial results by conducting a meta-analysis of all eligible case-control studies.
Materials and methods
To identify relevant studies eligible for the meta-analysis, we searched Medline, Embase, the Cochrane Library, Chinese Biomedical (CBM) and Chinese National Knowledge Infrastructure (CNKI) databases bases up to 1st July 2012, using the search terms'(ACE) and (erectile dysfunction or ED or male sexual dysfunction or impotence) and polymorphism'. The languages were limited to English and Chinese. The potentially relevant articles were read in their entirety to assess their appropriateness for inclusion in the analysis. All references cited in the articles were also scanned to identify possible additional relevant studies. Studies included in our meta-analysis had to meet all of the following criteria: (i) the study should evaluate the I/D polymorphism in ACE gene and ED risk; (ii) in a case-control design; (iii) the distribution of genotypes in the control groups should be consistent with Hardy–Weinberg equilibrium (HWE); and (iv) provided enough information to calculate the odds ratio (OR) with 95% confidence interval (CI). Abstracts,reviews and case reports were excluded. For studies with the same case series by the same authors, the most recently published studies or studies with the largest number of subjects were included.
Two of the investigators (Tao Zhang and Weili Li) checked and extracted the data from each article independently of each other to increase objectivity. A consensus meeting was then held to resolve any discrepancies. The following information was recorded from each study: first author's last name, year of publication, country of origin, ethnicity, genotyping methods, total number of cases and controls, genotype distributions in cases and controls, ED diagnostic criteria and clinical characteristics (age, smoking, cardiovascular comorbidities).
We used an exact test to evaluate the HWE of the genotype distribution in the control group for all studies (Zintzaras & Lau, 2008). OR and 95% CI was used to assess the strength of the association between the I/D polymorphism and ED risk. We calculated the OR and respective 95% CI by comparing the carriers of D-allele with the wild homozygotes (ID + DD vs. II).
The heterogeneity among studies in terms of degree of association was assessed using the Cochran's Q-statistic (Zintzaras & Ioannidis, 2005). If p < 0.10, the heterogeneity was considered significant. Heterogeneity was further tested by I2 metric (I2 = [Q − d.f./Q]), which is independent of the number of studies. The I2 metric takes values between 0 and 100%, with values >30% suggesting evidence for heterogeneity (Higgins & Thompson, 2002). When heterogeneity among studies was detected, the OR was pooled according to a random-effect model using DerSimonian and Laird method; otherwise, a fixed-effect model using Mantel–Haenszel method was selected (DerSimonian & Laird, 1986; Pereira et al., 2007). To evaluate the ethnicity-specific effects, subgroup analysis was performed by ethnic group (Asian, Latino and European). Comparisons of other genetic models were also performed (ID + II vs. DD, DD vs. II, DI vs. II and D vs. I).
Potential publication bias was assessed by visual inspection of the Begg's funnel plots in which the log ORs were plotted against their SEs. We also performed the Begg's and Egger's tests to evaluate the presence of publication bias (Begg & Mazumdar, 1994; Egger & Smith, 1998). Sensitivity analysis was carried out to evaluate the stability of the results. The influence of individual study was evaluated by estimating the pooled OR in the absence of each study (Tobias, 1999). All analyses were performed using Stata version 11.0 software (Stata, College Station, TX, USA). Statistical significance was defined as p < 0.05, except where otherwise specified.
Study selection and characteristics
We initially identified 346 results relevant to the search terms in these selected databases (Fig. 1). After reading the titles and abstracts, 12 potential articles were included for fulltext review. Further screening of these articles, three of them were excluded for being not relevant to ED risk with ACE gene polymorphism and two articles (Çam et al., 2011; Gamidov et al., 2011) were not included for the language are neither English nor Chinese. Thus, seven articles relevant to ACE I/D polymorphism and ED risk for more detailed evaluation. Of these, one article (Eisenhardt et al., 2003; Eisenhardt & Siffert, 2003) was excluded because it contained overlapping data from the same investigators. Thus, a total of six case-control studies were finally included in our meta-analysis (Park et al., 1999; Kim et al., 2001; Rosas-Vargas et al., 2004; Mazo et al., 2008; Andersen et al., 2010; Eisenhardt et al., 2010). Characteristics of the six case-control studies included are summarized in Table 1. Distribution of ACE genotype and allele frequency for each case-control study are shown in Table 2. These studies were published between 1999 and 2010. Studies were carried out in various ethnicities: two involved Asian (Park et al., 1999; Kim et al., 2001), two involved Latino (Rosas-Vargas et al., 2004; Andersen et al., 2010) and two involved European (Mazo et al., 2008; Eisenhardt et al., 2010). Assessment of ED was from a variety of sources, including colour doppler ultrasonography (Park et al., 1999; Kim et al., 2001; Eisenhardt et al., 2010), vasoactive drug-induced erection test (Rosas-Vargas et al., 2004), International index of Erectile Function-5 (IIEF-5) (Mazo et al., 2008) and an eight-item questionnaire (Andersen et al., 2010). The determination of the genetic polymorphisms used in all the studies are polymerase chain reaction (PCR). The distribution of genotypes in the controls was consistent with the HWE for all included studies.
