Cox regression analysis was performed to assess the association between overall and ER-specific PRS313 and CBC risk.We included women of European ancestry with a prevalent first primary invasive BC (BRCA1 heterozygotes the estrogen receptor (ER)-negative PRS313 showed the largest association with CBC risk, hazard ratio (HR) per SD = 1.12, 95% confidence interval (CI) (1.06–1.18), C-index = 0.53; for BRCA2 heterozygotes, this was the ER-positive PRS313, HR = 1.15, 95% CI (1.07–1.25), C-index = 0.57. Adjusting for family history, age at diagnosis, treatment, or pathological characteristics for the first BC did not change association effect sizes. For women developing first BC < age 40 years, the cumulative PRS313 5th and 95th percentile 10-year CBC risks were 22% and 32% for BRCA1 and 13% and 23% for BRCA2 heterozygotes, respectively.For 313 can be used to refine individual CBC risks for BRCA1/2 heterozygotes of European ancestry, however the PRS313 needs to be considered in the context of a multifactorial risk model to evaluate whether it might influence clinical decision-making.The PRS BRCA1 or BRCA2 (henceforth BRCA1/2 heterozygotes) have a higher risk of developing contralateral breast cancer than nonheterozygotes.1 The estimated cumulative 10-year contralateral breast cancer risk varies across studies between 18.5% and 34.2% for BRCA1 heterozygotes and between 10.8% and 29.2% for BRCA2 heterozygotes,6 compared to 4–6% in the population.8 Whether or not to undergo a risk-reducing contralateral mastectomy, which is an invasive intervention and associated with side effects such as postoperative surgical complications, inability to breast feed in the future, and psychosocial burden,9 is an important and difficult decision for BRCA1/2 heterozygotes who have been just confronted with their first breast cancer diagnosis. Precise individualized risk estimates could facilitate decision making for these women.Heterozygotes of germline pathogenic variants in BRCA1/2 heterozygotes are the age at diagnosis of the first breast tumor and a family history of breast cancer.10 The effect of family history on contralateral breast cancer risk suggests a role for other genetic factors. In the last decade, more than 180 common low risk variants have been associated with breast cancer risk in genome-wide association studies (GWAS).13 Individually, these variants are associated with small increases in risk, but when combined as polygenic risk scores (PRS) they may improve disease-related risk stratification for women of European and Asian ancestry in the population.16 A limited number of studies have shown that variants associated with the risk of a first primary breast cancer are also associated with the risk of contralateral breast cancer.19 Furthermore, the PRS derived from the general population has also been shown to be associated with breast cancer risk in BRCA1/2 heterozygotes.24Two important factors influencing contralateral breast cancer risk in 313); it showed an association with breast cancer in ten prospective studies with an odds ratio (OR) per standard deviation (SD) of 1.61 and an area under the receiver–operator characteristic curve of 0.630.14 Among BRCA2 heterozygotes, this same PRS313 was also associated with breast cancer risk, hazard ratio (HR) per SD = 1.31, 95% confidence interval (CI) (1.27–1.36).24 Among BRCA1 heterozygotes, the largest association with breast cancer risk was found using the estrogen receptor (ER)-negative PRS313 (which uses the same variants but with weights adapted to provide better prediction for ER-negative disease), HR = 1.29, 95% CI (1.25–1.33).24 Although these effect sizes were smaller than those for the general population, the 313 variant–based PRS could have a substantial impact on the high absolute risks24 associated with BRCA1/2 pathogenic variants.25 Whether variants associated with breast cancer are associated with contralateral breast cancer risk for BRCA1/2 heterozygotes as well, individually or combined in a PRS, has not been investigated previously. If so, the PRS may be useful to guide choices for risk management, especially regarding invasive risk-reducing contralateral mastectomy. In this study, we investigated whether the 313 variant–based PRS for breast cancer is associated with contralateral breast cancer risk among women of European ancestry with pathogenic variants in BRCA1/2 and explored the implications for contralateral breast cancer risk prediction for these women.The most predictive, well validated PRS for breast cancer in the general population is based on 313 breast cancer–associated variants (PRSBRCA1/2 (CIMBA).26 Briefly, CIMBA participants are heterozygotes of pathogenic variants in BRCA1 or BRCA2 who are 18 years or older at the time of inclusion and have phenotypic data available.26 CIMBA includes 81 individual studies of which the majority of the participants were ascertained through cancer genetics clinics.26 Although studies in CIMBA include individuals of non-European ancestry, our analyses were, due to power considerations , restricted to women of European ancestry with available array genotyping data .We used retrospective cohort data from heterozygotes participating in the Consortium of Investigators of Modifiers of n = 9,242 of whom 2,140 had a known negative lymph node status). Baseline age was defined as the age at local ascertainment (97%), or when this was not known, age at genetic testing (2%) or age at last follow-up (1%). Women were excluded if no information was available about the age at baseline or if they had developed synchronous contralateral breast cancer. Synchronous contralateral breast cancer was defined as contralateral breast cancer within one year after the first primary breast cancer, which was based on the exact date of cancer diagnosis or, if this was not available, on the age at diagnosis. A schematic overview of the selection is shown in Fig. SBRCA1 and 4,208 women with BRCA2 pathogenic variants were included in this study, among whom 1,402 BRCA1 heterozygotes and 647 BRCA2 heterozygotes have had contralateral breast cancer. The diagnosis of primary and contralateral breast cancer was confirmed by pathology records, tumor registry data, or medical records by the individual studies. Available phenotypic information for all participants is shown in Table Women were eligible for this retrospective analysis if they developed an invasive primary breast tumor without metastatic disease at least 1 year before the baseline