The association between induced abortion and birth control methods  and breast cancer may vary among countries, due to the different usage and frequency of birth control methods and induced abortion among countries. A better understanding of this association may help in determining safer birth control methods for Chinese women.A case-control study was conducted with a total of 794 cases and 805 controls. Standardized questionnaires were used to collect information on demographic characteristics, exposure to induced abortion, birth control methods, and other risk factors for breast cancer. Multivariate logistic regression was conducted to explore the association between birth control methods and breast cancer.Multivariate logistic regression analyses showed that having a history of medical abortions, ≥3 surgical abortions, or both medical and surgical abortions was associated with an increased risk of breast cancer in post-menopausal women . Pre-menopausal women who had used intra-uterine devices (IUDs) for more than 20 years tended to have a lower breast cancer risk than other age-matched pre-menopausal women . Both pre-menopausal and post-menopausal women who had <20 years exposure to IUDs and those who had used two or more birth control methods (with the exception of women who used IUDs for more than 20 years) tended to have much higher breast cancer risk.The relationship between induced abortion and birth control methods and breast cancer was complex, though being exposed to induced abortion and two or more birth control methods in one’s lifetime appeared to be risk factors for breast cancer in Chinese women. A multicenter clinical epidemiological study showed that the peak age of onset for Chinese female patients was 50–65 years, about 10 years earlier than that among women in western countries.2 Not coincidentally, women of this age were generally at reproductive age during the period when the Chinese birth-control policy (one child per family) was fully implemented.Breast cancer is the most common malignancy in Chinese women, whose incidence rate had risen from 23.37 per 100,000 in 2007 to 28.42 per 100,000 in 2013.3 Women who already had one child need to use birth control methods for a long period of time, and in some cases, women had induced abortion when other birth control methods failed. According to the China Health Statistics Yearbook 2016, the most common birth control method for Chinese women at childbearing age is induced abortion (IA), including surgical abortion (SA) and medical abortion (MA). In 2015, an estimated 9,851,961 women underwent IA in China, corresponding to a rate of 32.64 per 1,000 women aged 15 to 44.4 Use of an intrauterine device (IUD) is the second most common method, with an estimated 8,227,879 women having IUDs inserted in 2015 .5 Tcu220C is the most frequently used IUD type in China.6 The oral contraceptive (OC) usage rate among women of reproductive age in China is very low. In 2010, this prevalence rate was estimated to be 0.98%,7 which is much lower than that among American women aged 15–44 years.8 Some studies have focused on the relationships of the above risk factors with breast cancer in southeast China, but in mid-western China, where the one child birth control policy was implemented most stringently, there have been few relevant studies. Furthermore, Chinese women may use more than one birth control method over the course of their reproductive years, which leads to complex combinations of these exposures, making the relationships between birth control methods and risk of breast cancer difficult to discern. Zhen et al9 and Shi et al10 both found that there are positive relationships of MA and OC with breast cancer, while studies conducted by Wu et al11 and Li et al12 indicated no such relationship. As for studies focusing on IUD and breast cancer risk, few have been conducted in Chinese women.Breast cancer is estrogen-related, and it is believed that the policy of one child per family could be partially responsible for the increased incidence of breast cancer among Chinese women.This study is a matched case-control study conducted in Chengdu, with the purpose of exploring the effect of common birth control methods and IA on breast cancer in Chinese women. As a central city in southwest China, Chengdu is representative of the area in terms of economic level as well as composition of the population. In this study, breast cancer cases were recruited from three government-owned hospitals in Chengdu: Sichuan Provincial People’s Hospital, Sichuan Cancer Hospital, and West China Hospital. These three hospitals have reputations for providing high-quality cancer treatment, so patients come to them from across the southwest. Hence, the cases we recruited are representative of breast cancer patients from southwest China.13 Controls were recruited during the same period of time and were eligible if they had no previous diagnosis of malignancy or mental disorder, were confirmed breast cancer-free via breast ultrasound and mammography, had Han nationality, and had lived in Sichuan for more than 3 years. The numbers of urban and rural residents in our control group were 384 (47.7%) and 421 (52.3%), respectively.This was a matched case-control study with 794 cases and 805 controls. For the case group, 794 female patients with newly histopathologically diagnosed primary breast cancer were recruited from April 2014 through May 2015 from the hospitals listed above. Other inclusion criteria were Han nationality, had lived in Sichuan for more than 3 years, and had no history of mental disorder. We further excluded patients with metastatic breast cancer, as they had a low response rate. Among the 794 cases recruited, 424 (53.4%) were city dwellers and 370 (46.6%) lived in rural areas. For each case, a residence area-matched  female control was recruited from women who underwent annual physical examinations in two physical examination centers located in Wuhou district and Shuangliu county in Chengdu, to account for lifestyle differences caused by social economic level.The study protocol was approved by the ethics committee of Sichuan University. All subjects participated in the study voluntarily and signed informed consent forms.Women who met any of the following criteria were classified as post-menopausal: 1) absence of menstruation for at least 1 year (any age); 2) bilateral oophorectomy or estrogen deprivation therapy; 3) if there is not information on menstruation history, older than 60 years.Women who had at least one IA were categorized as having IA exposure.14,15 Therefore, we grouped women who have taken OC at any point within the past 10 years as OC users, and all others as non-users.Previous studies have found that OC may increase the risk of breast cancer, but there was no statistical difference in breast cancer risk between women who have not taken OC for the past 10 or more years and those who have never taken it, indicating that the effect of OC exposure on breast cancer risk may fades with withdrawal time.Women who had had an IUD implanted for at least 1 year were defined as having IUD exposure.2. We used 24.0 as the cutoff value for BMI, in accordance with the WGOC and The International Life Sciences Institute China Office.16BMI was defined as weight (kg)/height (m)Women who had previous diagnosis of diseases related to increased estrogen exposure, such as breast diseases, ovaries diseases, uterus diseases, and other chronic diseases , were defined as having a history of estrogen-related disease.Active smoking was defined as smoking for more than 6 months (including former smokers).We defined passive smoking as non-smokers who were exposed to tobacco smoking for more than 15 minutes at least 1 day per week.We defined alcohol drinking as participants who drunk alcohol at least one time each week.17 to calculate the amount of physical activity in the recent five years :17 The median MET in the control group was used as the cutoff value for categorization of high physical activity (>median MET) and low physical activity (≤median MET).We used the following formula from the Compendium of physical activitiesA structured questionnaire was used to collect information on demographics and risk factors for breast cancer, including general demographic characteristics , BMI , reproductive history , history of estrogen-related diseases, family history of cancer  and lifestyles . We also collected information on exposure to the following birth control methods in detail:IA: the number of medical abortions and surgical abortions.OC: the most recent OC use was collected to categorize women into OC users and non OC users. Women who had used OC in the last 10 years were defined as OC users, and others were defined as non-users. For OC users, further information on the types of OC used was collected, including long acting contraceptives (LOC), short acting contraceptives (SOC), and emergency contraceptives (EOC). As different types of OC have different components, their effects on breast cancer risk can be different. At present, the main components of LOC and SOC are estrogen and progesterone, while EOC contains progesterone only. Therefore, we classified OC exposure as either LOC or SOC exposure or as EOC exposure.IUD: the approximate dates of placing and removing the IUD were collected to calculate the total exposure time.All the information was collected by trained interviewers using in-person interviews. Data were entered into Epidata 3.0  and analyzed using SPSS 20.0 .t test or Chi-square test was used to describe and compare the distribution of demographic characteristics and breast cancer risk factors between cases and controls. Variables that were found to be statistically different between two groups were selected as covariates to be adjusted in the multivariate logistic regression. We further adjusted age in both the analyses of pre-menopausal and post-menopausal women. The key point of our analysis was clarifying the criteria of exposure to birth control methods. Based on the results of preanalysis and our previous research,18 we determined the cutoff values of times of IA use, times of OC use, and total time of IUD use. Then, in accordance with the results, women who met all the following conditions were defined as the reference group: never had IA or just had SA ≤2 times, and never used OC or IUD, and multivariate logistic regression was performed to explore the separate and combined effects of IA, OC, and IUD use on breast cancer risk and to calculate odds ratios (ORs) with 95% confidence intervals (CIs) after adjusting for confounders. Moreover, the dose-response relationship of IUD placing time was estimated using the Chi-square test for trend. Statistical tests were based on two-sided probability with a significance level of 0.05.Statistical analyses of pre-menopausal women were conducted separately from post-menopausal