Effects of isoflavones -LRB- soy phyto-estrogens -RRB- on serum lipids : a meta-analysis of randomized controlled trials Objectives To determine the effects of isoflavones -LRB- soy phyto-estrogens -RRB- on serum total cholesterol -LRB- TC -RRB- , low density lipoprotein cholesterol -LRB- LDL -RRB- , high density lipoprotein cholesterol -LRB- HDL -RRB- and triglyceride -LRB- TG -RRB- . Introduction In recent years , phytoestrogens have attracted a great deal of interests in the medical and scientific literature . It also appears in the lay press for its effects on cardiovascular and menopausal health , and even cancer prevention . These compounds are present in large quantities in soybeans , clover and some legumes . Because of resemblances to human estrogen and the observations that Asian populations who consume more isoflavones compared with women in western countries have less menopausal symptoms , isoflavones are postulated as natural products that may be beneficial to postmenopausal women in cardiovascular health . Commercial products containing different quantities and mixtures of isoflavones are now widely available . A meta-analysis of soy protein in 1995 found significant cholesterols lowering effects when compared with animal proteins -LSB- 1 -RSB- . The authors suggest that isoflavones may be the principal physiologically active components responsible for the lipid lowering effects . However , it is still controversial -LSB- 5 -RSB- . Many reviews and editorials -LSB- 2,3 -RSB- have discussed the relevance of phytoestrogens on cardiovascular health and hypocholesterolemic effects but there was no meta-analysis up to our searching . Methods Searching strategy We searched the databases from ACP Journal Club 1991 to Oct 2002 , Cochrane Controlled Trials Register 3rd Quarter 2002 , Cochrane Database of Systematic Reviews 4th Quarter 2002 , Database of Abstracts of Reviews of Effectiveness 4th Quarter 2002 , British Nursing Index -LRB- BNI -RRB- 1994 to Oct 2002 , CANCERLIT 1975 to Oct 2002 , CINAHL 1982 to Oct Week 4 2002 , CSA -- Life Sciences Collection 1982 to Oct 2002 , EMBASE 1980 to 2002 Week 45 , International Pharmaceutical Abstracts 1970 to Oct 2002 , PREMEDLINE Oct 27 , 2002 , MEDLINE 1996 to Oct Week 4 2002 . We searched the keywords with Ovid software version rel6 .2.0 : ` soy ' , ` soy protein ' , ` soybean ' , ` tofu ' , ` phytoestrogen ' , ` isoflavone ' , ` genistein ' , ` daidzein ' , ` formononectin ' and ` biochanin A ' by the method described by Dickersin 1994 -LSB- 4 -RSB- . We did not restrict any languages during the searching . Hand searching was made by retrieving relevant articles from the obtained studies and unpublished data were obtained through contacting experts . We identified on-going trials by searching ClinicalTrials.gov , the UK National Research Register and Meta-register of controlled trials on the internet . Selection of eligible trials We included both single and double blind randomized controlled trials with baseline and after treatment values for synthesizing risk -LRB- mean -RRB- differences . The outcome measures were differences of serum total cholesterol -LRB- TC -RRB- , low density lipoprotein cholesterol -LRB- LDL-C -RRB- , high density lipoprotein cholesterol -LRB- HDL-C -RRB- and triglycerides -LRB- TG -RRB- between post-randomization baselines and after treatments . Eligible interventions were isoflavones tablets of single isolated component or any mixtures of genisteins , daidzein , formononectin and biochanin A. Isoflavones interventions in forms of soy protein supplements or diets were also included as long as they compared isoflavones containing treatments with isoflavones depleting controls . Treatments with soy polysaccharides , fiber , and phytosterols as their primary interventions were excluded . Validity assessment and data abstraction Two independent investigators reviewed the articles obtained without masking . There were no scorings to the included trials . Data were entered twice to reduce input errors . Inter-rater reliability was not performed . Data were abstracted with a designed form before analysis . Duplicated trials or studies with the same population were counted once to reduce the duplicated publication bias . Data disagreement between the two reviewers was resolved by discussion . Study characteristics The characteristics of the obtained studies were tabulated with subtypes of isoflavones interventions , subjects ' serum lipids status , dosages of isoflavones and lengths of treatment . Sub-group analysis was performed with different forms of isoflavones interventions , such as isolated genistein or mixtures of isoflavones tablets versus placebo , isoflavones containing versus depleting soy protein diets . Sensitivity analysis was also made across different population characteristics and lengths of treatment . Funnel plots were used to detect possible publication bias or treatment heterogeneity across sample sizes . Quantitative data synthesis We obtained the risk differences -LRB- RD -RRB- from the post-randomization baselines and after-treatment values in each trial and calculated the pooled standard deviation of the RD as : where n1 and SD1 were sample size and standard deviation from treatment and n2 and SD2 were from control . Inverse variance method was used to pool all trial results -LSB- 6 -RSB- with software ` Review Manager 4.2 -LSB- 31 -RSB- . Fixed-effects model was used as the method of combination and it was supplemented with random-effects model if necessary . Since log odds ratios were not available in the trials , funnel plots were plotted with standard errors against risk -LRB- mean -RRB- differences . Results Trial flow The trial flow chart was illustrated in Figure 1 . Seventeen studies -LSB- 7-23 -RSB- -LRB- 21 comparisons -RRB- with 853 subjects were included in this meta-analysis . Reasons for exclusion were non-randomization , lack of control -LSB- 24-27 -RSB- , insufficient original data and baseline values -LSB- 28,29 -RSB- . Figure 1 Trial flow chart of including isoflavones studies . Databases generated 3542 potential eligible randomized controlled trials -LRB- RCT -RRB- . After careful review , seventeen RCT -LRB- reference -LSB- 7-23 -RSB- -RRB- were included in this meta-analysis . Study characteristics The characteristics of the trials included were shown in Table 1 . The populations being studied were adults of age between 18 and 73 . Soy phytoestrogens interventions varied from isoflavones tablets to isoflavones containing soy diets . Isoflavones tablets were introduced in 11 trials , soy capsule in 1 trial , isolated soy protein -LRB- ISP -RRB- in 3 trials , and soy foods in 2 trials . The average intake of isoflavones was 73 mg per day -LRB- ranging from 28.5 to 150 mg -RRB- and length of treatment was 10 weeks -LRB- ranging from 2 to 26 weeks -RRB- . Three trials recruited hyperlipidemic subjects while 8 included participants with normolipidemia . Thirteen studies recruited female , two recruited male and two recruited subjects with both genders . Table 1 Characteristics of the 17 included studies . There were 9 crossover and 8 parallel studies . The isoflavones tablets introduced were mainly in aglycone form . The abbreviations were listed below table . Study Treatment -LRB- Rx -RRB- † Isoflavones content in Rx -LRB- mg/d -RRB- ‡ Control Isoflavones content in control -LRB- mg/d -RRB- No . of subjects Lipid status § Subject gender | | Length of treatment -LRB- weeks -RRB- * Calvert 1981 -LSB- 7 -RSB- soy biscuits NA soy biscuits 0 10 H male 4 Clifton-Bligh 2001 -LRB- I -RRB- -LSB- 8 -RSB- T 57 T Clifton-Bligh 2001 -LRB- II -RRB- -LSB- 8 -RSB- T 85.5 28.5 46 B female 26 Dewell 2002 -LSB- 9 -RSB- T 150 G = 40 ; D = 50 ; glycosides = 60 placebo 0 36 H female 24 Hale 2002 -LSB- 10 -RSB- T 80 G = 40 ; D = 40 placebo 0 29 NS post 2 Han 2002 -LSB- 11 -RSB- soy protein capsule 100 G = 70 ; D = 18 ; glycitein = 12 soy protein capsule 0 78 N peri 16 Hodgson 1998 -LSB- 12 -RSB- T 55 G = 30 ; D = 1 ; B = 16 ; F = 8 placebo 0 59 N male & female 8 Howes 2000 -LSB- 13 -RSB- T 40 G = 1 ; D = 0.5 ; B = 26 ; F = 16 placebo 0 75 NS post 5 Mackey 2000 -LSB- 14 -RSB- ISP 65 ISP < 4 49 H post 12 * Merz-Demlow 2000 -LRB- I -RRB- -LSB- 15 -RSB- ISP 113 -- 144 ISP * Merz-Demlow 2000 -LRB- II -RRB- -LSB- 15 -RSB- ISP 55 -- 74 9 -- 11 13 N female 3 * Nestel 1997 -LSB- 16 -RSB- T 40 -- 80 G = 22 -- 43 ; D = 17 -- 33 ; glycitein = 1 -- 3 placebo 0 21 B female 5 * Nestel 1999 -LRB- I -RRB- -LSB- 17 -RSB- T 80 G = 8 ; D = 7 ; B = 49 ; F = 16 placebo * Nestel 1999 -LRB- II -RRB- -LSB- 17 -RSB- T 40 G = 4 ; D = 3.5 ; B = 24.5 ; F = 8 0 13 B female 5 * Samman 1999 -LSB- 18 -RSB- T 86 G = 8.6 ; D = 7.4 ; B = 51.4 ; F = 18.6 placebo 0 14 N pre 16 * Sanders 2002 -LSB- 19 -RSB- soy burger 56 G = 34.8 ; D = 21.2 soy burger 2 22 N male & female 2 * Simons 2000 -LSB- 20 -RSB- T 80 placebo 0 20 N post 8 Squadrito 2002 -LSB- 21 -RSB- T 54 G = 54 placebo 0 60 N female 24 * Urban 2001 -LSB- 22 -RSB- T 70 G = 42 ; D = 27 ISP 3 28 N elderly men 6 * Wangen 2001 -LRB- I -RRB- -LSB- 23 -RSB- ISP 110 -- 154 ISP * Wangen 2001 -LRB- II -RRB- -LSB- 23 -RSB- ISP 54 -- 76 6 -- 8.2 18 NS post 4 * crossover design † ` T ' = isoflavone tablet ; ` ISP ' = isolated soy protein ‡ ` G ' = Genistein ; 'D ' = Daidzein ; ` B ' = Biochanin A ; ` F ' = Formononectin § ` H ' = hyperlipidemic subjects , 'N' = normolipidemic subjects ; ` B ' = both hyper - and normolipidemic subjects ; ` NS ' = not specified | | ` pre ' = premenopausal women ; ` peri ' = perimenopausal women ; ` post ' = postmenopausal women Quantitative data synthesis We found that isoflavones tablets insignificantly increased serum TC by 0.01 mmol/L -LRB- 95 % CI = -0.17 to 0.18 ; heterogeneity p = 1.0 -RRB- -LRB- Figure 2 -RRB- ; LDL by 0.00 mmol/L -LRB- 95 % CI = -0.14 to 0.15 ; heterogeneity p = 0.9 -RRB- ; HDL by 0.01 mmol/L -LRB- 95 % CI = -0.05 to 0.06 ; heterogeneity p = 1.0 -RRB- ; and triglyceride by 0.03 mmol/L -LRB- 95 % CI = -0.06 to 0.12 ; heterogeneity p = 0.9 -RRB- . Isoflavones containing soy biscuits or burger had insignificant effects on serum TC with reference to isoflavones depleting soy biscuits or burger . Only isoflavones containing isolated soy protein -LRB- ISP -RRB- or soy protein -LRB- SP -RRB- capsule reduced serum TC when compared with isoflavones depleting ISP or SP capsule -LRB- Table 2 -RRB- . There was only one trial -LSB- 11 -RSB- introduced SP capsule as treatment . Mixtures of the four soy phytoestrogens -LRB- genistein , daidzein , formononectin and biochanin A -RRB- or isolated genistein did not have significant effects on serum TC . The significant result from intervening the mixtures of genistein and daidzein had shown to be statistically heterogeneous to combine -LRB- heterogeneity p = 0.06 -RRB- as genistein and daidzein tablets were introduced among 3 studies -LSB- 9,10,16 -RSB- while Sanders -LSB- 19 -RSB- chose genistein and daidzein soy burger and Urban -LSB- 22 -RSB- used genistein and daidzein ISP . In subgroup analysis , the effect of genistein and daidzein tablets on serum TC from the 3 studies was shown to be insignificant , 0.1 mmol/L -LRB- 95 % CI = -0.25 to 0.45 ; heterogeneity p = 0.87 -RRB- . Dose-response effect was not found in any forms of interventions in this meta-analysis . The results remained insignificant when the length of treatment increased . Although one trial -LSB- 22 -RSB- -LRB- without washout period -RRB- produced significant effect , it seemed that study design had no influence on the serum TC . There were insignificant decreases of serum TC among pre - and postmenopausal women . Interestingly , cholesterol lowering effects could only be found among normolipidemic subjects but not participants with hyperlipidemia and men were shown to be benefited with isoflavones treatment . Isoflavones in the forms of tablets and ISP were all insignificant on serum low density lipoprotein cholesterol -LRB- LDL -RRB- , high density lipoprotein cholesterol -LRB- HDL -RRB- and triglycerides -LRB- TG -RRB- . These results were shown in Table 3 . Funnel plots of isoflavones tablets were asymmetrical and the results of the published trials tended to favor treatment . Figure 2 Funnel plot of risk difference of isoflavones tablets versus placebo on serum total cholesterol . Total treatment size was 274 and total control was 208 . The pooled risk difference of isoflavones tablets versus placebo was 0.01 with 95 % confidence interval between -0.17 and 0.18 . Heterogeneity p-value was 1.00 . Note : 'N' = sample size ; ` SD ' = standard deviation ; ` RD ' = risk difference ; ' 95 % CI ' = 95 % confidence interval Table 2 Subgroup analysis of the effects of isoflavones on serum total cholesterol . Isoflavones were shown to have significant benefits when given in the form of ISP or soy protein capsule . Regardless the forms of prescription , isoflavones decreased serum total cholesterol among normolipidemic but not hyperlipidemic subjects . Subgroup outcome No . of comparisons No . of subjects | | Treatment effect on TC -LRB- mmol/L -RRB- ¶ Heterogeneity p-value § Form of intervention † : a. -RRB- isoflavones tablets 12 -LSB- 8-10,12,13,16-18,20,21 -RSB- 482 0.01 -LRB- -0.17 , 0.18 -RRB- 1.00 b. -RRB- ISP -LRB- + -RRB- 8 -LSB- 14,15,22,23 -RSB- 229 -0.11 -LRB- -0.21 , -0.01 -RRB- * 0.34 c. -RRB- soy protein capsule 1 -LSB- 11 -RSB- 78 -0.69 -LRB- -1.19 , -0.19 -RRB- * NA d. -RRB- soy biscuit 1 -LSB- 7 -RSB- 20 -0.05 -LRB- -0.47 , 0.37 -RRB- NA e. -RRB- soy burger 1 -LSB- 19 -RSB- 44 0.09 -LRB- -0.38 , 0.56 -RRB- NA f. -RRB- Overall 21 -LSB- 7-23 -RSB- 853 -0.09 -LRB- -0.18 , -0.01 -RRB- * 0.77 Isoflavones mixture ‡ : G&D&B&F 5 -LSB- 12,13,17,18 -RSB- 214 -0.06 -LRB- -0.34 , 0.21 -RRB- 0.96 G&D 6 -LSB- 9-11,16,19,22 -RSB- 285 -0.22 -LRB- -0.41 , -0.03 -RRB- * 0.06 G 1 -LSB- 21 -RSB- 60 0.10 -LRB- -0.35 , 0.55 -RRB- NA Isoflavones intake -LRB- mg per day -RRB- : < 50 3 -LSB- 8,13,17 -RSB- 132 -0.12 -LRB- -0.54 , 0.29 -RRB- 0.99 51 -- 100 12 -LSB- 8,10,12,14,15,17-23 -RSB- 484 -0.10 -LRB- -0.21,0.01 -RRB- 0.74 101 -- 150 4 -LSB- 9,11,15,23 -RSB- 176 -0.10 -LRB- -0.24 , 0.05 -RRB- 0.09 Design : parallel crossover 9 -LSB- 8-14,21 -RSB- 447 -0.08 -LRB- -0.27 , 0.10 -RRB- 0.48 a. -RRB- no washout 1 -LSB- 22 -RSB- 56 -0.43 -LRB- -0.73 , -0.13 -RRB- * NA b. -RRB- washout mentioned 6 -LSB- 15,19,20,23 -RSB- 208 -0.07 -LRB- -0.17 , 0.03 -RRB- 0.91 Gender : male 2 -LSB- 7,22 -RSB- 76 -0.30 -LRB- -0.54 , -0.05 -RRB- * 0.15 female 17 -LSB- 8-11,13-18,20,21,23 -RSB- 674 -0.07 -LRB- -0.17 , 0.02 -RRB- 0.89 Treatment length : 2 -- 10 weeks 14 -LSB- 7,10,12,13,15-17,19,20,22,23 -RSB- 541 -0.09 -LRB- -0.18 , 0.00 -RRB- 0.84 11 -- 20 weeks 3 -LSB- 11,14,18 -RSB- 155 -0.22 -LRB- -0.52 , 0.07 -RRB- 0.07 21 -- 30 weeks 4 -LSB- 8,9,21 -RSB- 157 0.02 -LRB- -0.28 , 0.31 -RRB- 0.96 Menopausal status : pre-menopausal 3 -LSB- 15,18 -RSB- 80 -0.06 -LRB- -0.17 , 0.06 -RRB- 0.58 peri-menopausal 1 -LSB- 11 -RSB- 78 -0.69 -LRB- -1.19 , -0.19 -RRB- * NA post-menopausal 13 -LSB- 8-10,13,14,16,17,20,21,23 -RSB- 516 -0.05 -LRB- -0.20 , 0.10 -RRB- 1.00 Subjects with : normolipidemia 8 -LSB- 11,12,15,18,19,21,22 -RSB- 377 -0.11 -LRB- -0.21 , -0.01 -RRB- * 0.08 hyperlipidemia 3 -LSB- 7,9,14 -RSB- 105 -0.05 -LRB- -0.36 , 0.26 -RRB- 1.00 * statistically significant † ` ISP -LRB- + -RRB- ' = isoflavone containing isolated soy protein ; ` ISP -LRB- - -RRB- ' = isoflavone depleting isolated soy protein ‡ ` G ' = Genistein ; 'D ' = Daidzein ; ` B ' = Biochanin A ; ` F ' = Formononectin § NA = not applicable | | Since subjects acted as their own controls in a crossover trial , the ` calculated ' total number was thus doubled in a single pair comparison . ¶ 95 % confidence interval in parenthesis Table 3 Effects of isoflavones on serum LDL-cholesterol , HDL-cholesterol and triglycerides levels . Isoflavones in the forms of tablets or isolated soy protein -LRB- ISP -RRB- did not show significant benefits over serum LDL-cholesterol , HDL-cholesterol and triglycerides levels . The results were not heterogeneous to combine . Type of intervention * No . of trials No . of comparisons No . of subjects Treatment effect -LRB- mmol/L -RRB- † Heterogeneity p-value 1 . -RRB- Isoflavones tablets LDL 9 11 -LSB- 8,10,12,13,16-18,20,21 -RSB- 446 0.00 -LRB- -0.14 , 0.15 -RRB- 0.94 HDL 10 12 -LSB- 8-10,12,13,16-18,20,21 -RSB- 482 0.01 -LRB- -0.05 , 0.06 -RRB- 0.98 TG 9 12 -LSB- 8-10,12,13,16,17,20,21 -RSB- 482 0.03 -LRB- -0.06 , 0.12 -RRB- 0.93 2 . -RRB- ISP -LRB- + -RRB- LDL 3 5 -LSB- 14,15,23 -RSB- 173 -0.06 -LRB- -0.16 , 0.03 -RRB- 0.84 HDL 3 5 -LSB- 14,15,23 -RSB- 173 -0.01 -LRB- -0.07 , 0.05 -RRB- 0.97 TG 3 5 -LSB- 14,15,23 -RSB- 173 0.02 -LRB- -0.05 , 0.09 -RRB- 1.00 * ` ISP -LRB- + -RRB- ' = isoflavones containing isolated soy protein ; ` LDL ' = Low density lipoprotein cholesterol ; ` HDL ' = High density lipoprotein cholesterol ; ` TG ' = Triglycerides † 95 % confidence interval in parenthesis Discussion Main findings Phytoestrogens with either isolated genistein or isoflavones mixtures of genistein , daidzein , formononectin and biochanin A were found to be statistically insignificant in lowering serum total cholesterol -LRB- Table 2 -RRB- . Resolving the heterogeneity from pooling the mixtures of genistein and daidzein , we found that tablets prescription with these two isoflavones -LSB- 9,10,16 -RSB- yielded insignificant benefits over serum TC , 0.1 mmol/L -LRB- 95 % CI = -0.25 to 0.45 ; heterogeneity p-value = 0.87 -RRB- . One trial -LSB- 19 -RSB- , introducing soy burger as the prescription of these 2 isoflavones , found insignificant benefits . Another trial -LSB- 22 -RSB- , prescribing them in the form of isolated soy protein -LRB- ISP -RRB- , had shown a decrease in serum TC by 0.43 mmol/L -LRB- 95 % CI = -0.73 to -0.13 -RRB- . It may be a result of bias as this trial lacked a washout period . When dose-response effect and treatment length were taken into account , phytoestrogens seemed to have insignificant effects over serum cholesterol -LRB- Table 2 -RRB- . Isoflavones in the forms of tablets , soy burger or biscuits seemed to have insignificant benefits over serum TC . Only Han -LSB- 11 -RSB- reported a significant benefit for isoflavones in the form of soy protein capsule . Isoflavones in the forms of tablets or ISP could not be shown to have significant benefits over serum low density lipoprotein -LRB- LDL -RRB- cholesterol , high density lipoprotein -LRB- HDL -RRB- cholesterol and triglycerides -LRB- TG -RRB- -LRB- Table 3 -RRB- . We could not demonstrate significant benefits with all types of prescriptions when the dosages or treatment lengths increased . No dose-response effect was detected nor benefits on serum cholesterol with any forms of prescription among pre - and post-menopausal women and hyperlipidemic subjects . The benefit over normolipidemic subjects was heterogeneous -LRB- p = 0.08 -RRB- . It may be again the result of prescription diversity . Separated analysis found that isoflavones tablets -LSB- 12,18,21 -RSB- had insignificant effect on serum TC among normolipidemic subjects , 0.05 mmol/L -LRB- 95 % CI = -0.23 to 0.33 ; heterogeneity p = 0.9 -RRB- . Only isoflavones in the form of soy protein capsule -LSB- 11 -RSB- or ISP -LSB- 22 -RSB- reported benefits on participants with normolidemia . In fact , some suggest that the beneficial effects of soy protein require synergistic reactions between isoflavones and other soy components -LSB- 32 -RSB- . This hypothesis was supported by some animal experiments -LSB- 33-35 -RSB- . It seemed that isoflavones in the form of tablets , up to 150 mg per day -LRB- genistein about 50 mg , daidzein about 50 mg -RRB- , had no significant benefits on serum cholesterols in this meta-analysis . Phytoestrogens treatments in forms of soy protein supplements or soy protein capsule may need further investigations , especially interactions between other chemically active components inside prescriptions . Validity and limitation In subgroup analysis , the sample sizes were inevitably reduced and there might be insufficient power to detect small significant differences . Isoflavones treatments with dosages beyond 150 mg per day or treatment lengths beyond 26 weeks could not be shown in this study . The effects on serum cholesterols among subjects with hypertension , diabetes mellitus or other cardiovascular risk factors were also beyond this meta-analysis . Generalizability over other phytoestrogens such as coumestans , lignans and resveratrol may need further trials to clarify the questions . No funnel plots were shown in this study because the choice of risk differences as the horizontal axes may lead to funnel plot asymmetry -LSB- 36 -RSB- . Publication bias was still possible although vigorous searching had been attempted . Biological plausibility It was suggested that isoflavones readily bind estrogen receptors -- beta -LRB- ER-beta -RRB- which are an important receptors in both the central nervous and cardiovascular systems -LSB- 37-39 -RSB- . Isoflavones may also have anti-oxidant effects on blood vessels -LSB- 40-42 -RSB- . However , compared with estradiol , isoflavones bind estrogen receptors with 100 and 1,000 times less affinity -LSB- 30 -RSB- . Both conjugated and aglycone forms of daidzein and genistein are rapidly cleared from the plasma following a single dose of isoflavones -LSB- 43 -RSB- and urinary excretion is virtually nil within 48 hours after ingestion -LSB- 44,45 -RSB- . Biological effects of soy phytoestrogens are still controversial . The hypothesis from an influential meta-analysis -LSB- 1 -RSB- stating soy estrogens may be responsible for the lipid reduction effects was criticized for the fact that the trials involved largely isoflavone-free soy protein products -LSB- 46 -RSB- . Apart from phytoestrogens , in fact , some other components such as phytic acid , saponins and fibers were potential candidates responsible for the hypocholesterolemic effects -LSB- 32,48,49 -RSB- . Suggestion to future trials Tablets of single isolated isoflavone with considerable dosages may be important in future randomized trials . Intervention with isoflavones mixtures may be helpful but interactions between components should be handled with extra cares . Introduction of phytoestrogens in forms of soy diets is not suggested because we found much heterogeneity inside the diets , ranging from soy biscuits to burger , which may be difficult to combine and analyze . Confounding factors such as fiber , fatty acids , amino acids and energy intake are especially hard to control . Complications associated with hyperlipidemia , such as coronary heart disease -LRB- CHD -RRB- or cardiovascular accident -LRB- CVA -RRB- , may be selected as other endpoints in future trials . It may help constructing convincing funnel plots with less heterogeneity as trials accumulate . Conclusions Isoflavones tablets , up to 150 mg per day , had insignificant effects in lowering serum total cholesterol , LDL-cholesterol and triglyceride . There was also insignificant benefit over serum HDL-cholesterol . The results were consistent when tablets were introduced as isolated genistein , mixture of genistein and daidzein , or mixture of genistein , daidzein , formononectin and biochanin A. No significant effects were found among participants with normo - or hyperlipidemia and women with pre - or postmenopausal status . Isoflavones interventions in the forms of soy proteins , such as isolated soy protein -LRB- ISP -RRB- , soy diets or soy protein capsule , were inconclusive due to inadequate sample size , heterogeneity and presence of potentially uncontrolled confounders . List of abbreviations ` ISP ' = isolated soy protein ; ` TC ' = total cholesterol ; ` LDL ' = Low density lipoprotein cholesterol ; ` HDL ' = High density lipoprotein cholesterol ; ` TG ' = Triglycerides Competing interests None declared . Authors ' contributions JY participated in the design of this manuscript . JY and YTF participated in abstracted the data and performed statistical analysis . All authors read and approved the final manuscript .

