Hadjisavvas et al. BMC Cancer 2010, 10:447 http://www.biomedcentral.com/1471-2407/10/447 last pregnancy (at the beginning of the term). Other than BMI (which was based on actual measurements of weight and height), all other factors investigated here, were self-reported. Only pregnancies with a gestation period of at least 24 weeks were considered (including those not resulting in a live birth). Only use of oral con- traceptives or HRT for at least a period of 1 month was considered. Exercise was taken to mean any activity involving a minimum of 30 minutes walk, or other form of regular exercise including cycling, swimming, jogging, etc at least 3 times a week for the past 6 months. Type of occupation was not taken into account as a form of exercise. Data Analysis We computed frequencies of categorical variables (or means and standard errors in the case of continuous variables such as age) separately for cases and controls. The frequencies were cross-tabulated and differences in participant characteristics between cases and controls were statistically assessed using the c2 test. The strength of association between each factor and breast cancer risk was estimated by odds ratios (ORs), calculated using logistic regression before and after adjusting for the confounding effect of all other risk factors, in multi- variable models. In the case of ordered categorical vari- ables (e.g. age at menarche), p-value for linear trend was reported. Due to the narrow age span of women invited for screening (ages between 50 and 69), women younger than 45 or older than 65 were under-represented in the study. To control for age differences between cases and controls, all model estimates (including associations with single risk factors) were therefore adjusted for age. Furthermore, the effect of restricting the investigation to women between 45-64 years of age was also tested in sensitivity analyses. In all cases, the fit of the models was assessed on the basis of the Pearson c2 and/or Hos- mer-Lemeshow goodness-of fit statistics (which compare the observed against the expected counts as estimated by the model) producing a non-significant result. A number of characteristics investigated here are (a) dependent on each other such as pregnancy and breast- feeding, or (b) are nested in each other such as breast- feeding status vs. breastfeeding duration or (c) are expected to display a high degree of collinearity i.e. num- ber of children and breastfeeding. The inter-relationships between these variables and the extent to which they are independently associated with breast cancer risk was firstly assessed in separate analyses (using stratification techniques or multivariable logistic models as appropri- ate), with the aim of identifying appropriate course of action e.g. include the variable with the strongest effect in order to avoid multicollinearity in the final model. For instance, the possible protective effect of pregnancy and Page 3 of 10 the added effect of breastfeeding over the effect of preg- nancy was investigated in a logistic regression model that included the main effect of pregnancy and an interaction term between pregnancy and breastfeeding (to capture the combined effect of the two variables) but no separate main effect for breastfeeding (since only women who have had a child breastfed). Similarly, as women with more children may be more likely to breastfeed (and in fact breastfeed for longer), we assessed the extent to which breastfeeding confounds or modifies any observed association between breast cancer risk and number of children by calculating Mantel-Haenszel odds ratios after stratifying by breastfeeding status along with a c2 test of heterogeneity of estimates. All data manipulation and statistical analyses were performed in STATA SE 9.0. Results A total of 2286 women - 1109 breast cancer cases and 1177 healthy controls (98% and 98% participation rate respectively) - from all five districts of Cyprus partici- pated in the study. The mean ± SD age of cases and controls were 56.1 ± 9.1 and 55.7 ± 7.0 years respec- tively (p-value of t-test for difference in means = 0.22). Nevertheless, differences in the age distribution of cases and controls recruited into the study were observed mainly due to the under-representation of younger and older age-groups amongst the controls. Table 1 presents the distribution of socio-economic characteristics and potential risk factors investigated among cases and con- trols separately. One in four Cypriot women have never breast-fed, as many as 80% have had at least two chil- dren and more than 70% reported having never used hormone replacement therapy. No statistically signifi- cant differences between cases and controls were observed in terms of level of education, marital status, number of children or use of oral contraceptives. In contrast, a higher proportion of cases than controls reported family history of breast cancer (14.1% vs. 9.1%) and early menarche (14.2% vs. 11.4%). Some strong associations were observed between the reproductive factors investigated and breast cancer risk. In general, cases were less likely than controls to have been pregnant (OR 0.69, 95%CI 0.49-0.97) or to have breastfed (OR 0.69, 95%CI 0.57-0.82). These variables are expected to exhibit a high degree of collinearity. Table 2 investigates the synergistic role of (a) breast- feeding and pregnancy as well as (b) breastfeeding and number of children in breast cancer risk. The combined effect of breastfeeding and pregnancy appeared stronger than the effect of pregnancy alone; the association with the latter attenuated after accounting for breastfeeding. Interestingly, no stepwise relationship was observed between breast cancer risk and reported duration of breastfeeding. In fact, risk of breast cancer appeared