women. The Student t tests showed that among pre-menopausal women, cases and controls were similar with the exception of per capita annual income, active smoking, and passive smoking (P < 0.05). Among post-menopausal women, cases and controls were significantly different in terms of their age, per capita annual income, active smoking, alcohol drinking and history of live birth  and 43.35  years, respectively, and 346 cases and 342 controls were post-menopausal women with average ages of 58.55  and 56.60  years, respectively. Chi-square tests and Student Among pre- or post-menopausal women, compared with those who never had IA, women who experienced at least one MA had a higher risk of breast cancer  tended to have much higher breast cancer risk.19 but the majority of the participants were western women, whose lifestyles and usage of birth control methods and IAs were different from Chinese women. Therefore, the results may have weak representativeness with respect to Chinese women. By contrast, a recent meta-analysis of 36 studies covering 14 Chinese provinces found that Chinese women with a history of IA had 49% higher odds of getting breast cancer than those with no history of IA , and the risk of breast cancer increased with the number of abortions.20 It is well known that women’s estrogen and progesterone levels increase during a pregnancy, which promotes the growth of the mammary glands. But when a pregnancy is terminated by SA, estrogen and progesterone levels suddenly drop, which consequently terminates the growth of breast cells, leading to acinar atrophy. Incompletely differentiated breast epithelial cells are more susceptible to stimulation by carcinogenic substances, and therefore become cancerous relatively easily.21,22 As for the relationship between MA and breast cancer, previous research showed that drug containing antiprogestin was used for terminating pregnancy at the early stages,23 and the antiprogestin contained in the drug may interfere with the internal hormone environment, making it conducive to the development of hormone-related tumors. Meanwhile, SA and MA can increase the risk of breast cancer by delaying the timing of a full-term pregnancy, which is considered to be a protective factor of breast cancer.24 In a departure from previous studies, we investigated the effects of MA and SA separately and found that having SA <3 times had no impact on breast cancer occurrence. However, for women with a history of both MA and SA, the risk of having breast cancer was 88% higher than for those exposed to MA or SA alone. But regretfully, the time of induced abortion was not included in the present study, which is meaningful and should be analyzed in later study. Our results indicated that it would be better for government to discourage IA as a method to control the birth number.As a method of terminating pregnancy, IA includes both MA and SA. Only a few studies have specifically investigated the relationship between different types of IA and breast cancer risk, with conflicting results. One meta-analysis of fifteen prospective studies showed an insignificant association between breast cancer and IA ,25 A systematic review of 15 case-control studies and 8 cohort studies found that a history of OC use slightly increases breast cancer incidence , but the results were no longer significant when only United States-based studies were included .26 Two recent case-control studies conducted in Norway27 and Saudi Arabia28 both indicated that breast cancer risk increased only if OC use continued for at least 10 years.The relationship between OC and breast cancer is still inconclusive, although most studies have found that OC increases the risk of breast cancer. A case-control study of 200 cases and 403 controls indicated that OC non-users had a lower risk of breast cancer .Although the main components of OC are hormones, different types of OC have different components, so their effect on breast cancer may be different. However, none of the above studies distinguished LOC, SOC, and EOC, whereas in our study, OC users were further categorized as LOC/SOC users and EOC users. Our results showed that compared with women who did not have a history of OC use, pre-menopausal women with a history of LOC/SOC or EOC use were more susceptible to breast cancer. However, we did not find a statistically significant association between either LOC/SOC or EOC use history and breast cancer risk in post-menopausal women. This may be due to the low percentage of OC users in the controls; only 8% of the participants in the control group had a history of OC use. What’s more, the definitions of OC users and non-users were dependent only upon the last time subjects had taken OC, and had nothing to do with the dosage. Therefore, whether OC use increases the risk of breast cancer is not clear from this study.29 Currently, T-shaped copper IUDs are the most commonly-used type among Chinese women.6 A survey about estrogen and breast cancer conducted in Finland found a positive association between breast cancer risk and previous hormonal IUD use in post-menopausal women ,30 while a cohort study of 66,661 Chinese women in Shanghai found that more than 14 years’ exposure to IUDs may be a protective factor for breast cancer .31 We found that at least 20 years’ exposure to IUD was associated with decreased risk of breast cancer. A possible reason for this difference is that the previous studies did not rule out the effects of other birth control methods. The underlying mechanism of the protective effect of using an IUD for more than 20 years is still unclear, though Curtis indicated that IUD did not increase the risk of neoplasia.32 However, we also found placing IUD for less than 20 years was associated with an increased incidence of breast cancer. A possible explanation is that copper-containing IUDs were found to be related to a reduction in estrogen receptors in animal study,33 which could provide free estrogen for tumor formation. Furthermore, we also found that exposure to two or more birth control methods could be a risk factor for breast cancer. It is clear that IA, OC, and copper- or contraceptive-containing IUDs can affect breast cancer incidence by influencing the body’s estrogen level. There may be interactions between these methods in affecting breast cancer incidence, but the mechanism of these interactions remains unclear.The most common types of IUDs are T-shaped copper, ring, γ, and uterine cavity-shaped.Compared with previous studies, exposure to different birth control methods was measured more precisely in our study. Ours was also the first study we know of to investigate the combined exposure of different birth control methods on breast cancer risk using women with relatively low risk as the reference group. Our study also has some limitations. Although we have matched the cases and controls by residence area, selection bias is still unavoidable due to the case-control study design. Second, we only collected information on the time of OC use, not dosage, so we could not explore the relationship between OC dosage and breast cancer risk. Moreover, due to the limited sample size of our study, we were not able to investigate any high-order interactions among birth control methods in breast cancer risk. Finally, our limited sample size may be partially responsible for our statistically insignificant results for some potential associations. Thus, cohort studies with detailed surveys and large sample sizes are needed to further investigate the relationship between birth control methods and breast cancer risk in Chinese women.Our results provide some epidemiological evidence that MAs, SAs, and less than 20 years IUD use could increase the risk of breast cancer for post-menopausal women. As for pre-menopausal women, less than 20 years IUD use could also increase the risk of breast cancer, while more than 20 years’ IUD use was associated with a decreased breast cancer risk. For both pre-menopausal and post-menopausal women who had been exposed to two or three kinds of birth control methods, the risk of having breast cancer was higher.


CASC16 polymorphisms and breast cancer susceptibility.Genetic variants play a critical role in the development of breast cancer. This investigation aimed to explore the association between We conducted a case-control study of 681 patients and 680 healthy individuals to investigate the correlation of five SNPs with breast cancer in a Northwest Chinese female population. Odds ratios (OR) and 95% confidence intervals (CIs) were used to assess the association.p = 0.022; OR 1.21, p = 0.026). While rs3803662 was a protective role in breast cancer risk . Stratified analyses indicated that rs4784227 and rs12922061 would increase breast cancer susceptibility at age >  50 years. Rs3803662 was a reduced factor of breast cancer risk by age ≤ 50 years. Rs4784227 was significantly increased risk of breast cancer in stage III/IV. The rs45544231 and rs3112612 had a protective effect on breast cancer with tumor size > 2 cm. Rs4784227 and rs12922061 could enhance breast cancer risk in lymph node metastasis positive individuals. CASC16 rs12922061 and rs4784227 polymorphisms correlated with an increased risk of breast cancer in BMI >  24 kg/m2. Haplotype analyses revealed that Grs45544231 Trs12922061 Ars3112612 and Grs45544231 Crs12922061 Ars3112612 haplotypes decreased breast cancer risk.Our study found that rs4784227 and rs12922061 were significantly related to an increased susceptibility to breast cancer (OR 1.22, CASC16 genetic variants were significantly related to breast cancer susceptibility, which might give scientific evidence for exploring the molecular mechanism of breast cancer.Our study revealed that  BRCA1, BRCA2, PTEN, TP53, CYP17 and other different genes have demonstrated that their polymorphisms were associated with risk of breast cancer  is one of the common malignant tumors in women , also termed LOC643714, is a kind of long non-protein coding RNA and located at chromosome 16q12.1. Data from one study showed that CASC16 gene had a higher expression in breast cancer cells compared with normal cells  in the In this present case-control study, 681 unrelated Chinese female breast cancer patients and 680 healthy subjects were recruited from the Shaanxi Provincial Cancer Hospital. All patients were newly diagnosed with histological examination and confirmed to be BC. Patients with a history of autoimmunity, secondary tumors, severe infections diseases, other types of cancer and family history of any cancers included breast cancer were excluded. Healthy individuals were matched with the case subjects based on age and ethnicity, who were randomly selected from the cancer-free female population with a routine health examination in the same hospital. Controls with the family history of any cancers were excluded. Each study participant was informed the