O O B O B B O O O B O O O O O O O O O O O O O B O B 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 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 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 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 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B O O O O B O O O O O O O O O O O O B 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B B O O O B O O B O B O O B O O O O O O O B 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 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 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 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 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 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 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 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 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 B O B O O O O O O O O O O O O B O B O O O O O B O O B 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 B 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 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 B 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 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 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 B 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 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 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 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 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B 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 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 B 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 B 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 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 B 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 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 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 B 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 B O O O O O B O O B 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 B 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 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 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 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 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 B O O O O O O O O O O O O O O O O O O O O O B 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 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 B 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B O O O O O B 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


Endovascular occlusion of high-flow intracranial arteriovenous shunts : technical note Endovascular closure of high-flow arteriovenous -LRB- AV -RRB- shunts in intracranial AV malformations or pial fistulas is technically challenging . In this paper , we illustrate two simple methods to occlude large high-flow AV shunts in a controlled manner . Introduction Endovascular closure of high-flow arteriovenous -LRB- AV -RRB- shunts in intracranial AV malformations -LRB- AVM -RRB- or pial fistulas is technically challenging -LSB- 1 -- 3 -RSB- . With the use of liquid embolic agents , the high flow through the shunt may cause migration of the embolic agent to the venous outflow tract with possible devastating consequences . In anatomical configurations with a very short arterial feeder , the shunt can only be occluded at the venous side . In this paper , we illustrate two simple methods to occlude large AV shunts in a controlled manner applied in three patients . Case 1 A 26-year-old man was referred for treatment after haemorrhage of a large callosal AVM -LRB- Fig. 1 -RRB- . Angiography demonstrated two high-flow AV shunts from the right anterior cerebral artery draining into enormously enlarged veins . Injection of Onyx 34 at the site of the largest shunt resulted in immediate migration to the venous side . Subsequently , a nondetachable microballoon -LRB- Magic B2 , Balt , Montmorency , France -RRB- was flow-directed into the main feeder just proximal to the shunt and inflated to block the flow . At that time , Onyx injection was continued and resulted in gradual occlusion of the fistula in 7 min . Next , the second high-flow shunt was occluded in a similar way . Control angiography confirmed complete occlusion of both AV shunts . A second embolization procedure was scheduled to be performed 3 months after the first . Fig. 1A 26-year-old man with large callosal AVM . a Frontal and lateral right internal carotid angiogram shows an AVM with two high-flow AV shunts from the anterior cerebral artery -LRB- arrows -RRB- . b Lateral subtracted fluoroscopy image obtained during injection of Onyx with a balloon catheter blocking the flow . The arrow points to the distal tip marker of the microcatheter delivering the Onyx . Onyx is deposited at the transition of artery to vein . c Lateral subtracted fluoroscopy image obtained during occlusion of the second shunt Case 2 A 12-year-old boy was referred for treatment of a left frontal AVM discovered on MRI performed for seizures . Angiography under general anaesthesia demonstrated , besides a plexiform part of the nidus , two large AV shunts from the anterior cerebral artery -LRB- Fig. 2 -RRB- . The largest shunt had a very short arterial feeder that precluded the use of Onyx , since reflux would have led to migration into the normal vasculature . A microcatheter was navigated through the shunt into the proximal venous part and detachable coils were inserted until the shunt was completely occluded . Next , the second AV shunt was occluded with simple injection of Onyx 34 and continued injection occluded the major part of the nidus . After embolization , still under general anaesthesia , the patient was transferred to the Gamma Knife unit and the small AVM remnant was irradiated . Fig. 2A 12-year-old boy with a left frontal AVM . a Lateral angiogram of the left internal carotid artery shows a large AV shunt with a very short arterial segment -LRB- arrow -RRB- . b Lateral angiogram obtained after occlusion of the venous and arterial side of the shunt with detachable coils Case 3 A 27-year-old woman was referred for treatment of an intracranial vascular disorder that had been discovered on CT scan performed for chronic headaches . Vertebral angiography revealed a high-flow single-hole side-wall pial fistula from the P3 -- P4 segment of the right posterior cerebral artery draining into a dilated basal vein of Rosenthal -LRB- Fig. 3 -RRB- . In addition , a flow aneurysm on the right superior cerebellar artery was present . A microcatheter was navigated through the shunt into the vein and the proximal part of the vein was occluded with detachable coils with preservation of continuity of the posterior cerebral artery . Subsequently , the flow aneurysm was occluded with coils . A follow-up angiogram obtained 6 months later showed continuing closure of the fistula and the aneurysm . Fig. 3A 27-year-old woman with pial fistula . a Frontal vertebral angiogram shows a high-flow single-hole side-wall pial fistula from the P3 -- P4 segment of the right posterior cerebral artery draining into a dilated basal vein of Rosenthal . In addition , a flow aneurysm of the superior cerebellar artery is present -LRB- arrow -RRB- . b Follow-up angiogram obtained 6 months after occlusion of the venous side of the fistula with detachable coils shows continuing occlusion of the fistula with preserved patency of the posterior cerebral artery Discussion The most commonly used embolic agent in brain AVMs is the fast polymerizing liquid adhesive n-butylcyanoacrylate -LRB- NBCA , Histoacryl ; Braun , Melsungen , Germany -RRB- . Pure NBCA polymerizes immediately after contact with blood . Slower polymerization is achieved when NBCA is mixed with iodinated oil -LRB- Lipiodol ; Guerbet , Roissy , France -RRB- . The use of a high concentration of NBCA or pure NBCA in high-flow shunts in brain AVMs may occlude the fistula within seconds , but this technique requires courage , experience and skill , since flow of NBCA is to some extent unpredictable -LSB- 3 -RSB- . Inadvertent migration of the glue into the draining veins may result in immediate haemorrhage by blocking venous outflow . Detachable coils may be inserted at the fistula site to decrease the flow and thereby facilitate the injection of NBCA -LSB- 2 -RSB- . Recently , the new liquid embolic agent Onyx has become available for embolization of brain AVMs -LRB- Onyx Liquid Embolic System ; Microtherapeutics , Irvine , Calif. -RRB- . Onyx is nonadhesive and polymerizes slowly . Onyx is available in several concentrations and the high-concentrated Onyx can be used to slowly occlude large AV shunts or pial AV fistulas in a more controlled way than that achieved with NBCA -LSB- 4 -RSB- . However , simple Onyx injection is not always feasible : in some very high-flow shunts Onyx may migrate through the fistula into the distal draining veins . We found the adjuvant use of a microballoon to block the flow to enable gradual occlusion of the large high-flow shunts with Onyx in the AVM of patient 1 a very useful approach . In the other patients , one with a very short arterial feeder of an AV shunt in an AVM and one with a side-wall pial fistula , the use of liquid embolic agents was not possible and controlled delivery of detachable coils to the venous side of the shunt was technically easy with complete occlusion of the fistula . This technique is helpful in shunts with a moderately dilated venous outflow tract in which placement of coils is possible . In conclusion , different types of high-flow AV shunts can safely be occluded with endovascular techniques tailored to the specific anatomical configuration of the shunt .