age. Women without information about metastatic disease were assumed to have no metastatic disease  and two ER-specific PRS, the ER-positive PRS313 and ER-negative PRS313 were calculated. The variants and their corresponding weights used in the PRS as published previously14 and the imputation quality are listed in Table 14 Using these SDs, the HR estimates for the associations of the standardized PRS313 in our study are directly comparable with the OR estimates reported in the BCAC population-based study14 and the HR estimates reported for primary breast cancer in BRCA1 and BRCA2 heterozygotes.24We used the 313 variant–based PRS for breast cancer developed in an independent study using data from the general population as described previously;BRCA1/2 heterozygotes, Cox regression analyses were performed. The time at risk was started one year after the first breast cancer diagnosis based on the exact date, or if not available, on the age of developing the first breast tumor. Time at risk of participants was censored at age at baseline, i.e., end of follow-up in these analyses, prophylactic contralateral mastectomy, or death, whichever was earlier  and heterozygotes of pathogenic variants that lead to mutant stable protein (class II). Finally, analyses were performed to test the association between a categorized PRS and contralateral breast cancer risk to establish whether the results were consistent with those under a continuous PRS model. The categories were defined on the basis of the distribution of the PRS in unilateral breast cancer cases, using PRS percentiles .The interaction between the PRS with the age at first breast cancer diagnosis was tested in the final model, treating the PRS as a continuous variable. Furthermore, the effect size of the PRS was evaluated for groups based on the age at first primary breast cancer diagnosis .BRCA1 and BRCA2 heterozygotes, using the log HR per SD and including an interaction term with the continuous age at first breast cancer diagnosis . For this purpose, we constrained the incidence of contralateral breast cancer, by age at first breast cancer and in years after the first breast cancer, and averaged over all PRS categories to agree with external contralateral breast cancer incidence estimates, as described previously.23 These external incidence estimates were based on prospective cohort data from three consortia on heterozygotes of pathogenic BRCA1 and BRCA2 variants,1 the International BRCA1/2 Carrier Cohort Study (IBCCS), the Breast Cancer Family Registry (BCFR), and the Kathleen Cunningham Foundation Consortium for Research Into Familial Breast Cancer (kConFab). Because the contralateral breast cancer incidences vary with the age of first breast cancer diagnosis, incidences were calculated for three different groups based on the age of the first breast cancer diagnosis .1Absolute contralateral breast cancer risks were calculated at percentiles of the best-performing continuous PRS for both 34 Statistical significance was defined as a two-sided p value <0.05.All statistical tests were performed with R version 3.5.0.BRCA1 and 4,208 BRCA2 heterozygotes of European ancestry who had developed an invasive first primary breast cancer before entry in CIMBA were identified. The median follow-up time was 6.0 and 5.4 years for BRCA1 and BRCA2 heterozygotes, respectively. In total, 1,402 BRCA1 and 647 BRCA2 heterozygotes were diagnosed with a metachronous contralateral breast cancer before enrollment in CIMBA. The cumulative 10-year risk of developing contralateral breast cancer in this cohort was 25%, 95% CI (23.5–26.4%) and 18.8%, 95% CI (17.1–20.5%) for BRCA1 and BRCA2 heterozygotes, respectively , p value = 6.0×10−5, C-index 0.53, 95% CI (0.51–0.55). There was no evidence of violation of the proportional hazard assumption, p value = 0.840.For 313 association more than 10% when compared with the model with no confounders , p value = 3.2×10−5.Considering only invasive contralateral breast cancer as the event of interest resulted in a similar association with the ER-negative PRS313, HR per SD = 1.12, 95% CI (1.06–1.18), p value = 4.9×10-5.Censoring at distant metastasis relapse, if applicable, did not change the effect size of the ER-negative PRS313, were consistent with the predicted HRs from the model using the continuous ER-negative PRS313 , p value = 0.002. For ER-negative contralateral breast cancer as event, only the ER-negative PRS313 showed a significant association, HR per SD = 1.07, 95% CI (1.01–1.15), p value = 0.036 , p value = 1.9×10−4, C-index 0.57, 95% CI (0.54–0.59). There was no evidence of violation of the proportional hazard assumption, p value = 0.300.For 313 association more than 10% when compared with the model with no confounders , p value = 6.0×10−4.Considering only invasive contralateral breast cancer as the event of interest resulted in a similar association, HR per SD for the ER-positive PRS313, HR per SD = 1.15, 95% CI (1.07–1.24), p value = 2.1×10-4.Censoring at distant metastasis relapse, if applicable, did not change the effect size of the ER-positive PRS313, were consistent with the predicted estimates using the continuous PRS313 , p value = 2.2×10−5 , p value = 0.346.The ER-positive PRS−5 Table . None of313 was found for BRCA1 heterozygotes: HR per year = 0.99, 95% CI (0.99–1.00), p value = 0.025. For BRCA2 heterozygotes a similar magnitude of interaction was observed with the ER-positive PRS313, although the interaction was not significant, HR per year = 0.99, 95% CI (0.99–1.00), p value = 0.09.A significant interaction between the age at first breast cancer diagnosis and the ER-negative PRSBRCA1 heterozygotes resulted in HRs per SD of the ER-negative PRS313 of 1.22, 95% CI (1.14–1.31); 0.99, 95% CI (0.90–1.09);, and 1.03, 95% CI (0.86–1.24) for ages <40 years, 40–50 years, and ≥50 year respectively. For BRCA2 heterozygotes the corresponding estimates for ER-positive PRS313 were 1.23, 95% CI (1.09–1.38); 1.19, 95% CI (1.05–1.34); and 0.97, 95% CI (0.81–1.15) respectively  compared with those leading to no protein or an unstable protein (class I). For BRCA2 heterozygotes, the ER-positive PRS313 effect size for the association with contralateral breast cancer risk was nonsignificantly smaller among heterozygotes of a pathogenic variant that lead to a stable mutant protein, although statistical power to detect these associations was low and the confidence intervals overlap with the overall estimate , although most of the confidence