purpose of the sample collection and their written consent were obtained. The participants’ basic information were obtained from the patients or their medical records including age, ethnicity, place of residence, tutor position, lymph node metastasis status, clinical stage, tumor size, estrogenic receptor (ER), progesterone receptor (PR) status, menopausal status, procreative times, age of menarche, and body mass index (BMI). All experiments were carried out depending on the guideline of Helsinki’s declaration and our study were approved by the ethics committee of the Shaanxi Provincial Cancer Hospital.CASC16 in the present study. Of the five SNPs, three polymorphisms  were chosen basing upon the published papers which they reported that these SNPs might be related to breast cancer susceptibility  to design PCR primers. SNP genotype was identified by Agena MassARRAY iPLEX platform, and Agena Bioscience TYPER version 4.0 software was used to manage and analyze the data  and 95% confidence intervals (CIs) in five inheritance models  using logistic regression analysis with or without adjustment for age or BMI. Linkage disequilibrium (LD) was constructed by Haploview V4.2 software and haplotype was analyzed by logistic regression. Besides, we also evaluated the relationship between CASC16 polymorphisms and BC patient subgroups with stratification analyses. All statistical analyses were performed using SPSS version 17.0 software  and PLINK software. All statistical tests were two-tailed and p-value < 0.05 was considered statistical significance.The differences in demographic characteristics between the case and control group were analyzed by continuous variable independent sample t-test and category variable Pearson’s chi-square test. Hardy–Weinberg equilibrium (HWE) of each SNP was tested by chi-squared test to assess genotype frequencies in controls. Comparisons of distribution in SNP allele and genotype frequencies between case and control were checked by a Pearson chi-squared test or Fisher′s exact test. The association between p = 0.930).The basic information of the study subjects was summarized in Table CASC16 gene were selected and analysed in this case-control study. The distribution of allele frequencies between cases and controls was compared using chi-square test (Table p > 0.05). It means appropriate SNP selection. And our results showed that the minor allele of two SNPs (rs4784227 and rs12922061) were significantly associated with increased BC susceptibility under allele model . We further examined the correlation between the genotypes of SNPs and BC risk by logistic regression analysis with adjustments for age under the codominant, dominant, recessive, and log-additive models (Table p = 0.048), dominant model  and the log-additive model . The rs12922061 also had a significant higher susceptibility to BC in codominant model  and log-additive model . In contrast, rs3803662 was associated with a reduced risk of BC in recessive model . Two SNPs (rs45544231 and rs3112612) were not observed association under any of the genetic models.Five SNPs in the st Table . All fivp = 0.007), codominant model , dominant model  and log-additive model  of the patients at age >  50 years (Table p = 0.012), codominant model , dominant model , and log-additive model  in subjects > 50 years old. However, the G/G genotype of rs3803662 played a reduced role in risk of breast cancer under the recessive model  of the patients ≤50 years. We also assessed the effect of CASC16 gene polymorphisms on BC risk by clinical characteristics including clinical stage, tumor size, lymph node metastasis, and BMI. As was displayed in Table p = 0.031) compared with stage I/II. The allele ‘C’ and C/C genotype of rs45544231, allele ‘G’ and G/G genotype of rs3112612 had protective effect on susceptibility of breast cancer with tumor size > 2 cm  than of tumor size ≤2 cm. The results further confirmed that TC + TT genotype of rs4784227 was significantly associated with an increased BC risk in lymph node metastasis positive individuals . Minor allele ‘T’ of rs12922061 was also noted to improve BC susceptibility in lymph node metastasis positive participants . In addition, the CASC16 polymorphisms correlations with breast cancer were carried out in accordance with BMI-based stratification .The association between five SNPs and BC susceptibility was analyzed by logistic regression under age and clinical characteristic subgroups Tables  and 5. OCASC16 polymorphisms in case and control subjects via Haploview software and logistic regression. The LD plot was shown in Fig. rs45544231 Trs12922061 Ars3112612 and Grs45544231 Crs12922061 Ars3112612 haplotypes in the CASC16 gene were found to reduce risk of breast cancer  and haplotype analyses of CASC16 variants and BC risk in a Northwest Chinese female population. The research showed that CASC16 polymorphisms  were significantly associated with BC susceptibility. Furthermore, rs4784227, rs12922061, rs3803662, rs45544231, and rs3112612 polymorphisms were associated with breast cancer patients with stratified subgroups including age, lymph node metastasis status, clinical stage, tumor size, and BMI. Taken together, these findings suggested an important role for the CASC16 gene in the occurrence of breast cancer.In the present case-control study, 681 breast cancer patients and 680 free-cancer subjects were recruited to evaluate the correlation between CASC16 gene association with breast cancer as previously published studies  in a Northwest Chinese population, and the same finding was showed in patients ≤50 years . However, Edward A et al. suggested that no relationship was found between rs3803662 and breast cancer in African-American population  who confirmed that rs4784227 could increase risk of breast cancer in a Southern Chinese population, while they hadn’t identified correlation under stratified analysis  and haplotype analyses of CASC16 polymorphisms in cases and controls. Haplotype analyses disclosed that Grs45544231 Trs12922061 Ars3112612 and Grs45544231 Crs12922061 Ars3112612 haplotypes reduced BC risk.Furthermore, our study firstly revealed that rs45544231 and rs3112612 in SCAC16 variants and breast cancer in a Northwest Chinese population. Further research in other areas or races in China is an essential step in supplementing the extant data. Besides, we determined the role of CASC16 SNPs in risk of breast cancer but there were still not detecting function of CASC16 in occurrence and evolution of breast cancer. Therefore, next work should focus on exploring the functions of CASC16 in breast cancer. In spite of its limitations, the study certainly adds to our understanding of the association between SNP variants and breast cancer. Moreover, our present work provided the possibility of using these SNPs to diagnose breast cancer in the future.The major limitation of this study was the fact that we just studied the association between CASC16 rs4784227 and rs12922061 were significantly related to increased susceptibility to breast cancer. Stratification analysis revealed that rs4784227 and rs12922061 would increase BC susceptibility in age > 50 years. Rs3803662 was a reduced factor of BC in age ≤ 50 years. Rs4784227 was significantly improved susceptibility to BC patients in stage III/IV. The rs45544231 and rs3112612 had protective effects on BC with tumor size > 2 cm. Rs4784227 and rs12922061 could increase BC risk in lymph node metastasis positive individuals. CASC16 rs12922061 and rs4784227 polymorphisms were correlated with increased BC risk in BMI > 24 kg/m2. We noted that Grs45544231 Trs12922061 Ars3112612 and Grs45544231 Crs12922061 Ars3112612 haplotypes reduced BC risk. These findings would give some new insights in the molecular mechanism of breast cancer occurrence.In summary,


C-peptide, insulin, leptin, and other metabolic hormones are assumed to play roles in breast cancer development; though, results are inconsistent. In this prospective case-control study nested within the Mano a Mano Cohort Study, we assessed the risk of breast cancer with regard to plasma levels of c-peptide, gastric inhibitory polypeptide, insulin, leptin, monocyte chemoattractant protein-1, pancreatic polypeptide, and peptide YY. Among women followed for a median of 8.5 years, 109 breast cancer cases were identified and frequency-matched to 327 controls at a ratio of 1:3. Overall, only c-peptide was observed significantly associated with breast cancer risk. High c-peptide levels (≥ the median level of controls) were significantly associated with increased breast cancer risk . In an analysis of participants stratified by age, the significant association between c-peptide levels and breast cancer risk was evident in only women age ≥51 years . Among women age <51 years, high leptin levels were significantly associated with decreased breast cancer risk . Our findings suggest that selected metabolic hormones are associated with breast cancer development in Mexican American women. Metabolic disorders are related to every step of breast carcinogenesis7. For example, in the National Institutes of Health–American Association of Retired Persons Diet and Health Study, Dibaba et al. found that women with metabolic syndrome had a 13% higher risk of breast cancer compared with their female counterparts 3. In addition, overwhelming evidence indicates that an excess of body fat is an independent risk factor for breast cancer, particularly among postmenopausal women9. Also, one recent meta-analysis of 28 cohort studies revealed that diabetic patients have a significantly higher risk of breast cancer than nondiabetic patients do10.Metabolic syndrome and its individual metabolic conditions, including increased blood pressure, high blood sugar, excess body fat around the waist, and abnormal cholesterol or triglyceride levels, are associated with increased risk of multiple chronic diseases, including cardiovascular disease and cancerin vitro and in vivo12. In addition, insulin deters sex hormone binding globulin production13, thereby increasing free estradiol and testosterone. C-peptide is a marker of pancreatic insulin secretion14. Leptin promotes breast cancer cell growth by hindering pro-apoptosis signaling pathways and by preferring sensitivity to estrogens15. PYY and PP, members of the neuropeptide Y family of peptide hormones, are neurotransmitters; both have roles in appetite regulation and obesity17. In breast tumors, neuropeptide Y can induce tumor cell growth in a dose-dependent manner18. GIP is an incretin hormone that is involved in regulating circulating glucose and insulin secretion19. Its relationship with breast cancer is unclear, but the GIP receptor has been identified as a therapeutic target in patients with neuroendocrine tumors20. MCP-1, a key pro-inflammatory chemokine that regulates monocyte activity, is involved in various diseases, including cancer21. Recently, MCP-1 