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


Treatment of the neurogenic bladder in spina bifida Renal damage and renal failure are among the most severe complications of spina bifida . Over the past decades , a comprehensive treatment strategy has been applied that results in minimal renal scaring . In addition , the majority of patients can be dry for urine by the time they go to primary school . To obtain such results , it is mandatory to treat detrusor overactivity from birth onward , as upper urinary tract changes predominantly start in the first months of life . This means that new patients with spina bifida should be treated from birth by clean intermittent catheterization and pharmacological suppression of detrusor overactivity . Urinary tract infections , when present , need aggressive treatment , and in many patients , permanent prophylaxis is indicated . Later in life , therapy can be tailored to urodynamic findings . Children with paralyzed pelvic floor and hence urinary incontinence are routinely offered surgery around the age of 5 years to become dry . Rectus abdominis sling suspension of the bladder neck is the first-choice procedure , with good to excellent results in both male and female patients . In children with detrusor hyperactivity , detrusorectomy can be performed as an alternative for ileocystoplasty provided there is adequate bladder capacity . Wheelchair-bound patients can manage their bladder more easily with a continent catheterizable stoma on top of the bladder . This stoma provides them extra privacy and diminishes parental burden . Bowel management is done by retrograde or antegrade enema therapy . Concerning sexuality , special attention is needed to address expectations of adolescent patients . Sensibility of the glans penis can be restored by surgery in the majority of patients . Introduction The incidence of spina bifida worldwide still ranges from 0.3 -- 4.5 per 1,000 births . Renal scarring and renal failure have been important issues in spina bifida literature over the years , with reported death due to renal failure up to 20 % in the first year of life . The incidence of renal damage is nearly 100 % in patients with an overactive pelvic floor -LRB- detrusor/sphincter dyssynergia ; DSD -RRB- when not adequately treated . It is important to realize that renal damage starts early in life , within the first 6 months . In contrast to a few decades ago , we can now assure parents of children with a neurogenic bladder that renal function can be preserved provided they comply with the treatment regime -LSB- 1 -- 14 -RSB- . To prevent renal damage , urological and nephrological treatment of a neurogenic bladder should start immediately after birth . Modern treatment provides the opportunity to convert a high-pressure bladder with functional urethral obstruction based on neuropathic detrusor/sphincter dyssynergia into a low-pressure reservoir that is safe for the upper urinary tracts -LSB- 15 , 16 -RSB- . The objectives in the urological management of patients with spina bifida are -LRB- 1 -RRB- preservation of renal function ; -LRB- 2 -RRB- quality of life , preferably with urinary dryness by school age ; and -LRB- 3 -RRB- independence at an older age with respect to bladder and bowel management . Finally , sexuality in the spina bifida patients is an underestimated problem . Initial approach for the management of spina bifida To preserve renal function , low bladder pressure must be maintained from birth . The status of pelvic floor activity must be assessed shortly after birth to ascertain whether a child is at risk for high detrusor pressures . At first presentation after birth , inspection of the anal sphincter , closed or open , gives an impression of the status of the pelvic floor : overactive or paralyzed . Approximately 50 % of children with spina bifida aperta and 25 % of children with occult spinal dysraphism have a detrusor/sphincter dyssynergia that carries a serious risk of early upper urinary tract damage by high bladder pressures and urinary tract infections -LRB- UTIs -RRB- . It is important to realize that after closure of the back , pelvic floor behavior can change from paralyzed to overactive in the first 2 -- 3 months of life . That is a reason to delay the first urodynamic study -LRB- UDS -RRB- until 2 months after birth . Clean intermittent catheterization In principle , all newborn patients are put on clean intermittent catheterization -LRB- CIC -RRB- , oxybutynin , and chemoprophylaxis -LRB- trimethoprim 2 mg/kg once daily -RRB- immediately after closure of the back . By doing so , safe pressures in the lower urinary tract can be obtained in most patients . In most patients , bladder volume and compliance remain satisfactory for years . In patients with DSD , this approach has also reduced the need for bowel augmentation of the bladder from 90 % to less than 5 % . Of course , it is still a matter of discussion whether it is necessary to perform CIC from birth onward in all patients . In patients with proven pelvic floor paralysis , it is also possible to wait until after surgery to make the patient continent . However , it can be a tedious affair to teach CIC to an 8-year-old . Parents of these children are advised to catheterize at least twice daily to maintain the habit of CIC . CIC was introduced for neurogenic bladders in 1972 -LSB- 17 -- 20 -RSB- . Parents and other caregivers carry out CIC in the first 8 -- 9 years of life : if the children show sufficient dexterity , they take up this task themselves after this age . After birth , CIC is carried out with self-lubricating 8-F catheters . The size of the catheter depends on the patient 's age ; the aim is always to use the largest possible catheter to obtain optimal bladder emptying . Antimuscarinic therapy Oxybutynin is best started together with CIC immediately after closure of the back . Other antimuscarinic agents have not yet been registered for pediatric use . This subject is more elaborately described in the section `` Overactive detrusor '' . Infection prophylaxis In principle , all patients are put on low-dose chemoprophylaxis , mostly trimethoprim 2 mg/kg per day . In case of breakthrough infections , nitrofurantoin or ciprofloxacin can be used . Symptomatic infections are treated intravenously with amoxicillin/clavulanic acid and gentamicin until the result of bacterial culture is known . Many centres stop prophylaxis after the age of 1 year , and approximately 50 % of patients seem to do well without prophylaxis . Scientific proof is lacking on this subject . To obtain such proof , we are doing a multicenter randomized study with half of the patients on prophylaxis and half without . Specific needs Overactive pelvic floor From birth , patients with an overactive pelvic floor generally become dry with the help of oxybutynin and CIC . Every year , UDS are carried out to check bladder activity , capacity , and compliance . Patients are given ultrasound scans for upper-tract dilatation and renal growth . A wait-and-see policy is adopted when bladder capacity is normal for age and end-filling detrusor pressures are 30 cms H2O or less . When bladder compliance is insufficient , with end-filling pressures of more than 30 cms , an autoaugmentation or detrusorectomy can reduce the pressure . Augmentation by ileocystoplasty or colocystoplasty is inevitable in patients with serious overactivity in combination with poor bladder compliance and low capacity . Both operations can be performed at any age and can be combined with other procedures to obtain better urinary continence to facilitate CIC and/or to reduce UTIs -LRB- reflux -RRB- -LSB- 21 -RSB- . Paralyzed pelvic floor Patients with a paralyzed pelvic floor are incontinent for urine . Their upper urinary tracts are safe as long as the paralytic pelvic floor is left untreated . They need bladder-neck surgery to become dry . If bladder capacity and compliance are insufficient , surgery can be combined with autoaugmentation of the bladder -LRB- detrusorectomy -RRB- or -LRB- rarely -RRB- with clam ileocystoplasty or colocystoplasty . Overactive detrusor muscle If UDS reveals detrusor overactivity , patients are treated with antimuscarinics to increase bladder capacity , even if detrusor pressures are safe . Life-long suppression of detrusor overactivity is required in patients with an overactive neuropathic bladder . In some patients , overactivity can be treated surgically by detrusorectomy or ileocystoplasty . In spina bifida , rhizotomies -LRB- interruption of spinal roots -RRB- are not yet being used routinely to cure overactivity , but they seem to be a promising alternative . Ideally , we would like to start a protocol with neonatal rhizotomies during the first back closure , but so far , practical and ethical considerations have prevented us from doing this . Antimuscarinic therapy is the gold standard for pharmaceutical therapy of neuropathic detrusor overactivity . Oxybutynin has proven to be inexpensive and effective and can be taken orally , intravesically , and transdermally . Several new antimuscarinic agents have been introduced , which may prove valuable in the future . Repeated injection therapy of the bladder with 300 U of botulinum toxin can be an alternative to antimuscarinic therapy . This therapy effectively suppresses detrusor contractions for 6 -- 9 months . Injections need to be repeated at a 6 - to 9-month interval . Adverse effects , even after repeated injection therapy , have not been reported . The long-term effects have yet to be established -LSB- 22 , 23 -RSB- . Surgical procedures Timing of surgery There is no age-related contraindication for any operation . Thus , indication for surgery is made in mutual agreement with the child 's parents or with the patients themselves after the age of 11 years . The combination of high bladder pressures and vesicoureteral reflux can sometimes force intervention as early as the first few months of life . We combined antireflux surgery , bladder autoaugmentation , and transvaginal sling suspension in a 3-month-old girl with febrile breakthrough infections , with good clinical and urodynamic result after follow-up for more than 10 years . Initially , reserves existed on doing sling suspensions before puberty , especially in boys , out of fear of introducing obstruction during puberty based on prostate growth . Over the years , it has been proven that puberty can safely be passed after sling suspension of the bladder neck , both in girls and boys . Parental burden can be an indication for performing a catheterizable stoma . If a child weighs 20 kg , five daily transfers for CIC can be too much for a parent with lower back pain . Finally , the patient 's privacy can be an important argumentation for construction of a stoma