Table 1. Characteristics of eligible case-control studies in the meta-analysis
As shown in Fig. 2, heterogeneity of ID + DD vs. II for all studies was analysed and the value of p = 0.244 in a fixed-effects model. In addition, I2 value is another index of the test of heterogeneity. The I2 was 25.4%, suggesting the presence of no heterogeneity. Thus, the fixed-effects model was chosen to synthesize the data. The results suggested that the variant D-allele carriers (ID + DD) do not have a significant increased risk of ED with those individuals without D-allele (II) (OR = 1.07, 95% CI = 0.84 − 1.37). Summary of the results of other genetic comparisons is listed in Table 3.
Table 3. Meta-analysis of ACE I/D polymorphism an ED association
Test of association
Test of heterogeneity
Random-effect model was used when p value of heterogeneity test (ph) < 0.10; otherwise, fixed-effect model was used; SZ: Sample size.
Significant between-study heterogeneity for all studies was detected in three genetic models (ID + II vs. DD: Q-statistic p = 0.0, I2 = 84.1%; DD vs. II: Q-statistic p = 0.007, I2 = 68.5; D vs. I: Q-statistic p = 0.0, I2 = 84.3) in Table 3. Subgroup analysis was performed after stratifications of the data by ethnicity (Fig. 3), no significant increased risk was observed in Asian, Latino and European. Thus, the polymorphism may not increase ED risk in different population.
Sensitivity analysis showed that the removal of any one case-control study for ID + DD vs. II comparative did not result in movement of the point estimate outside the 95% CIs (Table 4), suggesting no single study exhibited excessive influence and the results of this meta-analysis are stable.
Table 4. Sensitivity analysis for the current meta-analysis: ORs with 95% CIs and p value were results after excluding each case-control study for ID + DD vs. II
Visual inspection of the Begg's funnel plot did not identify substantial asymmetry (ID + DD vs. II). The Egger's test and Begg's test also indicated no evidence of publication bias among studies ACE I/D polymorphism and ED risk (Egger, p = 0.95 and Begg, p = 1.0 for ID + DD vs. II) (Fig. 4).
ACE is a key enzyme in the renin-angiotensin system, which is involved in the local and systemic regulation of blood flow and plays an important role in cardiovascular pathophysiology (for review, see ref. Sayed-Tabatabaei et al., 2006). Recent studies have pointed out that ACE is expressed in the corpora cavernosa, and there are hints that angiotensin II plays a role in the initiation of detumescence by activation of the angiotensin II receptor (Park et al., 1997; Becker et al., 2001a,b). Furthermore, other studies also revealed possible role of ACE in the aetiology of ED (Kifor et al., 1997; Llisterri et al., 2001). Thus, it is possible that genetic variations of the ACE gene may modulate the risk of ED. The most widely studied polymorphism I/D is located on intron 16 (Haiman et al., 2003). This polymorphism is based on insertion or deletion of a 287-bp Alu sequence, resulting in a change in the plasma ACE level. Many studies have been published to investigate the associations between this polymorphism and ED; however, the results were inconsistent and conflict. To derive a more precise estimation of association, we performed this meta-analysis of six case-control studies comprising 1966 individuals to assess the associations between the ACE I/D polymorphism and ED risk.