was found to be highly expressed in triple-negative breast cancers and consequently involved in tumor invasion and metastasis22.However, the ways in which metabolic syndrome and its individual metabolic conditions link to breast carcinogenesis is still largely undecided. The relationship may be partially mediated by certain metabolic hormones secreted by endocrine system organs, adipocytes, and/or the gastrointestinal tract in response to metabolic stimuli, as some of these hormones are suspected to play roles in breast cancer development. In the current study, we focused on 7 markers: pancreas-derived c-peptide, insulin, and pancreatic polypeptide (PP); gut-derived gastric inhibitory polypeptide (GIP) and peptide YY (PYY); and adipocyte-derived leptin and monocyte chemoattractant protein-1 (MCP-1). Experimental evidence suggests that insulin can promote breast cell growth 23. More Mexican American men have elevated fasting glucose levels than non-Hispanic white men do, and more Mexican American women have high waist circumference, reduced high-density lipoprotein cholesterol, and elevated fasting glucose than their non-Hispanic white counterparts do23. To the best of our knowledge, no prospective study has assessed the extent to which metabolic hormone levels are associated with breast cancer risk in Mexican American women. Therefore, in the present study, we measured pre-diagnostic levels of 7 metabolic hormones in plasma samples from 109 breast cancer patients and 327 healthy controls identified from the Mano a Mano Cohort Study and investigated their relationships with breast cancer risk.Investigation of metabolic hormones and their relationships with breast cancer are particularly relevant to Mexican American women, who are experiencing an epidemic of metabolic disorders24. In brief, participants of the Mano a Mano Cohort Study were recruited through community centers, local health clinics, and house-by-house canvasing in predominantly Mexican American neighborhoods in Houston, Texas, and through networking with currently enrolled participants. Eighty-eight percent of the identified eligible households agreed to participate in the study, and written informed consent was obtained from each participant. Trained bilingual research interviewers conducted structured, face-to-face interviews using each participant’s preferred language (either Spanish or English). A standardized and validated questionnaire, which captured information on basic sociodemographic characteristics, residential history, lifestyle behaviors, physical activity, medical history, family history of chronic disease, acculturation, and occupational exposure, was used in the interview. Participants were followed with annual telephone calls to obtain updated information regarding body weight, selected exposures, and diagnosis of selected chronic diseases, including cancer, type 2 diabetes, and hypertension. The cancer cases were further confirmed with the Texas Cancer Registry.The study participants were drawn from the ongoing Mano a Mano Cohort Study, a large population-based prospective cohort study of Mexican American households that was initiated in 2001 by the Department of Epidemiology at The University of Texas MD Anderson Cancer Center. Eligible participants in the Mano a Mano Cohort Study had to self-identify as Mexican or Mexican American. Detailed descriptions of the recruitment strategy and data collection procedures have been described previouslyThe women were followed until December 1, 2017 . A total of 126 new breast cancers were identified. Among them, 109 were validated through the Texas Cancer Registry and had blood samples that were collected at baseline. For each case, 3 matched controls were selected using an incidence density sampling protocol from appropriate risk sets consisting of cohort members who were alive and free of cancer at the time of diagnosis of the index case. Matching criteria were age at recruitment (±2 years), date of biospecimen collection (±1 year), and gender. Thus, the study included 109 cases and 327 controls. The study protocol was approved by MD Anderson’s Institutional Review Board.Plasma samples were analyzed using Luminex multiplex technology, which assesses multiple analytes in a single microwell plate, with the MILLIPLEX MAP Human Metabolic Hormone Magnetic Bead Panel (Millipore). This panel enabled the simultaneous analysis of the 7 metabolic hormones of interest: c-peptide, GIP, insulin, leptin, MCP-1, PP, and PYY. The MAGPIX System and an xPONENT 4.2 MAGPIX analyzer (Luminex) were used to analyze the samples. All samples were analyzed in triplicate. Negative controls, standards, and positive controls were included in each plate. Blinded duplicates (5%) were randomly inserted with the samples for quality control purposes. Samples from each case and its 3 matched controls were analyzed in the same plate. Any samples yielding results with an intra-assay coefficient of variation >10% were reanalyzed. The data were processed with xPONENT software using 5-parametric curve fitting and converted to pg/mL. The intra-assay variance was 7% for c-peptide, 5% for GIP, 5% for insulin, 4% for leptin, 8% for MCP-1, 4% for PP, and 6% for PYY.25. To assess the effect of plasma levels of metabolic hormones on breast cancer risk, we used unconditional multivariate logistic regression to estimate odds ratios (ORs) and 95% CIs. We ran a minimally adjusted model, adjusting for basic demographic variables  category, and education level), and a fully-adjusted model, with the following additional healthy behavior related variables . Metabolic hormone levels were designated “high” or “low” using the controls’ median levels of the hormones as cutoffs and were assessed as categorical variables. Finally, in an analysis in which participants were stratified by age group, we used similar multivariate logistic regression analysis to assess relationships between metabolic hormones and breast cancer risk.We used the statistical software package SAS version 9.4  for all analyses. First, we evaluated whether selected sociodemographic characteristics and lifestyle behaviors differed between breast cancer patients and healthy controls; the Student t test was used for 2-level dichotomous variables, and analysis of variance was used for variables with more than 2 levels. To assess relationships among metabolic hormones, we evaluated the pairwise correlations between all hormones among the controls. Hormones with pairwise correlations >0.5 were considered to be highly correlated and to have possible redundancy. Next, we used the Wilcoxon rank-sum test to evaluate whether the median plasma levels of metabolic hormones differed according to selected sociodemographic characteristics and lifestyle behaviors of the controls. To control for multiple comparisons, we set the false discovery rate at 0.05All procedures in this study were approved by MD Anderson’s Institutional Review Board and performed in accordance with the Declaration of Helsinki.Written informed consent was obtained from all participants.The basic sociodemographic characteristics and lifestyle behaviors of the 109 breast cancer cases and 327 healthy controls are summarized in Table We investigated the pairwise correlations among the 7 plasma metabolic hormones in the controls and found several significant correlations Table . C-peptiWe assessed relationships between the plasma levels of 7 metabolic hormones and sociodemographic characteristics and lifestyle behaviors among controls Table . As expeWe evaluated the associations between plasma levels of metabolic hormones and breast cancer risk Table . For eacWhen study participants were further stratified using the median age of the controls as a cutoff, higher c-peptide levels were significantly associated with breast cancer risk in only women age ≥51 years after adjustment for birthplace, language acculturation, parity, BMI category, and education level   but not women age <51 years. In addition, when younger and older women combined together, the association remained significant. Although the observed age difference in risk association is consistent the literature reports, we cannot exclude the likelihood of probable biases/measurement error that can influence the observation. Our study used BMI to identify obesity. However, a recent study has shown that BMI is a suboptimal marker for adiposity in the elderly30. Also, there may exist detection bias since tumor may be diagnosed later among obese women.Several previous studies have investigated the association between pre-diagnostic c-peptide levels and breast cancer risk31. Hyperinsulinemia with insulin resistance, which causes increased levels of insulin in circulation, has been linked to breast cancer34. Thus, the association of higher plasma levels of c-peptide with elevated risk of postmenopausal breast cancer is consistent with the notion that hyperinsulinemia is involved in in breast cancer development. Two hypotheses have been proposed to explore the underlying molecular mechanism. C-peptide may 1) potentiate the insulin receptor and/or 2) increase the concentration of bioavailable sex hormones, and thereby influence the action of insulin on breast cancer cell growth directly and/or indirectly35. Additionally, stimulation of the mitogen-activated protein kinase (MAPK) and the phosphoinositide 3-kinase (PI-3K) pathways has been proposed to be involved in the action of insulin and its receptor on promoting cell growth36.C-peptide, which is released into the blood as a byproduct of insulin, is considered to be a marker of insulin production and hyperinsulinemiaIn the present study, plasma c-peptide levels were significantly positively associated with BMI and BMI category. This finding is in line with the notion that obesity causes insulin resistance and hyperinsulinemia. This observation might also help explain the observed age difference in the association between c-peptide and breast cancer risk, as BMI is a protective factor for breast cancer in premenopausal women but a risk factor for breast cancer in postmenopausal women. Nevertheless, the observed risk continued fairly unchanged by adjustments for BMI, signifying that the influence of insulin on breast cancer risk was not related to excess weight.38. For example, in a prospective case-control study nested within the Nurses’ Health Study II cohort, after adjusting for BMI at age 18 years, weight change from age 18 years to blood draw, and other breast cancer risk factors, plasma leptin was a protective factor for breast cancer37. Leptin is thought to be a link between obesity and obesity-related complications including metabolic syndrome, type 2 diabetes, and cancer39. In breast epithelial cells, leptin can stimulate cell proliferation in obese women by accelerating the change of aromatizable androgens to estradiol. However, among postmenopausal