for CIC . Parents are increasingly reluctant for any caregiver to help their child perform CIC transurethrally , as this involves exposure of the genitalia to strangers several times a day . Surgery for incontinence Patients with a paralytic pelvic floor need bladder-neck surgery to achieve continence . There are many surgical options . Our standard approach is an abdominoperineal puboprostatic sling procedure in boys and a transvaginal sling procedure in girls . If there is some persistent leaking after a sling procedure , this is cured by injecting a bulking agent in the bladder neck . Optimal results are obtained if the injection needle is passed into the bladder neck by means of a suprapubic puncture with transurethral endoscopic visual control . So far , we have mainly used silicon grains in povidone -LRB- Macroplastique ® -RRB- as a bulking agent . The polymer Deflux ® offers a good alternative because it is easier to inject . However , in our experience , Deflux does not work if CIC needs to be carried out through the same channel . A recent survey , yet unpublished , of the results of 76 sling suspensions revealed an 80 % success rate . Sling suspension in boys There is a great variety in the reported results regarding sling suspension of the bladder neck in boys with neuropathic sphincter incompetence , especially when compared with girls . The plane between the bladder neck/prostate and the rectum must be developed to allow the sling to be wrapped around the bladder neck . There are three possible techniques , each with their specific advantages and disadvantages . First , from the abdominal wound , the pelvic diaphragm can be opened left and right so that a pathway can be bluntly dissected for the sling around the bladder neck . A disadvantage of this technique is the risk of passing into the prostatic urethra with the right angle that is used to feel the way around during surgery . Experienced urologic surgeons do not take this argument seriously , but in practice , most surgeons perform this operation with the relatively low frequency of only a few cases a year . The second possible technique is the abdominoperineal approach to develop the plane between the bladder neck and the urethra . The plane between the rectum and the bladder neck is exposed through a perineal incision . The most important advantages of this procedure are the short time , approximately 20 min , needed to develop the plane around the bladder neck and the fact that the bladder does not need to be touched at all -LSB- 24 -RSB- . A third method to find the path around the bladder neck has been proposed by Lottmann et al. . The complete bladder is dissected from the peritoneum and the rectum until only the urethra and both ureters with the vascular pedicles are left . The sling can easily be passed around the bladder neck , with direct visual control and exactly at the level of the bladder neck -LSB- 25 -RSB- . It is difficult to determine which of these three approaches is the best . All three have enthusiastic followers . At our department , we have had two urethral defects treated by two experienced surgeons in a small series of the abdominal approach going directly around the bladder neck . We have had one urethral defect leading to bladder-neck closure in 76 patients operated on abdominoperineally . Our experience with the Lottmann approach is limited to three successful cases and , because of operation time and for fear of limited bladder vascularization , we abandoned this approach . Another important subject is the amount of tension that needs to be put on the sling . In the past , we tried to regulate tension by measuring urethral and leak-point pressures during surgery , but there was no significant positive outcome . Nowadays , we determine sling tension by measuring the ability to pass a relatively large Foley catheter past the sling ; for example , a 12-F catheter in a 7-year-old boy . In some patients with anal atresia after prior rectal pull-through surgery , it may be necessary to adopt the abdominal approach to pass around the bladder neck . Abdominoperineal operations in 14 patients , published in 1999 , led to continence in all 14 patients ; one patient required a subsequent injection of a bulking agent to help achieve continence -LSB- 24 -RSB- . It is important to note that in this series , the erectile function of the penis proved to be preserved after sling suspension . False routes are a risk after sling suspension . Out of approximately 50 male patients , we had to construct a catheterizable stoma in five : four as a result of a false route , and one because of a huge congenital prostatic cyst . Sling suspension in girls In general , sling suspension of the bladder neck in girls leads to good results concerning urinary continence . There are also several ways to find the way around the bladder neck in girls . The standard well-known technique is to identify the level of the bladder neck by feeling the balloon of a transurethral Foley catheter , and , subsequently , developing the plane between the bladder neck and the anterior vaginal wall from above . This procedure can be very difficult in girls with severe spine deformities . There is always a certain risk of entering the urethra or the bladder neck when passing around . This is also the case in patients with a normal anatomy . To avoid this risk , in postpuberty female patients , many surgeons opt for incising the vaginal wall at the level of the bladder neck and bringing the sling around under direct visual control . We chose to put the sling through the vagina , first only in prepubertal girls and later in all patients . This method has two major advantages : there is no risk of bladder-neck lesions and only a small risk of sling erosion -LSB- 26 -RSB- . In spina bifida patients , the risk of sling erosion is higher than in patients with stress incontinence , because the sling must be tightened more strongly in patients with neuropathic sphincter incompetence . Alternatives There are many surgical alternatives for reaching urinary continence . Kropp and Pippi Sale developed operations to create a flap valve from the bladder neck . Young/Deese/Leadbetter , Tanagho , and Mitchell procedures lengthen the urethra into the bladder . Several authors prefer American Medical Systems -LRB- AMS -RRB- artificial sphincters to help reach continence . The majority of patients still need CIC after AMS sphincter prosthesis -LSB- 27 -RSB- . We found that Burch-type colposuspension was insufficient for spina bifida girls to reach urinary continence . This is related to the huge intra-abdominal pressure that can result from transfers from the wheelchair . Several groups do not opt for urinary continence at an early age and try to maintain safe leak-point pressures by regular dilatation of the female urethra , sometimes also of the male urethra , after making this possible with a perineal urethral stoma . In some patients , they construct a cutaneous vesicostomy for the first few years and perform an ileocystoplasty when the stoma is closed . Alternatives in dealing with a neurogenic bladder When cerebral function is bad and the patient has no dexterity , diapers can also be chosen . For this choice , a low-pressure bladder is necessary . Good results are obtained by cutaneous vesicostomy . In girls , low pressures can be achieved by making a vesicovaginal fistula by endoscopic cutting into the vagina between the ureteric orifices . Temporary low pressures can be achieved by overdistension of the female urethra . In boys , endoscopic external sphincterotomy in the 12 o'clock position produces low outlet resistance for 2 -- 3 years and needs to be repeated when bladder pressures rise . Catheterizable stomas The need for transfers from a wheelchair to do CIC is the most important indication to make a catheterizable stoma . Sometimes in boys , the impossibility of carrying out CIC transurethrally after false routes results in the need for a stoma . Parents increasingly indicate their child 's privacy as a reason for asking for a catheterizable stoma . In patients with a large bladder capacity for their age , a continent stoma can be constructed from a bladder tube . Patients with a normal bladder capacity can be treated by cutaneous appendicovesicostomy or by an ileal tube -LRB- Monti procedure -RRB- -LSB- 28 -RSB- . The direction of peristalsis from the appendix -LRB- Fig. 1 -RRB- needs to go in the direction of the bladder to avoid mucus spotting in the clothes . When ileal or colonic bladder augmentation is performed at the same time , the continent stoma can be constructed from the side or middle part of the gut used for the augmentation . In some patients , a ureter can be used as a catheterizable stoma . The intravesical tunnel of the tube in the bladder should be at least 2 cm long , as there is a risk that the tunnel will end up being too short , especially in case of an extravesical implantation of an appendix . Fig. 1The appendix can be used to make a stoma to the bladder for intermittent catheterization In the literature , after construction of a catheterizable stoma , complications are described in as many as 50 % of patients . These complications are mostly temporary . Stomal stenosis , mostly at skin level , is a frequently occurring temporary complication that can be avoided with a silicon ace stopper left behind in the skin portion of the stoma for 6 months between catheterizations . Stomal leakage of urine can be a frustrating complication . Endoscopic treatment with bulking agents can be tried , but lengthening of the intravesical tunnel or reimplantation of the stoma will often be needed . Leakage of the stoma occurs more often in midline stomas -LRB- umbilicus -RRB- than in stomas placed on the right side of the lower abdominal wall . The reason is probably that the lateral stoma passes through the rectus muscle and is thus occluded during increases in abdominal pressure due to rectus contraction . In midline stomas we now also pass the stoma through the median margin of one rectus muscle . Stoma stenosis at the level of entrance into the bladder can sometimes be treated endoscopically with success . In those patients , formal reimplantation of the stoma into the bladder will frequently be needed as well . Autoaugmentation or detrusorectomy of the bladder In general , not too much extra capacity should be expected , but high-end filling pressures based on low bladder compliance can be reduced to safe values by detrusorectomy . In selected patients that undergo surgery to become dry , we also do an autoaugmentation -LRB- Fig. 2 -RRB- to try and get patients off antimuscarinic therapy , with success in approximately 50 % of cases . An important factor for success is identification , opening , and marking