intervals overlapped. The effect sizes for the PRS are also larger for ER-positive disease in the general population, perhaps because ER-positive disease is commoner and the power to identify genetic variants has been greater for ER-positive disease. With larger data sets, it should be possible to develop better subtype specific PRS for contralateral breast cancer.For both BRCA1 heterozygotes, the HR per SD for the association between the ER-negative PRS313 and breast cancer was 1.29, 95% CI (1.25–1.33),24 compared with 1.12, 95% CI (1.06–1.18) for contralateral breast cancer in this study. For BRCA2 heterozygotes, the HR per SD for the association between the ER-positive PRS313 and breast cancer was 1.31, 95% CI (1.26–1.36),24 compared with 1.15, 95% CI (1.07–1.24) for contralateral breast cancer in this study. This lower relative risk is consistent with a general pattern of a lower relative risk in a higher risk population, as seen in the lower relative risk for contralateral breast cancer than first breast cancer in the general population,19 and the lower relative risk for the first cancer in BRCA1/2 heterozygotes than in the general population.24 The attenuated estimate might be explained by several factors, some of which are speculative. BRCA1/2 pathogenic variant heterozygotes in this study were selected based on having a first breast cancer; these women will have on average a higher PRS, but also higher frequencies of other genetic and nongenetic risk factors than women who do not develop breast cancer at all. This can lead to a weaker association with the PRS as women with the largest PRS may have lower risks due to other factors, a phenomenon related to index event bias.35 There could also be negative interactions between the PRS effect and other risk factors . However, in this study, we have shown that adjustment for the known contralateral breast cancer risk factors did not change the effect size of the PRS, which was also shown in population-based studies.19 Finally, although we tried to exclude potential early metastases misdiagnosed as second primaries by excluding women who developed a contralateral breast cancer the first year after the primary diagnosis, it is possible that a small percentage of contralateral breast cancers were metastases.36Although we found clear associations between the PRS and contralateral breast cancer risk, the magnitude of these associations (expressed in terms of HRs) were smaller than previously reported for the first breast cancers. For BRCA1/2 heterozygote breast cancer population. Indeed, the estimated 20-year risks of developing contralateral breast cancer in this study were higher compared to a previously published study with a prospective design:1 47% versus 40% for BRCA1 heterozygotes and 40% versus 26% for BRCA2 heterozygotes, respectively. While this is unlikely to introduce a significant bias in the relative risk estimates, a prospective cohort would clearly be preferably, although this will take several years to achieve. Finally, the PRS was developed using data sets of women of European ancestry, since our data set included insufficient samples of women of other ancestries, and our results were exclusively based on women of European ancestry. Therefore, caution is required when applying this to non-European ancestry populations. However, a population study found clear associations between the PRS, based on the same 313 variants or a subset of these variants, and  breast cancer also in women of Asian ancestry. The effect size of these associations were slightly weaker, possibly reflecting the fact that this PRS was developed in a cohort of women of European ancestry.19 These results suggest that there might be an association with the PRS as well in BRCA1/2 heterozygotes of Asian ancestry. Future studies including a sufficient number of individuals of Asian ancestry are needed to confirm this statement.A limitation of this study is that participants were recruited through clinical genetic centers, resulting in ascertainment bias, as individuals are more likely to have a strong family of breast cancer and/or be affected at a young age to be referred for testing. This was a historical cohort in which follow-up was prior to entry into CIMBA, so that all cases are prevalent. Therefore, the breast cancer patients included in the analyses are likely to be at higher contralateral breast cancer risk when compared with the general BRCA1 and BRCA2 heterozygotes under age 40 at first breast cancer, at the 5th and 95th percentile of the PRS, differed by 10% in 10-year contralateral breast cancer risk. These absolute risk differences are modest, but might be of relevance for the choices regarding preventive surgery if incorporated into a multifactorial model that includes other predictive factors, such as family history and adjuvant systemic treatment of the first breast cancer.38 In the context of such a comprehensive model, further research is needed to investigate whether the PRS would contribute to the choices that women make for follow-up or preventive surgery.Although the relative risks of the PRS for contralateral breast cancer were modest, differences in the PRS may still have an important effect on the absolute risk, which is high. BRCA1/2 heterozygotes. We found that the PRS is associated with contralateral breast cancer risk in both BRCA1 and BRCA2 heterozygotes of European ancestry and that PRS can be used to refine estimates of contralateral breast cancer risks in these women. However, for women with a first breast cancer after the age of 50, PRS may be of less value in the prediction of the contralateral breast cancer risk. Incorporating risk factors other than PRS and including ER-specific estimates may further improve contralateral breast cancer risk prediction. Before implementation in a diagnostic setting, our results should be validated in a prospective cohort of BRCA1 and BRCA2 heterozygotes.To summarize, we have investigated the associations between PRS based on 313 variants with contralateral breast cancer risk in a large international series of Supplementary informationSupplementary table S2