women, their levels of circulating estrogens decline41. That is probably why we didn’t see significant association between leptin and breast cancer risk in older women. On the other hand, leptin is involved in the regulation of ovarian folliculogenesis42 and at high levels may reduce follicular estradiol secretion43. Such reduction is particularly relevant to premenopausal or younger women since it can help explain why high leptin may lower breast cancer risk among them.In our study, the relationship between leptin and breast cancer risk differed depending on age; increased plasma leptin levels were significantly associated with decreased breast cancer risk in younger participants (<51 years old) but not older participants (≥51 years old). This finding is in line with the results of several other cohort studies44.In the present study, GIP, insulin, leptin, and MCP-1, in addition to c-peptide, were significantly associated with BMI, even after adjustment for multiple comparisons. This finding is consistent with these hormones’ roles in metabolic syndrome and its individual metabolic conditions. We also found that levels of c-peptide and insulin were significantly increased with the time spent sitting per day after multiple comparison adjustment. This is consistent with the observation that being less physically active is a risk factor for insulin resistance27. Finally, we don’t have data on estrogen status so we cannot assess whether the relationships between plasma metabolic hormones and breast cancer risk differ by tumor subtype. A few studies have suggested that the relationship between obesity and breast cancer risk differs between estrogen receptor positive (ER+) and ER− breast tumors in postmenopausal women47. Given the strong correlations between those metabolic hormones and obesity, it is likely that the observed associations between plasma metabolic hormones and breast cancer risk are more evident among for ER+ than ER- breast tumors. However, in an analysis from Nurse Health Study, the association between plasma c-peptide and breast cancer was stronger among ER− than ER+ breast tumors48. Clearly, more studies at here filed are needed.Our study had several potential limitations. For example, we measured metabolic markers at only one time point, which prevented us from evaluating the value changes over time. Data on menopausal status at the time of diagnosis were lacking. Hence, we chose to use age at diagnosis as an estimation of menopausal status at the time of diagnosis when stratifying participants. In addition, the blood samples used in this study were collected from patients who had not been fasting. Fasting plasma metabolic markers may be better biomarkers than non-fasting onesDespite these potential limitations, our results provide the first evidence that higher c-peptide levels are significantly associated with increased breast cancer risk among older Mexican American women and that higher leptin levels are significantly associated with decreased risk of breast cancer among younger Mexican American women. Large prospective studies to validate our results are warranted.


Oxidative stress (OS) is a key characteristic feature in cancer initiation and progression. Among multiple cancers, NADPH oxidase (NOX) dependent free radical production is implicated in oxidative stress. P22phox, a subunit of NADPH oxidase encoded by the CYBA gene has functional polymorphisms associated with various complex diseases. The present study was aimed to examine the importance and association of the functional polymorphisms of CYBA gene (-930 A/G and 242 C/T) with the oxidative stress in breast cancer (BC) development and progression.We have performed a case-control study on 300 breast cancer patients and 300 healthy individuals as controls to examine the role of CYBA gene -930 A/G and 242 C/T single nucleotide polymorphisms (SNPs) using As-PCR and PCR-RFLP assays and its association with OS as measured by plasma MDA levels. Linkage disequilibrium (LD) plots were generated using Haploviewtool and Multifactor dimensionality reduction (MDR) analysis was applied to assess high-order interactions between the SNPs. The Insilco analysis has been performed to predict the effect of SNPs on the gene regulation using online tools.p < 0.05). The haplotype combination -930G/242C and -930G/242T were associated with 1.44 & 1.56 folds increased risk for breast cancer respectively. Further, the MDA levels were higher in the patients carrying -930G/242C and -930G/242T haplotype (p < 0.001). Our results have been substantiated by Insilco analysis.We have found that genotype frequencies of CYBA gene -930 A/G and 242C/T polymorphism were significantly different between controls and BC patients (Results of the present study suggest that GG genotype of -930 A/G polymorphism, -930G/242C and -930G/242T haplotypes of CYBA gene polymorphisms have shown association with higher MDA levels in breast cancer patients, signify that elevated oxidative stress might aid in increased risk for breast cancer initiation and progression. Breast cancer (BC) is one of the most frequent malignant tumors, and its morbidity and mortality rates have been increasing in developing countries such as, India . The breOxidative stress (OS) is a key risk factor for cancer initiation and progression  resultinIntracellular compartments such as mitochondria, is the major site of ROS production . Enzymes+ catalyzed by NADPH oxidase generates superoxide radical from oxygen. NADPH oxidase enzyme present in cytoplasmic membrane of phagocytic cells was described first as an enzyme involved in the generation of ROS in the phagocytic cells  located in the promoter and the 242 C/T polymorphism (rs4673) located in the exon 4 of the CYBA gene, and the level of oxidative stress in BC patients. Therefore, the present study was aimed to examine the importance and association of the functional polymorphisms of CYBA (-930 A/G and 242 C/T) with the oxidative stress in BC development and progression.To date little is known about the association of -930 A/G polymorphism , was assayed by the method described previously . BrieflyGenomic DNA was isolated from blood sample using a non-enzymatic method . PolymorP-value lower than 0.05 was considered statistically significant. For comparison of continuous variables in demographic data between controls and breast cancer patients, Student’s t-test was performed. Observed genotype frequencies were tested for deviation from Hardy-Weinberg equilibrium with the chi-square goodness-of-fit test (χ2). Risk estimates were calculated for co-dominant, dominant and recessive genetic models using SNPStats. Odds ratios (OR) and their 95% confidence intervals (CI) were estimated using a univariate analysis. Linkage Disequilibrium (LD) plots were generated using Haploview (v.4.2) software. Multifactor dimensionality reduction (MDR) analysis was performed to identify high-order interaction models that were associated with BC risk using open-source MDR software (v.2.0 beta 8.4).Demographic, clinical, and biochemical variables are expressed as the mean ± SD. All statistical tests were two-sided, a http://gene-regulation.com/pub/programs/alibaba2/index.html) (http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi) online tool (https://zhanglab.ccmb.med.umich.edu/I-TASSER/) (Prediction of presumptive changes in transcription factor binding sites (TFBS) caused by nucleotide alterations in the promoter region was performed with AliBaba software 2.1 (ex.html) . Pre-mRNine tool . The 3D TASSER/) .p < 0.05).The baseline characteristics are summarised in p = 0.034). The allelic distribution has revealed that the prevalence of the G-allele was significantly higher in cases and conferred increased risk for breast cancer compared to A-allele .The genotypic and allele frequency distribution of the CYBA -930 A/G polymorphism is represented in p = 0.036). The allelic association revealed that the minor allele T of 242 C/T polymorphism was associated with an increased risk of BC .The genotype and allele frequency distribution of CYBA 242 C/T polymorphism among the controls and patients with breast cancer is presented in p = 0.05) and 1.56  respectively compared with the common haplotype (-930A/242C).We further have analysed the haplotype frequencies with respect to CYBA gene polymorphisms in association with risk of breast cancer. Our analysis has revealed a total of four haplotypes as shown in D′ = 56) between the markers in BC patients and a weak LD (D′ = 31) between the markers in controls as shown in the Pairwise LD was computed for CYBA -930 A/G and 242 C/T polymorphism in cases and controls separately. LD plots revealed a moderate LD  compared to the control (2 ± 0.69 nmoles/µl) group as depicted in Breast cancer is a common disease worldwide and also one of the leading cause of cancer deaths in India . Breast In the present study, a higher frequency of breast cancer patients with smoking or alcohol habits was observed. Multiple reports have also shown that habit of smoking and alcohol consumption were associated with increased risk for breast cancer as they are more exposed to free radicals leading to oxidative damage to lipids, proteins and DNA that may aid in cancer progression . In contAlteration in expression of enzyme system that produces ROS such as NADPH oxidase (NOX) has been shown to be an important susceptibility factor for cancer . The mosThe C242T polymorphism has been demonstrated to be related to multiple diseases . ResultsMDA is a naturally occurring endogenous product of lipid peroxidation and prostaglandin biosynthesis, but is mutagenic and carcinogenic. Oxidative stress as measured by an increase in MDA levels was established in gastric, colorectal adenomas, prostate and oral cancer . In thisThere are several limitations in this study. The foremost limitation to our study concerns the use of limited sample size, which prevented us from drawing causal relationships. Owing to its importance as an oxidative stress indicator, we have measured MDA levels; however, MDA levels alone are not a sole indicator of oxidative stress and we have not directly quantified the NADPH oxidase activity. Further studies on CYBA gene polymorphisms/haplotypes, along with different oxidative stress markers, should be done in a multicenter, multi-ethnic population and with a large number of patients to strengthen our findings.In conclusion, our results suggest that the individuals with GG genotype of -930 A/G polymorphism, -930G/242C and/or -930G/242T haplotypes of CYBA gene may predispose to increased oxidative stress. Therefore, more attention should be paid to oxidative stress-related pathological manifestations in individuals with the risk genotype/haplotype, as it plays an important role in development and progression of breast cancer.10.7717/peerj.5509/supp-1Supplemental Information 1Click here for additional data file.