When all the eligible studies were pooled into this meta-analysis, there was no evidence of significant association between ACE I/D polymorphism and ED risk. Moreover, in other comparative genetic models, no significant associations were found in any genetic models. These results indicated that this polymorphism may not contribute to ED risk. Although previous studies revealed possible roles of ACE in ED aetiology, our results suggested that these roles may not account by the variant of ACE gene. The exact mechanism of this enzyme in ED aetiology remains unclear, but our results may indicate that ACE I/D polymorphism may not influence ED risk. In addition, considering the possible role of this polymorphism in serum ACE level, it is possible that the ED risk may be modified by plasma ACE level, but not by the variant. Hence, future studies are warranted to identify the associations between ACE polymorphism, ACE levels and ED risk.
Considering the property of genetic background may affect the results of genetic association studies, we performed subgroup analysis by ethnicity. Three subgroups were included in this meta-analysis: Asian, Latino and European. In this meta-analysis, we did not find a significant association between this polymorphism and ED risk in any subpopulations. Moreover, no significant associations were found in any other genetic models. Interestingly, this polymorphism and ED risk in Latino and non-Latino were all inversely associated, although they were not statistically significant. These results may suggest that this polymorphism may exert varying effects in different populations. However, all included studies were from Asian, Latino and European populations, further studies are necessary to validate these findings for other ethnic populations, especially in Africans.
When conducting meta-analysis, one of the important issues is heterogeneity. We found statistical significant between-study heterogeneity for all studies in three genetic contrasts (ID + II vs. DD: p = 0.0, I2 = 84.1%; DD vs. II: p = 0.007, I2=68.5; D vs. I: p = 0.0, I2=84.3). After subgroup analysis by ethnicity, the heterogeneity was effectively decreased or removed in Latino, suggesting that certain effects of genetic variants are ethnic specific. The stability of this meta-analysis was analysed by sequentially excluding individual studies, and our results indicated stability of results. Apart from ethnicity, other potential factors may influence the genetic associations. ED is age dependent, suggesting that prevalence and severity of ED increase with age (Selvin et al., 2007). A young control group may include some individuals who later develop ED and thus influence the relationship between gene variants and phenotypes (Wang et al., 2010). Socioeconomic status and ED risk factors, such as diabetes mellitus, hypertension and myocardial infarction, may modulate the prevalence of ED, and thus also contribute to heterogeneity in these studies.
Publication bias is another important issue which should also be discussed in meta-analysis. After evaluating the publication bias by Egger's funnel plots, Begg's test and Egger's test, we did not detect a publication bias, indicating the strength of the results.
It is worthy to mention a previous published study by Wiwanitkit in 2005 (Wiwanitkit, 2005). He also evaluated the association between ACE I/D polymorphism and ED risk. After reading the article with great interest, we found that there were some differences between these two studies. First, Wiwanitkit reported that subjects with the DD genotype have 1.2 times higher risk of having erectile dysfunction, which is inconsistent with our study. Second, evidence was limited because only three case-control studies were available in Wiwanitkit's study. Third, some issues which may affect the results of meta-analysis were addressed in our study, such as publication bias, sensitivity analysis and HWE analysis.
There are several limitations in our meta-analysis. First, it was much difficult to obtain the all articles published in various languages. We only included the studies published in English or Chinese which were included in the selected databases for data analysis, and thus some relevant studies which included by other languages or published with other databases or unpublished could be missed. Second, the results of our meta-analysis should be interpreted with caution because the number of studies included and the sample size of individual studies were relatively small. Third, this meta-analysis included data from Asian (korea), Latino (Mexico, Brazil) and Europe (Germany, Russia), hence, our results are applicable to only these ethnic groups. Fourth, because of the lack of original data, we were also unable to examine the interactions of gene–gene and gene-environment, which may be an important component of the association between ACE I/D polymorphism and environment and ED risk. Fifth, some other important factors may bias our results, such as age, smoking status, socioeconomic status and other unknown function of the renin-angiotensin system.
In Conclusion, this meta-analysis indicates that the I/D polymorphism in the ACE gene may not contribute to susceptibility to ED. The results of our meta-analysis should be interpreted with caution. Larger and well-designed studies are warranted to validate these findings. Moreover, future study should also evaluate gene–gene and gene-environment interaction on I/D polymorphisms in ACE gene and ED risk.
This study was supported by a grant from National Natural Science Foundation of China (81170566).