Aims: The antenatal period provides an important opportunity for giving advice on healthy lifestyle choices. However, the prevalence of maternal obesity is increasing, and women report that they do not receive counseling. We investigated the information given to pregnant women on gestational weight gain, physical activity, and nutrition during pregnancy in relation with their initial weight status, current gestational weight gain and diagnoses of either pre-pregnancy overweight/obesity or excessive gestational weight gain.Methods: Cross-sectional survey using a questionnaire. Pregnant participants (n = 141) were recruited from a midwife center. They completed a structured questionnaire on the information they received during their pregnancy and we assessed its relationship with their weight.Results: We found that many pregnant women did not receive advice about physical activity, gestational weight gain and nutrition . Women with weight problems  were less targeted for counseling, although more than 80% of the women viewed receiving information on these topics as positive. Also, being informed of a weight problem was associated with a greater chance of receiving information about physical activity, gestational weight gain and nutrition . However, verbalization of the weight problems was low (14.0% of women with pre-pregnancy overweight were informed of their status).Conclusion: Health professionals should dispense more information, especially on PA and particularly for women with weight problems. Verbalization of the weight problem seems associated with more frequent transmission of information. Physical activity (PA) prevents excessive gestational weight gain (excessive GWG)  and manyThe pregnancy weight risk may be due to pre-pregnancy overweight  and/or eThe objective of this study was to investigate the information given to pregnant women on gestational weight gain, physical activity, and nutrition during pregnancy in relation with their initial weight status, current GWG and diagnoses of either pre-pregnancy overweight/obesity or excessive GWG. We hypothesized that weight problems would increase the frequency of counseling for pregnant women. Secondly, we investigated pregnant women’s beliefs and attitudes toward PA with the objective of improving the targeting and content of prenatal PA counseling.This was a cross-sectional study design, with 141 pregnant women from a midwife center in Guadeloupe. After giving informed consent, the women responded to a structured questionnaire under their midwife’s supervision on the counseling they had received and their beliefs about PA during pregnancy. Inclusion criteria were being pregnant; able to read, write and speak French; followed throughout pregnancy in the center where the recruitment took place; and with no contraindication to PA according to the ACOG. All experimental procedures conformed with the Declaration of Helsinki and were approved by the local ethics committee.The study was conducted in accordance with the Declaration of Helsinki and the current local regulations. All participants provided written informed consent. It was emphasized that participation was voluntary and that they could withdraw from the project at any time with no explanation required. No financial support was given.On the day of data collection, all eligible and available women were invited to participate in the study. The progress of pregnancy in weeks of amenorrhea and trimesters of pregnancy, stature in centimeters, current weight status, age in years, GWG in kilograms at the time of the study, and weight in kilograms as beginning pregnancy were collected from medical records. The women were also interviewed during visits to the midwife center using a questionnaire built for the current study. The information from the participants about PA, nutrition, and GWG was examined. We developed the questionnaire based on questions and results from similar studies . AlthougThe questionnaire was composed of a mix of closed-ended questions, semi-closed-ended questions, and 5-point Likert scale questions. It required approximately 10 min to complete.The frequency of advice received  was examined on participants on a scale of 0–5, where 0 represented “never” and 5 “very frequently.” Did you receive advice during your consultations on nutrition, GWG and PA?To assess whether women had received information about their weight problems, closed questions were used. “Were you informed about an overweight/obesity problem before pregnancy by your midwife?”, “Were you informed about excessive GWG during pregnancy by your midwife?”2), normal weight (BMI: 18.5–24.9 kg/m2), overweight (BMI: 25.0–29.9 kg/m2) or obese (BMI: ≥30.0 kg/m2). The total GWG was calculated from the maternal weight. Based on the ACOG recommendations  was explored by Chi2 tests. All questionnaire data were entered into Excel software and imported into SPSS (v24.0). The results were considered significant when the p-value was ≤ 0.05.The demographic characteristics were analyzed as means and standard deviations (SD) for quantitative variables and confidence intervals for qualitative variables. Average and frequency comparisons were used for qualitative and quantitative variables, respectively. Variables evaluated by Likert scales were recoded into dichotomous variables (“expresses an agreement” and “does not express an agreement”) because of the small sample size. Odds ratios were calculated when Chip < 0.05, One hundred and forty-one 30.3 ± 6.2-year-old women between 10 and 42 weeks of gestation participated in the present study. They were predominantly married (61.0%) and employed (63.1%). The majority had completed secondary school (70.2%) and 51.1% had at least one child. Before pregnancy, 9.3% (95%CI 4.5–14.1), 50.0% (95%CI 41.7–58.3), 26.4% (95%CI 19.1–33.7) and 14.3% (95%CI 8.5–20.1) were lean, normal weight, overweight and obese, respectively. The expected maximum weight gain throughout the pregnancy was 8.2 ± 7.7 kg and by the time of the study, 13.5% (95%CI 7.8–19.2) showed excessive GWG. Nevertheless, 77.3% (95%CI 70.4–84.2) considered themselves in good health. The distribution of BMI into classes before pregnancy was as follows: 9.3% (95%CI 4.5–14.1), 50.0% (95%CI 41.7–58.3), 26.4% (95%CI 19.1–33.7), and 14.3% (95%CI 8.5–20.1) in lean, normal, overweight and obese women, respectively. A significant relationship between being pre-pregnancy overweight or obese and excessive GWG was evidenced (The women most frequently reported that the main source of information was press/books/internet [51.8% (95%CI 43.6–60.0)], followed by family and friends [28.4% (95%CI 21.0–35.8)] and last by the medical professions [19.8% (95%CI 13.1–26.4)].Most of the women [88.6% (95%CI 83.4–93.8)] thought PA was an important topic and 83.2% (95%CI 77.0–89.4) and 84.4% , respectively, thought