polycystic ovary syndrome ( pcos ) is characterized by oligo - anovulation , clinical or biochemical hyperandrogenism and/or polycystic ovaries ( franks , 1995 ; knochenhauer et al . , 1998 ; fauser , 2004 ) . insulin resistance ( ir ) accompanied by compensatory hyperinsulinaemia constitutes another major biochemical feature of pcos ( nestler and jakubowicz , 1996 ; dunaif , 1997 ; nestler , 1997 ; jakubowicz et al . , 2001 ; fleming et al . , 2002 ) . the syndrome affects 510% of women during reproductive age ( asuncion et al . , 2000 ) . because of the link between ir and pcos , metformin has been put forward as a drug to induce ovulation in women with pcos . in fact , metformin either alone or in combination with clomifene citrate is now the most widely used insulin - sensitizer for ovulation induction in women with pcos ( nestler , 2002 ; glueck et al . , 2003 ; the rationale of this treatment was based on only a few small studies with conflicting results ( nestler et al . , 1998 ; recently , we demonstrated in an rct among newly diagnosed , therapy naive women with pcos that there were no significant differences in either ovulation rate , ongoing pregnancy rate or spontaneous abortion rate in women using clomifene citrate plus metformin compared with women using clomifene citrate ( moll et al . , 2006 ) . since then , our results considering ongoing pregnancy rates have been confirmed in another large rct ( legro et al . , 2007 ) . however , pcos is a heterogeneous condition and thus both studies included a heterogeneous group of women . surprisingly , there is only one small study involving 32 patients with pcos that has investigated the effect of metformin in specific subgroups . this study showed in a multivariable analysis that higher insulin , lower androstenedione and less severe cycle abnormalities appeared to be independent significant parameters for better response to metformin ( moghetti et al . , 2000 ) . increasing age and high waist  hip ratio ( whr ) are known risk factors in developing ir and , as such , these factors may also affect clinical response to metformin ( despres , 1993 ; legro et al . , 1999 ; pasquali et al . , 2000 ; ncep expert panel , 2001 ; laine and wilson , 2007 ) . apart from these parameters , some authors suggest that metformin would most likely be beneficial in women with high bmi ( lord , et al . , 2003 ) . thus , although many investigators claim a beneficial effect of metformin in specific subgroups of women , the evidence is limited . we therefore wanted to determine whether co - treatment with metformin improves pregnancy rates compared with the standard treatment of clomifene citrate alone in subgroups of women based on clinical and biochemical variables . the data used in this study were collected in an rct that has been reported elsewhere ( moll et al . , 2006 ) . this double blinded trial took place from june 2001 to may 2004 in 20 dutch hospitals . all patients with chronic anovulation ( a menstrual cycle 35 days ) , world health organization type ii criteria ( normogonadotrophic normoestrogenic oligo- or anovulation ) , polycystic ovaries diagnosed by transvaginal ultrasonography and a desire to conceive were randomly allocated to clomifene citrate ( 50150 mg a day for five consecutive days in the beginning of a menstrual cycle , per os ) plus metformin ( 500 mg 4/day ) or to clomifene citrate plus placebo . primary exclusion criteria were other causes of anovulation , age over 40 years and liver , kidney or heart disease / failure ( i.e. abnormal results on liver function tests , serum creatinine concentration > 95 mol / l or a history of heart disease / failure ) and sperm quality indicating male subfertility ( total motile count < 10  10 ) . randomization was carried out in the coordinating centre ( amc , amsterdam ) , using computer - generated blocks of four . the randomization was stratified per centre , and the centres received blinded , numbered containers with medication . patients continued to take the study medication until a positive pregnancy test ( 4 weeks after the first day of menstruation ) , six ovulatory cycles or clomifene citrate resistance occurred , whichever came first . women had to discontinue their medication as soon as they had a positive pregnancy test as the safety and benefit of using metformin during pregnancy have not yet been proven . the study was approved by the institutional review boards of all hospitals . written informed consent was obtained from all participants . at the start of the study , we measured height , weight , whr and plasma testosterone . total plasma testosterone concentrations were determined using an in - house radioimmunoassay , without extraction and chromatography and with tritiated testosterone as label ( pratt et al . , 1975 ) . intra - assay and inter - assay coefficients of variation were 4 - 7% and 59% , respectively . subgroup analysis was performed on age , bmi , whr , testosterone , 2 h glucose and homa ( homeostatic model assessment for assessing insulin sensitivity ) as a measure of ir ( ehrmann et al . ncep expert panel , 2001 ; fauser , 2004 ; samaras et al . , 2006 ) . since not all data were available for all women , in some analyses only a proportion of the women the parameters to be analysed were chosen , after completion of the trial , according to the most recent literature ( despres , 1993 ; legro et al . , 2000 ; ncep expert panel , 2001 ; lord , et al . , 2003 ; laine and wilson , 2007 ) . for this subgroup analysis , we explored differences in treatment effect on ongoing pregnancy rate in subgroups defined on age , bmi , whr , plasma testosterone , 2 h glucose level and ir evaluated by homa - ir . for this purpose , we performed logistic regression analysis including treatment , the subgroup indicator and the interaction between treatment and subgroup . we assessed the linearity of the association between the continuous variables age , bmi , whr , testosterone , 2 h glucose level and homa and the ongoing pregnancy rate using spline functions ( harrell et al . , 1988 ) . non - linear associations were accommodated by redefining the corresponding variables . by testing the interaction in logistic regression , if the interaction is statistically significant , this is a clear indication that the effect of treatment differs between subgroups ( matthews and altman , 1996 ) . next , to explore potential interesting subgroup features , we combined subgroups for which the interaction with treatment was p < 0.20 . in all analyses , for this subgroup analysis , we explored differences in treatment effect on ongoing pregnancy rate in subgroups defined on age , bmi , whr , plasma testosterone , 2 h glucose level and ir evaluated by homa - ir . for this purpose , we performed logistic regression analysis including treatment , the subgroup indicator and the interaction between treatment and subgroup . we assessed the linearity of the association between the continuous variables age , bmi , whr , testosterone , 2 h glucose level and homa and the ongoing pregnancy rate using spline functions ( harrell et al . , 1988 ) . non - linear associations were accommodated by redefining the corresponding variables . by testing the interaction in logistic regression , if the interaction is statistically significant , this is a clear indication that the effect of treatment differs between subgroups ( matthews and altman , 1996 ) . next , to explore potential interesting subgroup features , we combined subgroups for which the interaction with treatment was p < 0.20 . in all analyses , baseline characteristics are presented in table  i. out of the 111 women in the clomifene citrate plus metformin group , 44 ( 40% ) reached an ongoing pregnancy versus 52 ( 46% ) of the 114 women in the clomifene citrate group , a difference which was not significant [ relative risk ( rr ) 0.87 ; 95% confidence interval ( ci ) 0.641.2 ] . a significantly larger proportion of patients in the metformin group discontinued treatment because of side effects ( 18/111 versus 6/114 ; rr 2.9 ; 95% ci 1.27.1 ) . the total dropout rate in both treatment arms was not statistically different ( 28/111 versus 21/114 ; rr 1.3 ; 95% ci 0.822.2 ) . baseline characteristics of patients with pcos who were treated with clomifene citrate plus metformin or clomifene citrate alone . homa , homeostatic model assessment for assessing insulin sensitivity ; whr , waist  hip ratio . the data for 2 h glucose level were available in 80% of patients , and for homa , in 50% of patients . in women 28 years of age , the effect of metformin versus placebo showed a small trend towards a different effect compared with the effect of metformin versus placebo in women < 28 years ( p - value of interaction 0.11 ) ( table  ii ) . in women with a whr  0.85 , the effect of metformin versus placebo was significantly different from the effect of metformin versus placebo in women with a whr < 0.85 ( p - value of interaction 0.012 ) ( table  ii ) . pregnancy rates for subgroups of women with pcos treated with clomifene citrate plus metformin or clomifene citrate alone . we did not find significant differences in effect of treatment for groups based on bmi , plasma testosterone , 2 h glucose values or homa . on the basis of these findings , we combined age and whr and created four subgroups . we found a significantly different chance of reaching an ongoing pregnancy of metformin versus placebo between these subgroups ( p - value of interaction 0.014 ) ( table  iii ) . a positive effect of metformin versus placebo on pregnancy rate was found in older women ( 28 years ) with a whr  0.85 , a negative effect of metformin versus placebo in young women regardless of their whr and no effect in the older , not viscerally obese women ( fig .  