The revised-Goldberg method was used to characterize EI misestimation. Four strategies assessed the influence of EI misestimation: Retaining individuals with EI misestimation in the cluster analysis (Inclusion), excluding before (ExBefore) or after cluster analysis (ExAfter), or reassigning into ExBefore clusters using the nearest neighbor method (InclusionNN). Misestimation of EI affected approximately 50% of participants. Cluster analysis identified three patterns: Healthy, Meats/Pizza and Sweets/Dairy. Cox proportional hazard regression models assessed associations between the risk of cancer and dietary patterns. Among men, no significant associations (based on an often-used threshold of p < 0.05) between dietary patterns and cancer risk were observed. In women, significant associations were observed between the Sweets/Dairy and Meats/Pizza patterns and all cancer risk in the ExBefore (HR (95% CI): 1.28 (1.04–1.58)) and InclusionNN (HR (95% CI): 1.14 (1.00–1.30)), respectively. Thus, strategies to address misestimation of EI can influence associations between dietary patterns and disease outcomes. Identifying optimal approaches for addressing EI misestimation, for example, by leveraging biomarker-based studies could improve our ability to characterize diet-disease associations.The objective of this study was to determine the influence of strategies of handling misestimation of energy intake (EI) on observed associations between dietary patterns and cancer risk. Data from Alberta’s Tomorrow Project participants ( Cancer continues to exert a large toll on morbidity and mortality globally . Cancer Epidemiological studies investigating associations between eating patterns and disease risk are typically reliant on self-reported intake captured using tools such as food frequency questionnaires (FFQ) . HoweverEvidence from validation studies indicates that estimation of energy intake (EI) is particularly affected by measurement error . Given tFor the purpose of population-based research, alternative methods have been developed to assess the plausibility of reported EI derived from self-reported food and beverage consumption in relation to EE based on basal metabolic rate (BMR) and physical activity level (PAL) . For exak-means clustering, and risk of all cancers, a subgroup of cancers with strong evidence of association to diet, and digestive system cancers, among adults.Once energy misestimation is characterized, researchers must determine how to handle it in their analyses. Excluding individuals determined to have implausible EI estimates is not recommended but it has been suggested that analyses are stratified based on energy reporting status  or that Alberta’s Tomorrow Project (ATP) is a prospective cohort of ~55,000 Albertans established in 2000 to facilitate studies into the etiology of cancer and chronic diseases. Recruitment, enrollment, and data collection methods are described in detail elsewhere ,24,25. Bn = 29), had a prior cancer diagnosis, except for non-melanoma skin cancer, assessed via ACR linkage (n = 71), were recruited as the second ATP member in their household (n = 342) , were pregnant (n = 63), or were characterized as underweight (body mass index (BMI) <18.5) based on self-reported heights and weights (n = 18)  were excluded since these values are required to calculate BMR for the purpose of the revised Goldberg method. The final sample sizes were n = 9,847 men and n = 16,241 women.Inclusion in the current study was limited to participants who consented to administrative data linkage and completed the HLQ, PYTPAQ, and CDHQ-I. Participants were excluded if they resided outside of Alberta ( disease ). AdditiThe CDHQ-I is a 257-item past-year FFQ based on the Diet History Questionnaire developed by the U.S. National Cancer Institute  and modiThe PYTPAQ collects domain-specific  information on frequency, duration, and intensity of physical activity in the past 12 months . The PYTFor EI estimation, participants were classified as EI under-reporters, plausible-reporters, or over-reporters using the revised Goldberg method ,32. Brie). Another subgroup of 11 primary cancers were chosen based on the World Health Organization (WHO) classification of digestive system cancers [Primary incident cancer cases  were obtained by linkage to ACR in July 2017. The International Classification of Diseases for Oncology 3rd edition (ICD-O-3) was used to identify individual cancers. A subgroup of 21 primary cancers were identified based on a matrix from the World Cancer Research Fund/American Institute for Cancer Research Continuous Update Project (WCRF/AICR CUP) reporting on dietary components with convincing or probable evidence for increased or decreased risk of cancer  Dietary. Another cancers  . In participants who remained cancer-free but died before linkage to ACR, follow-up time was calculated from age at enrollment to age at death.Linkage with the ACR identified 2276 primary cancer cases  over 33,6524.5 person-years follow-up (median (IQR)= 13.1 (5.0) years). For Dietary-Cancers and Digestive-Cancers, there were 1169 (264 men and 905 women) and 392 cases (191 men and 204 women), respectively.k-means cluster analyses [k = 1) is a pattern classification method that measures the Euclidean distance between a test example  and the data set and assigns the test example to the cluster of the nearest neighbour [analyses  were peranalyses   and initial cluster seeds . Subsequently, each additional participant was automatically assigned to the nearest cluster on the basis of Euclidean distance, forming temporary clusters. Seeds were then replaced by the centroid of each temporary cluster, with the “centroid” referring to the mean observation of a cluster. Each participant was then reassigned to the nearest centroid, updating the location of the centroids. The process was repeated until centroids did not significantly change location. For these analyses, between two and seven cluster solutions were tested to balance feasibility and robustness. To reduce the impact of local optima [All analyses were stratified by sex as self-reported by participants. The percentages of total rEI contributed by each of the 55 food groups were used as input variables. The k-means cluster analyses method started with the researcher selecting l optima , clusterz score [Before cluster analysis, each input variable was standardized by subtracting the minimum input value and then dividing by the range. This standardization method, known as the range method, has been reported to give consistently better recovery of cluster structure in different error conditions, separation distances, clustering methods, and coverage levels when compared with other standardization methods, such as the z score .Multivariable Cox proportional hazard regression models were used to assess the associations between observed dietary patterns and cancer risk, including All-Cancers, Dietary-Cancers, and Digestive-Cancers. Adjusted hazard ratios (AHR) were estimated in comparison to the association of a reference pattern with cancer outcomes. Competing risk analysis was performed, with the standard multivariable Cox proportional hazard regression model applied to the cause-specific hazard of interest and competing events treated as censored observations . Regressp-value threshold of <0.05, though the consistency of estimates across methods of accounting for EI misestimation is also considered more holistically given that this p-value threshold is arbitrary [Means and SD are presented for continuous variables, and counts and percentages for categorical variables. For interpretation purposes, comparisons examined whether associations would be considered significant based on the often used rbitrary . All anaThree dietary patterns, or clusters, were identified for both men and women: Healthy, Meats/Pizza, and Sweets/Dairy. Baseline sociodemographic characteristics stratified by dietary pattern and EI reporting status are presented in The greatest contributors to total rEI in each dietary pattern across different methods of accounting for EI misreporters is summarized in p-value threshold of <0.05.For All-Cancers, no significant associations were observed between dietary patterns and cancer risk in men, regardless of the method used to account for misestimation of EI. However, the point estimates for the Sweets/Dairy and Meats/Pizza patterns and All-Cancer were higher in the ExAfter and Inclusion methods, respectively, compared to the other methods of accounting for EI misreporting. In women, a significant increased cancer risk was associated with the Meats/Pizza pattern in the InclusionNN (AHR (95%CI): 1.14 (1.00–1.30)) method and in the Sweets/Dairy pattern for the ExBefore (AHR (95%CI): 1.28 (1.04–1.58)) method . Among wFor Dietary-Cancers, the Meats/Pizza pattern was associated with increased cancer risk among men under both the Inclusion (AHR (95%CI): 1.42 (1.00–2.02)) and ExAfter (AHR (95%CI): 1.92 (1.12–3.29)) methods . Also amFor Digestive-Cancers, no significant associations were observed with dietary patterns among men. Among women, a significantly increased risk of digestive cancers was observed for the Meats/Pizza pattern under the InclusionNN method (AHR (95%CI): 1.43 (1.02–2.01)) and the Sweets/Dairy pattern under the ExBefore method (AHR (95%CI): 1.73 (1.03–2.89)) .Competing risk analysis to account for deaths before ACR linkage date in participants who were cancer-free during follow-up did not significantly change the observed hazard ratios .The findings of this study suggest that misestimation of EI, ascertained using a prediction equation and self-reported physical activity and body weight and height, was prevalent among adults whose dietary intake was characterized using a FFQ within the context of a cohort study. Further, differing methods to account for this misestimation appear to impact observed associations between dietary patterns and cancer risk. Among men, there were no significant associations between dietary patterns and risk of all cancers regardless of the method of handling EI misestimation. However, the point estimates for All-cancers risk associated with the Sweets/Dairy and Meats/Pizza patterns were higher in ExAfter and Inclusion methods, respectively, compared to the other methods of accounting for EI misreporting. Among women, the Meats/Pizza pattern was associated with a 14% increased risk of all cancers in the