they had enough time and felt comfortable enough to ask questions about PA in the consultations. However, 74.5% (95%CI 67.3–81.7) would have liked to know more. Regarding the advice given, 25.5% (95%CI 18.2–32.7) and 31.2% (95%CI 23.6–38.8) reported difficulties in following the advice on PA and nutrition, respectively.Gestational weight gain was considered an important topic by 65.2% (95%CI 57.3–73.1) of the women. Most [87.2% (95%CI 81.7–92.7)] thought receiving information on their weight was positive, and 56.1% (95%CI 47.9–64.3) reported trying to control their weight gain.p ≥ 0.323.In sample, 53.2% (95%CI 45.0–61.4) reported receiving information on GWG, 66.2% (95%CI 58.4–74.0) on nutrition, and 37.5% (95%CI 29.5–45.5) on PA from their health professionals. Respectively, 67.2% (95%CI 59.5–74.9) and 65.0% (95%CI 57.1–72.9) of the women said they were aware of the impact of PA and nutrition on GWG. With regard to weight gain, 26.7% (95%CI 19.4–34.0) were informed of a weight gain not to be exceeded. When asked about the maximal weight gain that they had been advised not to overpass, 88.7% (95%CI 83.4–93.9) reported a gain that was in the range of the IOM recommendations. Among the women who received PA information, 89.4% (95%CI 84.3–94.5) reported that their doctor spoke positively about PA. PA was assumed to be positive for the mother and the fetus by, respectively, 90.1% (95%CI 85.2–95.0) and 80.7% (95%CI 74.2–87.2). The frequency of reporting the PA benefits for their baby’s health was higher in women who had received information about PA, whereas the frequency of reporting PA as dangerous was lower . Resultsp = 0.005).Fourteen percent (95%CI 8.3–19.8) of the women who were overweight or obese before pregnancy had been informed of their status, and 31.6% (95%CI 28.1–44.0) of the women who had gained too much pregnancy weight by the time of the study had been informed about it. Not all the women received information about GWG but giving information tended to increase with pregnancy progression: 27.8% (95%CI 20.4–35.2), 48.3% (95%CI 40.0–56.6) and 65.1% (95%CI 57.2–73.0) for the first, second, and third trimesters, respectively , 32.8% (95%CI 25.0–40.5) and 48.3% (95%CI 40.0–56.5) of the women being informed for the first, second and third trimesters, respectively: The analyses failed to evidence any tendency of PA information to be more frequently received by women with pre-pregnancy overweight or excessive GWG . HoweverThe objective of this study was to investigate the information that pregnant women receive with regard to their initial weight status, pregnancy weight gain and weight diagnosis. We hypothesized that weight problems would increase the frequency of counseling for these women. The results highlight that many pregnant women did not receive advice about PA, GWG and nutrition. In addition, women who had a pre-pregnancy weight issue were less targeted for counseling.The proportion of women who declared that they had received information on gestational weight gain or physical activity by a medical professional was quite low, even though it increased as the pregnancies progressed. In the third trimester, only 65.1 and 48.3% of the women said they had heard of gestational weight gain and physical activity, respectively. These frequencies may be surprising, given the importance of these two parameters over the course of a normally progressing pregnancy and childbirth, but they approximately reflect the frequencies found in the literature, with values ranging from 21 to 52% for gestational weight gain  and fromOne explanation is that health professionals might be apprehensive about raising the issue of weight with pregnant women and being perceived as judgmental, especially when the women are overweight . This idMoreover, our results indicated that very few women who were overweight or obese before pregnancy declared that they had been informed of their weight status, although BMI calculation seems to be a common practice . The oveAlthough health professionals believe that gestational weight gain, nutrition, and physical activity are important and are likely to have an impact on the health of women and their babies , the litStudies have reported that health professionals might perceive their advice on weight control as ineffective . For exaThis question of confidence is important since it appears that the most confident physicians are more likely to inform their patients that excessive weight gain increases the risk of pregnancy complications and has possible consequences for their baby . Also, oIt has also been observed that accessible and consistent information about the positive effects of PA is likely to contribute to adequate PA behaviors during pregnancy , particuHealth professionals may also think that women do not have much control over their weight and that PA would only put them in a situation of probable failure , but ourProfessionals might also assume that pregnant women are uninterested  in issueFurthermore, our results indicated that although 65% of the pregnant women declared knowing the impacts of PA and nutrition on gestational weight gain, almost three quarters of them reported that they would have liked to know more. It should be noted that in our study, gestational weight gain was considered as an important topic by 65.2% of the women. Our results are in accordance with those of Health professionals may think they are not sufficiently trained for counseling. They have reported a lack of training in antenatal PA counseling , and a lOur results showed that the women who had the most chance of receiving information were those who felt comfortable asking questions. This indicates that in order to obtain information, women should be able to ask for it; however, they are generally less proactive in seeking advice from health professionals .The literature suggests that a non-negligible proportion of health professionals might not be familiar with the PA recommendations in pregnancy . This isIn addition, some women may experience only limited counseling from midwives because these latter lack sufficient knowledge on this topic . Thus, tSpecial attention to women with weight problems has been signaled in the current recommendations . We thusFinally, although pre-pregnancy overweight diagnosis was not associated with more frequent weight management counseling, it was associated with lower excessive gestational weight gain. This diagnosis thus seems to be associated with