1 ) . relative risks and 95% confidence intervals for combined subgroups of women treated with clomifene citrate ( clomid ) plus metformin or clomifene citrate alone with respect to pregnancy rates . pregnancy rates for combined subgroups in women with pcos treated with clomifene citrate plus metformin or clomifene citrate alone . in the present analysis , there was a significantly different chance of an ongoing pregnancy of metformin versus placebo between subgroups , based on age and whr . a positive effect of metformin versus placebo on pregnancy rate was found in older women with a high whr , a negative effect of metformin versus placebo in young women regardless of their whr and no effect in older , not viscerally obese women . we have included women with pcos , who had never used clomifene citrate and were seeking treatment for their fertility problems for the first time . our study group reflects the largest group of women with pcos a fertility clinic will see and treat . our results contradict those from the only other subgroup analysis , where no differential effect of metformin was shown in subgroups of women based on age and whr ( moghetti et al . , 2000 ) : this study did show that in multivariable analysis higher insulin , lower androstenedione and less severe cycle abnormalities appeared to be significant parameters for better response to metformin , variables we did not assess in our study . however , our data may be more valid , because we analysed over 200 patients , 10 times more than in the moghetti et al . ( 2000 ) subanalysis and because our study was based on a double - blinded rct as opposed to the open protocol used in the previous subanalysis . the most likely reason why metformin has a beneficial effect on pregnancy rates in older and viscerally obese women is that ir is common in these women ( cowie et al . , however , we did not find a significant interaction between pregnancy rates and the outcome of other surrogate markers of ir , including bmi , plasma testosterone , 2 h glucose value and homa . this is probably due to the fact that whr is the most powerful predictor of ir of all surrogate markers ( rexrode et al . , 1998 ; lord et al . , 2003 ; alberti et al . , 2006 ; lord et al . , 2006 ) . we did not measure ir directly by means of the gold standard , the glucose clamp technique , since this is considered to be too invasive and burdensome to be part of a large clinical trial in outpatients ( legro et al . the lack of effect of metformin in patients with high bmi , increased plasma testosterone , higher 2 h glucose levels and homa may be real , if confirmed by further studies . however , another possibility is that the present study did not have enough power to show a difference . this might especially hold true for homa , since we only had data on half of the patients . a limitation of the study is that we did not collect all parameters in all patients . in particular , fasting insulin is missing in 49% of the patients . when comparing baseline characteristics of the patients for whom data on insulin were available with those for whom data were not available , we found no significant differences ( data not shown ) . the lack of data appears , therefore , to be due to a random effect of not executing the complete study protocol rather than a deliberate omission of patients with certain clinical or biochemical characteristics . after our study protocol was written ( 1999 ) and our study performed ( 20012004 ) , it became clear that testosterone assays show a wide inter - laboratory variability ( kane et al . , 2007 ; rosner et al . , 2007 ) . when we had finished the trial , it was impossible to adjust for this . the calculations performed in this trial were therefore performed without correcting for this inter - laboratory imprecision , as is the case in all previous literature . it is possible that the variation is sufficiently high to render the result not significant . this relatively new understanding of the variability of testosterone assays may have great impact , not only on existing but also on future literature on pcos , especially in multicentre studies . our rct was not powered for these subgroup analyses ; therefore , the data need to be interpreted with great caution . nonetheless , we feel that our data argue for performing a trial comparing metformin and clomifene citrate with clomifene citrate alone in older visceral obese women with pcos . although our initial randomized trial showed no benefit for metformin , our current findings suggest that metformin may be an effective addition to clomifene citrate in infertile , older and visceral obese women with pcos . randomized studies should be performed specifically for this subgroup , or data from other existing trials should be re - analysed before we can consider implementing these findings in clinical practice .