method that included all participants regardless of EI misestimation (similar to that observed in the InclusionNN method). The Sweets/Dairy pattern was associated with a 28% increased risk of all cancers in the method that excluded women whose EI estimates were deemed to be affected by misestimation following the cluster analyses. Similarly, associations between dietary patterns and risk differed based on how EI misestimation was addressed for the subgroup of primary cancers for which there is evidence of the influence of dietary risk factors (men and women) and for digestive cancers (women). However, given that there is no marker of true dietary patterns, it is not possible to ascertain which method for accounting for EI misestimation results in observed associations that are the closest to truth.Other studies have similarly suggested that analytical approaches used to account for potential EI misestimation can impact observed associations between dietary intake and disease outcomes among adults. A cross-sectional study of Norwegian women aged 50–69 years , which up < 0.05, only one of the two would be interpreted as significant. Thus, the findings highlight the need to consider not only how EI misestimation is accounted for across studies, but also to improve the reporting and interpretation of findings within nutritional epidemiology [Despite slight differences in methodology and design, the findings of this study are in line with previously published results indicating that estimated diet-disease associations can be influenced by measurement error . Associaemiology .Prior analyses have highlighted the importance of considering measurement error and identified the need for caution in terms of the interpretation of diet-disease associations that have not been, at least partially, corrected for this error ,46,48. FHowever, many current sources of data on diet and disease outcomes, with sufficient time elapsed from baseline data collection for cases of cancer and other conditions to accrue, may not provide opportunities for regression calibration. In this study, no reference data are available and we opted to use the revised Goldberg method to attempt to account for measurement error exhibited as EI misestimation. However, this method has challenges. The use of EI/BMR for evaluating EI depends on knowledge of energy requirements or EE . For theSeveral statistical techniques are available for identifying dietary patterns and the choice of method depends largely on the research question at hand . For exaThis study made use of an existing cohort with a large sample size and careful validation of data and few missing values . One excThe results of this study suggest that observed associations between dietary patterns and health outcomes vary in relation to strategies for addressing EI misestimation. It is possible cohort studies that include the administration of biomarkers such as DLW in a subset of participants can shed light on misreporting of different dietary components and optimal strategies for accounting for it. Advances are also needed to enable improved characterization of dietary patterns, which inherently involve intake of many different foods and beverages that may be reported with different levels of accuracy. In the meantime, researchers should carefully consider how misestimation and other sources and symptoms of measurement error are characterized and accounted for and carefully report these details to enable appropriate interpretation of their findings.


Identification of modifiable risk factors for breast cancer is critical for primary prevention of the disease. The aim of this study was to evaluate how certain lifestyle variables modify the chances of developing breast cancer based on menopausal status. A case-control study was performed in a group of 542 women, 197 who were diagnosed with breast cancer and 344 control individuals. The groups were matched by age, body mass index, and menopausal status. Participants were evaluated for level of physical activity, alcohol consumption, smoking habit, weight, height, and waist circumference (WC). A multivariate logistic regression model was used to estimate odds ratios and 95% confidence intervals (95% CI). Regular consumption of alcoholic beverages  and sedentary behavior  were associated with breast cancer risk in pre- and postmenopausal women, respectively. High WC  was associated with an increased risk of developing breast cancer in premenopausal women. While in postmenopausal women, current smoking  or previous history of smoking  increased the chances of developing breast cancer. Sedentary behavior and current consumption of alcoholic beverages were more likely to increase the risk of developing breast cancer regardless of menopausal status. Breast cancer represents a public health problem on a global scale . ApproxiDespite the high incidence and prevalence of this cancer, the overall survival of Brazilian women is increasing. Five-year survival estimates were 76.9% and 75.2% from 2005 to 2009 and 2010 to 2014, respectively . The latIdentification of the etiological factors for breast cancer is important for the primary prevention of the disease ,6. Only In this context, identification of modifiable risk factors may help to elaborate specific strategies for the control and management of breast cancer. The aim of this study was to evaluate the effects of certain lifestyle variables on the chances of developing breast cancer based on menopausal status.This case-control study was conducted from August 2014 to January 2018. In total, 542 women were enrolled in the study. There were 197 participants diagnosed with breast cancer and 344 control individuals. Participants were matched by age, body mass index, and menopausal status. A post hoc sample size analysis was performed using G*Power 3.1.9.2 software . An odds ratio of 1.81 for the variable postmenopausal physical activity was obtained. The proportion of the outcome for the unexposed obtained was 55.56, type I error 0.05, returning a test power (1−β) of 99.9%, and demonstrating that a suitable sample for the analysis was determined. The study was approved by the Ethics Committee of the Federal University of Goias (protocol number 751.387/2014). All study participants received oral and written information about the study and gave their written informed consent to participate in the study.Women aged between 30 and 80 years with new primary breast cancer diagnoses were recruited as cases for the study. Breast cancer originating in the breast parenchyma and that had not spread beyond the breast or the lymph nodes under the arm was considered as a primary breast cancer. The participants were evaluated immediately after histological diagnoses and before systemic treatment. Women diagnosed with metastases, cutaneous neoplasms (melanoma and squamous cell carcinoma), lymphomas, ductal carcinoma in situ or with history of other cancers were not eligible for this study.Healthy participants with similar values for body mass index (BMI), age, and menopausal stage to those in the case group were recruited as control individuals. The criteria used to determine eligibility for the control group were mammography and breasts clinical examination without pathological changes up to one year before recruitment, and absence of a personal history of breast cancer or other malignant neoplasia. The exclusion criteria for both groups were pregnancy, lactation, cognitive impairment, psychiatric illness, limb amputation or loss of mobility that would make it impossible to collect the necessary information.A personal interview was conducted by a trained researcher using a structured and standardized questionnaire. Age (full years), first-degree family history of breast cancer, marital status (with or without partner), and level of education attained  were obtained using the questionnaire.Intensity of physical activity was measured using the short version of the International Questionnaire of Physical Activity (IPAQ) . Women wSmoking status was determined based on the response to the question, “Do you now or have you ever smoked cigarettes, at least one a day for one year’s time?” If the answer was “yes,” the participant reported the average number of cigarettes smoked per day . Consump2 [To ensure data reliability, measurement techniques recommended by Habicht  were app2 . WC was 2 , and cla2 .All interviewers and kinanthropometrists were previously trained and standardized in a pilot study to ensure data quality. In addition, study data were checked on three occasions; by the interviewer, by the study coordinator, and at the moment of database entry.® software . The Shapiro–Wilk test was used to test normality and the Mann–Whitney test was used to compare groups. Data were reported as mean ± standard deviation.Data were processed under the double entry method using Excel software 10.0  and consistency was verified by Epi-Info™ 2014 software . Statistical analysis was performed using STATAp < 0.20) and adjusted by BMI according to the automatic selection of the statistical program. The data were expressed in the form of a Forest Plot. The level of significance used for all tests was 5%.Categorical variables were expressed in absolute and relative frequencies. The Pearson’s chi square test or Fisher’s exact two-tailed test were used to assess the degree of homogeneity or comparability between groups. Odds ratios and 95% confidence interval (95% CI) for developing breast cancer based on menopausal status were calculated. Binary logistic regression and multiple logistic regression calculations were performed using the backward method (p = 0.541) and postmenopausal stages .Demographic characteristics are shown in p = 0.03, Considering the variables associated with lifestyle, women in the case group were mostly sedentary and reported higher consumption of cigarettes and alcoholic beverages, regardless of menopausal status . In premp = 0.005; p = 0.03) or a previous history of smoking  increased the risk of breast cancer in postmenopausal women  gene mutations in Ashkenazi Jewish women ,50. HoweOur study concluded that a sedentary lifestyle and regular consumption of alcoholic beverages were considered risk factors for breast cancer in women, regardless of menopausal status. In addition, increased abdominal adiposity and smoking posed a risk for premenopausal and premenopausal breast cancer, respectively.