different behaviors in pregnant women  that theTalking about weight problems and addressing PA and healthy eating should be included in antenatal care , and womWomen would like advice and information on the recommendations for PA , becauseRegarding weight information, women want to know if they gain too much weight during their pregnancy. In addition, the recommended weight gain based on their initial weight also seems an important topic for them . Women dAlthough nutrition appears to be the most frequently provided information in our study, pregnant women generally seem a little disappointed to see that the issue is rarely discussed . GeneralThis study has a number of limitations that should be taken into consideration when interpreting and extrapolating the findings. First, the cross-sectional design intrinsically weakens the search for in-depth understanding and causal links. The second important limitation is a non-validated questionnaire, although structured on the basis of the existing literature and pre-tested. It should be recalled that all the data regarding the information received by the women were self-reported. Essentially, this meant that the data reflected the women’s points of view and it cannot be assumed that they reflected the practices of the medical professionals . In addiOverall, pregnant women receive little counseling on PA and GWG, and this is particularly so for those with weight problems, who are more exposed to excessive GWG. Our results provide additional evidence that the diagnosis of a weight problem impacts behavior and beliefs. Better training for health professionals would increase their knowledge and the frequency of the counseling they give pregnant women to convince them to engage in PA . It seemThe original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. The study was conducted in accordance with the Declaration of Helsinki and the current local regulations. The patients/participants provided their written informed consent to participate in this study. It was emphasized that participation was voluntary and that they could withdraw from the project at any time with no explanation required.SR and SS designed and set up the study protocol. SR, SS, and SA-J analyzed and interpreted the data and contributed to successive drafts and the revising of the manuscript. SR, SS, OH, EJ, and SA-J read and approved the final manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Metastasis remains the main challenge to overcome for treating ovarian cancers. In this study, we investigate the potential role of the Cdc42 GAP StarD13 in the modulation of cell motility, invasion in ovarian cancer cells. StarD13 depletion does not affect the 2D motility of ovarian cancer cells. More importantly, StarD13 inhibits matrix degradation, invadopodia formation and cell invasion through the inhibition of Cdc42. StarD13 does not localize to mature TKS4-labeled invadopodia that possess matrix degradation ability, while a Cdc42 FRET biosensor, detects Cdc42 activation in these invadopodia. In fact, StarD13 localization and Cdc42 activation appear mutually exclusive in invadopodial structures. Finally, for the first time we uncover a potential role of Cdc42 in the direct recruitment of TKS4 to invadopodia. This study emphasizes the specific role of StarD13 as a narrow spatial regulator of Cdc42, inhibiting invasion, suggesting the suitability of StarD13 for targeted therapy. The AmeMetastasis is a result of the dysregulation of molecules that regulate actin polymerization and cell migration, leading to the acquired ability of cancer cells to metastasize and invade . InitialRho GTPases are small monomeric G proteins that belong to the Ras superfamily . These aSteroidogenic acute regulatory protein-related lipid transfer domain-containing protein 13 (StarD13) is a GAP for RhoA and Cdc42 with potential tumor suppressor functions . Indeed,The results obtained support the tumor suppressor role of StarD13 and that StarD13 is a GAP for Cdc42 in ovarian cancer cell lines, as was previously shown in other tumor types . Moreove2.2.1.Cdc42 and RhoA biosensors were kind gifts from Dr. Louis Hodgson (Albert Einstein College of Medicine). RhoA/Rac1/Cdc42 Activation Assay Combo Kit was purchased from Cell BioLabs . QCM Gelatin Invadopodia Assay was obtained from Millipore . Primary antibodies against Cdc42, StarD13, actin, vinculin, Arp2, cortactin (Mouse and Rabbit) and TKS5 antibodies were obtained from Abcam . Primary mouse anti-TKS4 was obtained from Merck Millipore. Primary mouse anti-StarD13 and rabbit anti-Cdc42 were obtained from Santa Cruz Biotech. HRP-conjugated secondary antibodies were obtained from Promega . Fluorescent secondary antibodies Alexa Fluor 488-green and Alexa Fluor 594-red as well as Rhodamine-phalloidin stain were purchased from Invitrogen . DAPI stain, and cell proliferation reagent were acquired from Roche Diagnostics . Hiperfect transfection reagent, luciferase and human Flexi Tubes siRNA for luciferase, StarD13, Cdc42 and RhoA were obtained from Qiagen . Lipofectamine LTX was from Waltham  and crystal violet was from SCP Science .2.2.2.Metastatic SKOV-3 (Human ovarian adenocarcinoma derived from ovaries and from metastasis in ascites), Caov-3 (Human ovarian adenocarcinoma) and PA-1 (Human ovarian teratocarcinoma derived from metastasis in ascites) cell lines were purchased from ATCC (American Type Culture Collection). All cell lines were culture in DMEM medium supplemented with 10% fetal bovine serum and 100U penicillin/streptomycin at 37 °C and 5% CO2.3.https://www.oncomine.org/resource/login.html). Data was plotted using the normal versus ovarian cancer data sets and parameters and the threshold was set at p-value of 0.001. The analysis reflects the expression of StarD13 in normal ovarian tissue as well as different groups of ovarian cancers . StarD13 gene expression from two different microarray data sets was plotted.To determine the expression of StarD13 in human ovarian tumors, we mined the publicly available Repository Oncoming gene expression microarray database .2.5.Normal ovarian and ovarian adenocarcinoma paraffin-embedded tissues were purchased from OriGene technologies . Paraffin was removed by soaking the slides in xylol before rehydrating the tissues with PBS for 20 min at room temperature. Next, 200–300 ml of pre-heated antigen retrieval buffer (1 mM EDTA pH=8) were added to the slides before heating twice for 5 min in the microwave (700 W). Following, the tissues