the adventitial layers of the bladder separately before the detrusorectomy starts , and the meticulous closure of this layer at the end of the procedure . This means that before the start of the detrusorectomy , the detrusor has been freed of all adventitial layers . If a mucosal leak occurs during the operation , this is closed with Tissuecoll ® . To prevent shrinkage and scarring , cycling of the bladder has to start immediately after detrusorectomy . This can be done by maintaining a 20-cm H2O pressure on day 1 , 30 cm on day 2 , followed by clamping the catheter on day 3 for 2 h , and finally by opening the catheter every 3 h to empty the bladder on following days . In this way , optimal expansion of the detrusorectomized bladder can be obtained -LSB- 21 -RSB- . Several authors have reported success by expanding the bladder over a balloon for several days while ensuring drainage of urine by ureteral catheters . We have no experience with this procedure but assume it could work well if there is leakage . Others have successfully covered the bare part of the mucosa with a demucolized patch of sigmoid colon . Covering with peritoneum or omentum has been reported to fail . Fig. 2The bladder can be augmented by removing the detrusor muscle . A sling is used to improve bladder outlet resistance Clam cystoplasty Clam cystoplasty -LRB- Fig. 3 -RRB- is indicated when the bladder capacity is too small to reach acceptable numbers of catheterization per day and/or end-filling pressures are too high to be safe for the upper tracts . Clam cystoplasty is performed with either ileum or colon . In general , 25 cm of ileum are used , opened antimesenterically , and constructed into a U-shaped cap . The last 20 cm of the ileum are not used , to prevent malabsorption of vitamins . If there is a short ileal mesentery , the sigmoid colon can easily be used . It is important to avoid the formation of an hourglass bladder by opening the clam anteriorly up to the bladder neck and posteriorly up to the trigone . In children , we prefer to bring the augmented bladder extraperitoneally by closing the peritoneum around the vascular pedicle . This extraperitonealization of the bladder may initially make it more difficult to reach sufficient bladder capacity , but it seems to reduce the risk of spontaneous perforation of the bladder , which is a prominent risk in children because of their tendency toward bad CIC compliance during puberty . During a period of several years , we had two spontaneously leaking ileal bladders out of six that had not been placed extraperitoneally , and no spontaneously leaking/rupture in more than 40 other patients with extraperitoneal ileal bladder . Fig. 3The bladder can be augmented by using a bowel segment Bowel management and surgery For the first 2 years of life , a wait-and-see policy is adopted in bowel management , often supported by chronic use of laxatives . At the age of 3 years , bowel management can be performed successfully in the vast majority of patients with retrograde colonic enemas . The rectum is filled with 20 ml/kg tap water through a plastic cone that passes approximately 2 cm into the anus . In the rare case where patients have trouble emptying their rectums with retrograde enemas , we offer an antegrade colonic enema stoma -LSB- 29 , 30 -RSB- . A patient 's choice can be an indication for an antegrade colonic enema stoma . As a separate procedure , the simplest way to carry out an antegrade colonic enema stoma is to use laparoscopy to bring the tip of the appendix to the abdominal skin -LRB- Fig. 4 -RRB- . Alternatives are an open procedure with open abdomen for other surgery or , in the absence of an appendix , to create a transverse tube from the colon -LRB- left or right -RRB- and a submucosal tunnel to prevent leakage . Many pediatric surgeons and pediatric gastroenterologists have had good experiences with several types of buttons that are put into the colon . Fig. 4The appendix can be used to make an antegrade colonic enema stoma If these procedures are carried out , treatment of spina bifida patients can be very successful . In our experience between 1988 and 2001 , the incidence of renal scarring in 144 patients was 2.1 % , and urinary dryness in patients who opted to be dry was nearly 80 % -LSB- 14 -RSB- . Surgery to improve sexuality in male patients Sexuality can be frustrating for male spina bifida patients , because in most cases , they can have intercourse but penis sensibility is absent . To overcome this , in patients with a lesion under the level of L1 , we have connected the ilioinguinal nerve with the dorsal nerve of the penis , resulting in glans sensibility and erogenous feeling in most patients . Patients report a positive effect on sexuality . Our experience with the first three patients has been published , and by now , of 15 cases operated , 12 report normal sensibility of the glans penis -LSB- 31 -RSB- . Conclusion Kidney-function preservation and early urinary dryness are important factors for optimal quality of life for spina bifida patients . Another important factor is patient independence regarding his or her bladder and bowel management . For optimal treatment of this difficult group of patients , a multidisciplinary team is needed consisting of members from all the necessary medical specialties , including rehabilitation specialists , physical therapists , specialized nurses , and social workers . Questions -LRB- Answers appear following reference list -RRB- Concerning renal scarring and renal failure in spina bifida literature : Reported death rate due to renal failure is up to 20 % in the first year of lifeReported renal failure rate is up to 20 % in the first year of lifeReported death rate due to renal failure is up to 20 % in pubertyReported renal failure rate is up to 20 % in pubertyConcerning clean intermittent catheterization -LRB- CIC -RRB- : CIC is best started immediately after closure of the spinal defectCIC is best started immediately after the age of 8 -- 9 yearsCIC is best started not earlier than after bladder neck surgeryCIC is best started not earlier than after several urinary infections and reported renal scarringInfection prophylaxis in spina bifida patients : All patients need low-dose chemoprophylaxisFifteen percent of all patients need low-dose chemoprophylaxisScientific proof of the need for infection prophylaxis is still lackingNo patients need low-dose chemoprophylaxisIn case of hyperactive detrusor muscle in a patient with spina bifida : Central side-effects occur less frequently if antimuscarinic therapy is administered orallyAntimuscarinic therapy is the gold standard for pharmaceutical therapyRepeated injection therapy of the bladder with only 3 U of botulinum toxin can be an alternative to antimuscarinic therapyAntimuscarinic therapy has positive effects on perspiration and body-temperature regulationIn modern management of spina bifida , endpoint targets are : Kidney-function preservationDrynessFecal continenceAll the above Electronic supplementary material Below is the link to the electronic supplementary material . 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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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 B 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 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 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 B I 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 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

spina bifida
incontinence
renal function
bladder augmentation
dryness
antimuscarinic


EGFR Inhibitor Enhances Cisplatin Sensitivity of Oral Squamous Cell Carcinoma Cell Lines Epidermal growth factor receptor -LRB- EGFR -RRB- is involved in multiple aspects of cancer cell biology . EGFR has already been identified as an important target for cancer therapy , with various kinds of EGFR inhibitors currently used in treatment of several human cancers . Recently , EGFR and its downstream signaling pathways were identified as being associated with cisplatin sensitivity . In addition , EGFR inhibitors have shown significant promise for patients who failed cisplatin-based therapy . In this study , we investigated whether treatment with an EGFR inhibitor improves cisplatin sensitivity in oral squamous cell carcinoma -LRB- OSCC -RRB- cell lines . The effects of a combination of AG1478 , a specific EGFR tyrosine kinase inhibitor , with cisplatin were evaluated in cultured OSCC cell lines and cisplatin-resistant sublines . Higher expression of EGFR and p-EGFR was found in the two cisplatin-resistant cell lines compared with the corresponding parental cell lines . In addition , augmented inhibition of OSCC cell growth by the combination of AG1478 with cisplatin was found in both cell lines . These results suggest that the combination of an EGFR inhibitor and cisplatin may be useful as a rational strategy for the treatment of patients with oral cancer with acquired cisplatin resistance . Introduction Epidermal growth factor receptor -LRB- EGFR -RRB- is expressed at high levels in a variety of solid tumors including oral cancers -LSB- 1 , 2 -RSB- . EGFR and its downstream signaling pathways are involved in multiple aspects of cancer cell biology , including tumor cell proliferation , inhibition of apoptosis , invasion , metastasis , and angiogenesis -LSB- 1 -- 4 -RSB- . EGFR has already been identified as an important target for cancer therapy , and various kinds of EGFR inhibitors are currently used in the treatment of several human cancers -LSB- 5 -- 10 -RSB- . Cisplatin-based combination chemotherapy displays significant anti-tumor activity against solid tumors of oral squamous cell carcinoma -LRB- OSCC -RRB- . However , the effectiveness of cisplatin in the treatment of recurrent/metastatic tumors is limited because of acquired or intrinsic resistance . EGFR and its signaling pathways are involved in the mechanism of cisplatin resistance . Cells that are resistant to cisplatin have an altered response to the EGF ligand and enhanced activation of the protein kinase -LSB- 11 -RSB- . In addition , several studies have suggested that enhanced expression of EGFR may be associated with cisplatin resistance in a variety of solid tumors including oral cancers -LSB- 12 , 13 -RSB- . Increased EGFR expression may be a survival response by some tumors exposed to chemotherapeutic agents -LSB- 14 -RSB- . Increased availability of EGFR inhibitors in cisplatin-resistant cells has also been reported previously -LSB- 13 -RSB- . EGFR inhibitors have shown significant activity in cases failing cisplatin-based therapy -LSB- 15 , 16 -RSB- . Therefore , EGFR blockade may be a useful therapeutic tool in the treatment of patients with acquired cisplatin