The association between the use of hormonal contraceptive and pancreatic cancer among premenopausal women has until now been unclear. This is the first study to investigate the risk of pancreatic cancer in pre-menopausal women.To determine whether hormonal contraception increases the risk of developing pancreatic cancer in pre-menopausal women.A nationwide prospective cohort study followed all women in Denmark in the age range of 15–49 years without previous cancer or venous thrombosis from 1995 to 2014. The Danish National Prescription Registry provided individually updated exposure information on use of hormonal contraception. The Danish Cancer Registry provided cancer diagnoses, and the Danish National Patient Register containing clinical diagnoses and surgical codes at discharge from public and private hospitals.Population-based cohort study.All women living in Denmark aged 15–49 years at January 1st, 1995, and those subsequently reaching age 15 years up to December 31st, 2014 were eligible for the study.Among 1.9 million women who were followed on average for 11.4 years, 235 pancreatic cancers occurred. Compared to never users, ever users of any type of hormonal contraception had a relative risk (RR) of pancreatic cancer of 0.90  0.68–1.19). No overall association between duration of hormonal contraceptive use and pancreatic cancer risk was found. Neither was long-term use of hormonal contraception associated with pancreas cancer, RR 0.83 (95% CI 0.47–1.50). The risk did not vary between users of combined and progestogen-only products. All models were adjusted for age, completed or ongoing education, polycystic ovary syndrome, endometriosis and among parous women; parity, age at first birth, smoking and body mass index.Compared to never users the risk of pancreatic cancer is not significantly higher among current and recent users of contemporary hormonal contraception and does not vary between users of combined and progestogen-only products. In conclusion, our study suggests no risk of pancreatic cancer with use of any type of hormonal contraception. The worldwide incidence rate of pancreatic cancer is 10.9 per 100,000 women versus 13.9 per 100,000 men; and pancreatic cancer is relatively rare in young women , reflectSex-steroid hormone receptors have been detected in both normal and neoplastic human pancreatic tissue . EstrogeSince 1960 all citizens in Denmark have had a personal identification number recorded in the Civil Registry that holds records of age, death, and emigration. Data from The Danish Cancer Register  provided information on any malignant disease; the Danish National Patient Register contains clinical diagnoses and surgical codes at discharge from public and private hospitals since 1976, and the National Birth Register information about births since 1973. The National Register of Medicinal Product Statistics contains details of redeemed prescriptions for hormonal contraception and was linked using the personal identification number as the key identifier. Data from the National Register of Medicinal Product Statistics is considered complete from January 1, 1995 and this was consequently the start date of this study. Data was stored and analyzed on servers at Statistics Denmark. The unique personal identification numbers were encrypted ensuring anonymity during the data analyses.All women living in Denmark aged 15–49 years at January 1st, 1995, and those subsequently reaching age 15 years up to December 31st, 2014 were eligible for the study.From the National Register of Medicinal Product Statistics, we obtained information on the date of redeemed prescriptions, the specific Anatomical Therapeutic Chemical code, dose, number of packages, defined daily doses, and route of administration . The use of hormonal contraception was updated daily, with women changing status at time of cessation or switch in type of hormonal contraception used. Gaps between prescriptions of less than 28 days were filled out prospectively.Hormonal contraceptive use was categorized as current or recent use (cessation within last six months), previous use (cessation more than six months ago) or never use. The levonorgestrel-intrauterine system (IUD) was used for four years, unless pregnancy or another hormonal product was prescribed before the IUD expired.The National Birth Register and Statistics Denmark provided information on potential confounders; parity , age at first birth among parous women , smoking status among parous women , body mass index among parous women  and completed or ongoing education . The Danish National Patient Register provided information on diagnosis of polycystic ovary syndrome (yes/no) or endometriosis (yes/no). All models were adjusted for these conditions since women with these diseases more often use hormonal contraception.Data was analyzed by Poisson regression using SAS statistical software, version 9.1  to calculate incidence rate ratios (referred to as relative risk) and 95% confidence intervals. Age was used as a timescale in the Poison regression analyses with 5-year age bands. The study population was followed until a first diagnosis of pancreatic cancer, death, emigration, 50 years of age, or end of follow-up on December 31, 2014, whichever came first. Women were censored permanently at time of cancer diagnosis, infertility treatment, or venous thrombosis. Women were censored temporarily during pregnancy and for six months after delivery.The following time-dependent variables were updated daily: hormonal contraception variables, age, calendar year, parity, age at first birth, and education. The simple adjusted models included hormonal contraception, age, and calendar year. Calendar year was included to account for possible changes in the use of specific types of contraception and in pancreatic cancer incidence during the study period. The fully adjusted models included all the time-dependent and the time-constant variables; polycystic ovary syndrome (ever), and endometriosis (ever). Additional adjustment for body mass index and smoking status and the time of first birth was conducted among parous women with this information. The reference group in each analysis was women who had never used hormonal contraception at a given time during follow-up. Calculated risk ratios will be referred to as relative risks throughout the manuscript, this includes all tables.st of February 2018. A flow chart further describing the inclusion of studies can be found in the supporting information. .Data was made available through Statistics Denmark and approval obtained from the Danish Data Protection Agency and Health Data Board . According to the Danish Research Ethics Committee Law , ethical approval is not required for register-based studies. Data must be requested from the Danish National eHealth Authority due to legal restrictions on sharing data publicly. Data requests are sent through the application form at: From 1995 to 2014 women aged 15–49 years old accumulated 12.9 million person-years of hormonal contraceptive use and 235 pancreatic cancers incidents were recorded.Compared to never users, ever users of any type of hormonal contraception had a relative risk of pancreatic cancer of 0.90  0.68–1.19) .Current or recent users of any type of hormonal contraception had a relative risk of 1.01 (95% CI 0.72–1.42). No overall association between duration of hormonal contraception use and pancreatic cancer risk was found . NeitherCompared to never users, current or recent use of progestin only contraception showed a relative risk of 1.16 (95% CI 0.79–1.89) and combined oral contraception a relative risk of 0.92 (95% CI 0.62–1.36) . The simIn accordance with most of the current evidence we found no association between hormonal contraception use and pancreatic cancer risk ,17,19,20The exposure of both exogenous estrogens and endogenous estrogens (i.e. parity and menopause) and risk of pancreatic cancer have been explored in epidemiological studies and results are not consistent 19]. A s. A s19].No prior study has assessed the risk of pancreatic cancer with progestin-only products in pre-menopausal women. We found no influence on risk of pancreas cancer with use of these products.We were not able to adjust for alcohol consumption and dietary factors 22]. If . If 22].Our study adds to the sparse evidence on the influence of newer hormonal contraceptive products on the pancreatic cancer risk in women of reproductive age. Our nationwide study is a large-scale prospective cohort study with 1.9 million Danish women followed for an average of more than ten years. We had complete follow-up until diagnosis of cancer, venous thrombosis, infertility treatment, emigration, death or end of study. The validity of pancreatic cancer diagnoses is high because the Danish Cancer Register cross-check their records with histology records to ensure both a high level of completeness and validity of diagnoses. ,2526]26]The validity of exposure is also likely to be high in our study because recall bias has been eliminated. Information about redeemed hormonal contraception prescriptions is transferred electronically from the pharmacies using bar codes. Thus, we were able to account for switch in use and cessation of use. Our information about both exposure and relevant confounders was updated daily. Information about hormonal contraception and pancreatic cancer was recorded in the relevant national registers without the intention of exploring the association between hormonal contraceptive use and clinical outcomes known, making differential ascertainment of exposure and cancer incidence unlikely.Our results were adjusted for age, year, educational attainment, number of births, endometriosis, and polycystic ovary syndrome. Additionally, for parous women, smoking and BMI. Adjustment for smoking and BMI did not change our estimates noticeably indicating no major confounding influence of these factors.The novelty of this study is based upon the quality and quantity of our data utilized for our analysis. This dataset is unprecedented compared to other studies in this area and our focus has solely been on premenopausal women. It is reassuring that we do not find an increased risk of pancreatic cancer in premenopausal women using hormonal contraceptives due to the serious nature of the disease and high mortality rate.In conclusion our study suggests no risk of pancreatic cancer with use of any type of hormonal contraception in premenopausal women. Thus, the risk of pancreatic cancer is not a factor to be considered when assessing the risks and benefits of hormonal contraceptive use.S1 Fig(DOCX)Click here for additional data file.S1 TableStudies assessing the risk of pancreatic cancer in users of hormonal contraception stratified according to study design as case-control studies.(DOCX)Click here for additional data file.S2 TableStudies assessing the risk of pancreatic cancer in users of hormonal contraception stratified according to study design as cohort studies.(DOCX)Click here for additional data file.S3 TableStudies assessing the risk of pancreatic cancer in users of hormonal contraception stratified according to meta analyses.(DOCX)Click here for additional data file.