were delimited using a clear nail polish and blocked using a 4% BSA blocking solution before incubation with the primary StarD13 antibody for 2 h. After washing the slides with ice-cold PBS1X, the tissues were incubated with the appropriate secondary antibody coupled to Alexa fluor-488 fluorophore for 30 min before mounted using a mounting solution mixed with DAPI. Fluorescent cell images were taken using the 20X objective lens of the fluorescent Zeiss Observer Z1 microscope operated by the Zen software .2.6.SKOV3, Caov-3 and PA-1 ovarian cancer cells were transfected with 10 nM of control Luciferase siRNA or RhoA siRNA, Cdc42 siRNA, Rac1 siRNA and StarD13 siRNA, alone or in combination, using the Hiperfect transfection reagent. Where indicated, 48 h after transfection with the siRNA, the cells were co-transfected with 5 μg of the RhoA or Cdc42 biosensors or with 5 μg of the constitutively active RhoA construct. All assays were performed 72 h after transfection with the siRNA.2.7.Proteins extracted from ovarian cancer cells by scraping with Laemmeli sample buffer, were run by SDS-PAGE under standard conditions as previously described . After t2.8.Proteins were extracted from ovarian cancer cells using the cell lysis buffer provided with the RhoA/Rac1/Cdc42 Activation Assay Combo Kit. The cell lysate was divided in two parts: the first part (500 μl) was incubated with GST-RBD beads or GST-PBD-PAK1 beads, respectively. After incubation on a shaker for 1 h at 4 °C, the samples were centrifuged, and the pellet was washed several times before resuspension in Laemelli sample buffer as described previously . The sec2.9.2). Cell proliferation was finally quantified at 450 nm using the Varioskan microplate reader from ThermoFisher scientific .Ovarian cancer cell Proliferation was determined by adding 10 μl of the WST-1 reagent to control and transfected ovarian cancer cells which were plated in the different wells of a 96 well plate (dilution of WST-1 to media 1:10). The cells were then incubated with WST-1 for 2 h in the incubator  presented in the figures was obtained in ImageJ by measuring the distance between the cells at 11 different points of the wound region, and calculating and averaging the speed of wound closure by dividing the distance over time (48 h).2.11.2). Phase-contrast images of the randomly moving cells were collected every 60 s for 120 min using the 20X objective of the Zeiss Observer Z1 microscope. The total distance traveled by at least 50 randomly moving cells was determined using the ROI tracker in ImageJ. To obtain the average speed of migration in μm/min, the distance was divided over time.Random cell motility assay was performed as previously described . Briefly2.12.5 cells/ml were added to the wells before placing the plates back in the incubator 30 min. Non-adherent cells were then removed by washing the wells 3 times. Adherent cells in the wells were fixed with 4% paraformaldehyde solution for 10 min, before staining with crystal violet (5 mg/ml in 2% ethanol) for 10 min. Finally, the plates were thoroughly washed and dried before solubilizing crystal violet in DMSO for 30 min. Cell adhesion was quantified at 550 nm using the Varioskan microplate reader from ThermoFisher scientific .Cell adhesion was measured as previously described. Briefly, the wells of a 96-well plate were coated with collagen type I overnight, before washing with washing buffer (0.1% BSA in DMEM media) and blocking in 0.5% BSA blocking solution in the incubator for 1 h. After that, 50 μl of control or transfected ovarian cancer cells suspension containing 4 × 102.13.Invasion assay was performed as previously described using the collagen-based invasion assay kit from Millipore  . Briefly2.14.The invasion of control and transfected ovarian cancer cells was examined using the QCM Gelatin Invadopodia Assay kit from Millipore . Briefly, ovarian cancer cells were plated on fluorescently labeled gelatin matrix for 24 h before fixing the cells with 4% paraformaldehyde solution for 10 min at 37°C, and permeabilizing them with 0.5% Triton-X 100 for 15 min on ice. All cells were blocked in 1% BSA solution for 1 h, before incubation with cortactin or TKS4 primary antibodies overnight at 4°C, and with the appropriate fluorophore-conjugated secondary antibodies for 1 h. The cells were imaged using a 63X objective lens on Zeiss Observer Z1 fluorescent microscope and the degradation of the matrix was measured upon quantification of dark areas lacking cortactin or TKS4 signaling using ImageJ.2.15.FRET analysis was performed as previously described . Briefly2.16.The area and number of focal adhesion were quantified in ImageJ using the CLAHE and Log3D plugins as described previously . CLAHE e2.17.p-values were calculated by two way ANOVA or t-test to check if the changes observed in the results were significant.The results reported represent average values from three independent experiments. The error estimates are given as ± SEM. The 3.3.1.To understand the role of StarD13 in ovarian cancer cells, we determined the expression profile of StarD13 in normal ovarian tissues as well as in tissues obtained from patients with ovarian adenocarcinoma . Immunoh3.2.After having established the anti-proliferative potential of StarD13 in ovarian cancer cells, we investigated its ability to modulate cell motility using two approaches: wound healing and time-lapse assays. The results show similar wound closure areas in control and StarD13-depleted SKOV-3, Caov-3 and PA-1 cells . QuantitThis was surprising since previous reports from studies performed in astrocytoma, breast cancer, colon cancer and lung cancer, showed that StarD13 depletion inhibited cancer cell migration due to the dysregulation of RhoA activation . Indeed 10.1016/j.ejcb.2021.151197.Supplementary material related to this article can be found online at doi: 3.3.To confirm the GAP function of StarD13, we performed a pull-down assay to investigate the differences in GTP loading of RhoA and Cdc42 in SKOV-3 and Caov-3 cancer cells in the presence or absence of StarD13. Western blot analysis revealed an approximate 3-fold increase in GTP-Cdc42 levels in both SKOV-3 and Caov-3 cells upon StarD13 depletion, compared to controls . This st3.4.Previous studies from our laboratory and others reported an effect of StarD13 on cell adhesion and a potential localization of StarD13 to focal adhesions . Here, w3.5.Knowing that Cdc42 is the main effector of StarD13 in these cells, we then looked