resistance . In this study , we established a cisplatin-resistant cell line from an OCSS-derived cell line and investigated the differential EGFR and phosphorylated EGFR -LRB- p-EGFR -RRB- expression between OSCC cell lines and the cisplatin-resistant sublines . In addition , we examined the effect of combination therapy with an EGFR inhibitor and cisplatin on the growth of OSCC cells . Materials and Methods Cell Lines Two human OSCC cell lines have been established at Wakayama Medical University , Wakayama , Japan . The H-1 line was derived from a biopsy specimen of moderately differentiated OSCC in the lower gingiva . The Sa-3 line was derived from a biopsy specimen of well-differentiated OSCC in the upper gingiva . Both cell lines were cultured in Dulbecco 's modified Eagle 's medium -LRB- DMEM ; Nissui , Tokyo , Japan -RRB- supplemented with 10 % fetal bovine serum -LRB- FBS ; Equitech-Bio , Kerrville , TX , USA -RRB- , 100 units/ml penicillin , and 100 μg/ml streptomycin -LRB- Gibco BRL , Grand Island , NY , USA -RRB- in a highly humidified atmosphere of 5 % CO2 at 37 °C . In accordance with previously described methods -LSB- 17 , 18 -RSB- , the cisplatin -LRB- CDDP -RRB- - resistant sublines H-1 / CDDP and Sa-3 / CDDP were established by repeated subculture in the presence of increasing concentrations of cisplatin -LRB- Nippon Kayaku Corporation , Tokyo , Japan -RRB- , from 0.1 μg/ml until cells became fully resistant to cisplatin and could grow exponentially ; in each case the final cisplatin concentration was 0.5 μg/ml . The drug-resistant cell lines were passed in drug-free medium , and there was no loss of resistance during the two-month testing period . Cell Growth Analysis with MTT Assay Cells were seeded in 96-well plates at 2000 cells per well in DMEM containing 10 % FBS . After 24 h , cells were exposed to one of nine concentrations -LRB- 0.05 , 0.1 , 0.25 , 0.5 , 1 , 1.25 , 2.5 , 5 and 10 μg/ml -RRB- of cisplatin or five concentrations -LRB- 1 , 5 , 10 , 20 and 30 μM -RRB- of AG1478 -LRB- Calbiochem San Diego CA , USA -RRB- . After cells were incubated with cisplatin or AG1478 for 24 h , medium was changed to drug-free DMEM and cells were incubated for an additional 72 h. Thereafter , the number of cells per well was quantified with a MTT cell growth assay kit -LRB- Funakoshi , Tokyo , Japan -RRB- according to the manufacturer 's instructions . Briefly , after 10 μl MTT solution was added to each well , the well was incubated for 4 h and scanned at 550 -- 630 nm by a MTP-300 microplate reader -LRB- Corona , Tokyo , Japan -RRB- . Six wells were used for each drug concentration , and the experiment was repeated three times . The 50 % inhibitory concentration -LRB- IC50 -RRB- was calculated from the survival curve . In another experiment , to test whether the combination of cisplatin and AG1478 would achieve higher growth inhibition than the single agent at a concentration lower than the IC50 , fixed concentrations of each drug were then tested in combination treatment of OSCC cell lines . All cells were exposed to AG1478 for 1 h before cisplatin . The statistical significance of differences was analyzed by applying Mann -- Whitney U-test . The level of significance was set at p < 0.05 . Western Blot Analysis Subconfluent cells were scraped from culture dishes , washed twice with phosphate buffered saline -LRB- PBS -RRB- , and suspended in 700 μl Western blot lysis buffer containing 62.5 mM Tris -- HCl -LRB- ph 6.8 -RRB- , 25 % glycerol , and 2 % sodium dodecyl sulfate -LRB- SDS -RRB- . Samples were centrifuged at 15,000 rpm for 20 min at 4 °C , after which supernatants were collected . After heating at 95 °C for 5 min , equal amounts of proteins were separated with the use of 10 % SDS-PAGE . After electrophoresis , proteins were transferred to a PVDF membrane in Tris-glycine buffer containing 20 % methanol . The membrane was blocked with 3 % skim milk containing 0.01 % polyoxyethlene sorbitan monolaurate for 60 min , and incubated overnight with the corresponding primary antibodies -LCB- a 1:750 dilution of anti-rabbit polyclonal EGFR antibody -LSB- Santa Cruz Biotechnology , Santa Cruz , CA , USA -RSB- , and a 1:750 dilution of anti-goat polyclonal p-EGFR -LRB- Tyr 1173 -RRB- antibody -LSB- Santa Cruz Biotechnology -RSB- -RCB- at 4 °C . The membrane was washed three times for 5 min each with PBS containing 0.05 % polyoxyethlene sorbitan monolaurate and horseradish peroxidase-conjugated anti-rabbit or anti-goat antibody for 1 h at room temperature , respectively . Protein signals were visualized by enhanced chemiluminescence using ECL Western blotting detection reagents -LRB- Amersham , Arlington Heights , IL , USA -RRB- for 1 min and exposed to Kodak Biomax XAR film . Results Single Agent Effects The sensitivities of all cell lines to various concentrations of cisplatin and AG1478 were determined by MTT assay and plotted on a survival curve -LRB- Fig. 1 -RRB- . The IC50 values of cisplatin and AG1478 are shown in Table 1 . The IC50 values of the H-1 / CDDP and Sa-3 / CDDP lines to cisplatin were about 10-fold greater than the values for the parental H-1 and Sa-3 lines . Resistance of all cell lines to AG1478 was similar , but the IC50 values for parental cell lines tended to be slightly higher . Fig. 1Inhibitory effects of cisplatin and AG1478 on growth of OSCC cell lines . The four lines , H-1 , H-1 / CDDP , Sa-3 , and Sa-3 / CDDP , were treated with cisplatin -LRB- 0.05 -- 10 μg/ml ; a -RRB- or AG1478 -LRB- 1 -- 30 μM ; b -RRB- for 24 h as described in the Materials and methods section . Cell viability was measured by MTT assay and is expressed as a percentage relative to control cells . The results showed that cisplatin and AG1478 inhibited OSCC cell growth in a dose-dependent mannerTable 1Cisplatin and AG1478 sensitivity of OSCC cell linesCell lineIC50IC50Cisplatin -LRB- μg/ml -RRB- AG1478 -LRB- μM -RRB- H-10.4925.3 Sa-30.4328.5 H-1 / CDDP6.523.3Sa-3 / CDDP5 .921.2 Combination Effects All cell lines were treated with AG1478 -LRB- 10 μM -RRB- for 1 hour before cisplatin -LRB- 0.25 μg/ml -RRB- for 24 h . The combination of AG1478 with cisplatin inhibited cell growth > 60 % in parental cell lines > 50 % in cisplatin-resistant cell lines -LRB- Fig. 2 -RRB- . In both cell lines , we showed augmented effects of inhibition and showed a large effect of augmentation in cisplatin-resistant cell lines . Fig. 2Effect of a combination of AG1478 with cisplatin on growth of OSCC cell lines . The four lines , H-1 , H-1 / CDDP , Sa-3 , and Sa-3 / CDDP , were treated with AG1478 -LRB- 10 μM -RRB- for 1 h before addition of cisplatin -LRB- 0.25 μg/ml -RRB- for 24 h. Cell viability was measured by MTT assay and is expressed as a percentage relative to control cells . In both cell lines , we showed augmented effects of inhibition and showed large-effect augmentation in cisplatin-resistant cell lines . * p < 0.05 , compared with cisplatin-treated cells Expression of EGFR and p-EGFR To investigate the molecular basis for the increased sensitivity of chemoresistant tumors to cisplatin , we used Western blot analysis to determine the level of EGFR and p-EGFR expression in parental and cisplatin-resistant cell lines . Higher EGFR and p-EGFR expression was found in cisplatin-resistant cell lines compared with the corresponding parental cell lines -LRB- Fig. 3 -RRB- . Fig. 3Total expression of epidermal growth factor receptor -LRB- EGFR -RRB- and phosphorylated receptor -LRB- p-EGFR -RRB- . Total expression of EGFR and p-EGFR was determined by Western blot analysis . Ten micrograms of total cellular lysates prepared from parental and cisplatin -LRB- CDDP -RRB- - resistant cells were resolved by electrophoresis , transferred to a PVDF membrane , and blotted with anti-EGFR and anti-p-EGFR antibodies as described in the Materials and methods section Discussion Our studies suggest that changes in the expression of EGFR play an important role in regulating the drug-resistant phenotype of cells in response to cisplatin . Cisplatin is widely used for chemotherapy of many malignancies , especially of oral squamous cell carcinoma -LRB- OSCC -RRB- . However , the effectiveness of cisplatin in the treatment of recurrent/metastatic tumors is limited because of acquired or intrinsic resistance . Increased EGFR expression may be a survival response by some tumors exposed to chemotherapeutic agents -LSB- 14 -RSB- . In our study , higher EGFR expression was found in two cisplatin-resistant cell lines compared with the corresponding parental cell lines -LRB- Fig. 3 -RRB- . EGF receptors initiate cytoplasmic signaling through autophosphorylation of their intracellular domains -LSB- 19 -RSB- . Phosphorylated EGFR -LRB- p-EGFR -RRB- cytoplasmic tyrosine residues initiate the activated protein kinase pathway -LSB- 20 -RSB- . This mitogen-activated protein kinase -LRB- MAPK -RRB- pathway is involved in the mechanism of cisplatin resistance -LSB- 21 -- 23 -RSB- . Activation of JNK and p38 by cisplatin has been shown to promote apoptotic cell death -LSB- 22 , 23 -RSB- . In addition , activation of the ERK pathway by cisplatin was reported to promote induction of cell death -LSB- 21 -RSB- . In our study , higher p-EGFR expression was found in two cisplatin-resistant cell lines compared with the corresponding parental cell lines . These findings offer further evidence that activation of EGFR downstream signaling pathways is involved in cisplatin sensitivity . The effect of combining EGFR inhibitors with cytotoxic agents has been studied in various types of cancer -LSB- 16 , 24 -RSB- . The quinazoline derivative AG1478 is a specific reversible inhibitor of EGFR . The cancer cell growth depression effect of AG1478 has been reported in various carcinomas -LSB- 24 -- 26 -RSB- . In our study , the combination of AG1478 and cisplatin showed augmented effects of inhibition in both cell lines and showed large-effect augmentation in cisplatin-resistant cell lines . These results suggest that acquired resistance to cisplatin is associated with enhanced sensitivity to an EGFR tyrosine kinase inhibitor , which correlates with increased EGFR and p-EGFR expression . Augmented effects of inhibition of OSCC cell growth through the combination of AG1478 and cisplatin provide a potential and novel strategy for patients with oral cancer with acquired cisplatin resistance . In addition , it would be very advantageous if an equal chemotherapeutic effect could be obtained with a smaller dosage of cisplatin . The combination of AG1478 and cisplatin against OSCC definitely deserves additional in vivo and clinical studies .

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epidermal growth factor receptor (egfr)
oral squamous cell carcinoma (oscc)
cisplatin-resistant oscc cell line
cisplatin sensitivity and resistance