at the effect of StarD13 depletion on cell protrusion, a main event during cancer migration regulated by Cdc42 . In orde10.1016/j.ejcb.2021.151197.Supplementary material related to this article can be found online at doi: 3.6.Cdc42 is directly implicated in the regulation of cancer cell invasion, therefore we investigated the role of StarD13 in regulating the invasion of ovarian cancer cells . Using t3.7.Matrix degradation is a critical step in cancer invasion and metastasis. We therefore next tested the ability of StarD13 to modulate ovarian cancer cell matrix degradation using the gelatin invadopodia assay. StarD13 and Cdc42 were silenced alone and in combination cells before plating SKOV-3 and Caov-3 cells on fluorescently labeled gelatin matrix for 24 h and subsequent staining with cortactin to the mark invadopodia. Fluorescent micrographs show that StarD13 depletion increases the matrix degraded area in SKOV-3 and Caov-3 cells  and 6F. 3.8.In a recent study, we showed that Cdc42 localizes to TKS4-labeled invadopodia and to the sites of matrix degradation in lung cancer cells. We also showed that StarD13 depletion leads to an increase in these potential invadopodial structures and that this is mediated through an increase in GTP-Cdc42 levels . In addi10.1016/j.ejcb.2021.151197.Supplementary material related to this article can be found online at doi: 10.1016/j.ejcb.2021.151197.Supplementary material related to this article can be found online at doi: 10.1016/j.ejcb.2021.151197.Supplementary material related to this article can be found online at doi: 10.1016/j.ejcb.2021.151197.Supplementary material related to this article can be found online at doi: 3.9.RhoA has been previously shown to regulate matrix degradation through a mechanism that involves delivery of matrix metalloproteinases to the invadopodia . While t10.1016/j.ejcb.2021.151197.Supplementary material related to this article can be found online at doi: 10.1016/j.ejcb.2021.151197.Supplementary material related to this article can be found online at doi: 3.10.Having isolated a subset of invadopodia which lacked the accumulation of GTP-Cdc42 , we sougFinally, we show that StarD13 depletion has no effect on the number of TKS5 positive invadopodia but that it significantly increases the number of TKS4 positive invadopodia as compared to the control . The opp4.This study provides an in-depth understanding of the role of StarD13 in ovarian cancer cells, as well as uncovers the key targets mediating StarD13 effects on cell proliferation, motility, invadopodia formation and invasion. Most, importantly, the data provide a model for the recruitment of an ordered set of molecules and signaling events required for the formation and the maturation of invadopodia in ovarian cancer cells. This suggests StarD13 as a main regulator of invadopodia and reveals, for the first time, its clear role in the spatial regulation of Cdc42 activation. Through the use of different markers of invadopodia, this study distinguishes stages of formation of invadopodia and the potential role of StarD13 and Cdc42 during these stages.Immunohistochemistry and data microarray analysis revealed a lower expression of StarD13 in ovarian cancer tissues as compared to the normal ovarian tissues, which is similar to the results observed in lung and hepatocellular cancers . StarD13However, unlike in previous studies performed on breast, lung, astrocytoma and colorectal cancers , where SKnock down of StarD13 however, showed an increase in GTP-Cdc42, strongly suggesting that StarD13 is a GAP for this Rho GTPase in ovarian cancer cells, similarly to findings in other tumor types . In addiStarD13-depleted cells also showed a dramatic increase in ruffles and in protrusion (in quiesced cells as well as cells stimulated with EGF), which is mediated by Cdc42. This is consistent with previous reports highlighting the role of Cdc42 in actin polymerization and cell protrusion in cancer cells . As mentIn addition to the effect on actin-rich protrusion, StarD13 silencing increased matrix degradation and ovarian cancer cell invasion. This was previously observed in other tumor types . More imInterestingly, monitoring the dynamics of StarD13, Cdc42, TKS4 and TKS5 expression in cells and in invadopodia uncovered a set of organized events required for the formation and maturation of invadopodia . The modIn an earlier study, we reported StarD13 to always be absent in invadopodial structures . In thatIn summary, both RhoA and Cdc42 activation persist in invadopodia while StarD13 is only expressed in early invadopodia along with TKS5 and never coincides with TKS4  and E. IAltogether this study showed that StarD13 is a GAP for Cdc42 and emphasized the importance of StarD13 and Cdc42 in regulating the formation of invadopodia in a temporal and functional manner.Supplemental Movie S5Supplemental Movie S7Supplemental Movie S6Supplemental Movie S8Supplemental Movie S3Supplemental Movie S2Supplemental Movie S1Supplemental Movie S4Supplemental Figure S5Supplemental Figure S1Supplemental Figure S4Supplemental Figure S2Supplemental Figure S3


The inability to assess PM2.5 concentrations have limited the assessment of the health impact of these exposures, both in the native populations and previously deployed military personnel. As part of a Department of Veterans Affairs Cooperative Studies Program health study of land-based U.S. military personnel who were previously deployed to these countries, we developed a novel approach to estimate spatially and temporarily resolved daily PM2.5 exposures 2001–2018. Since visibility is proportional to ground-level particulate matter concentrations, we were able to take advantage of extensive airport visibility data that became available as a result of regional military operations over this time period. First, we combined a random forest machine learning and a generalized additive mixed model to estimate daily high resolution (1 km × 1 km) visibility over the region using satellite-based aerosol optical depth (AOD) and airport visibility data. The spatially and temporarily resolved visibility data were then used to estimate PM2.5 concentrations from 2001 to 2018 by converting visibility to PM2.5 using empirical relationships derived from available regional PM2.5 monitoring stations. We adjusted for spatially resolved meteorological parameters, land use variables, including the Normalized Difference Vegetation Index, and satellite-derived estimates of surface dust as a measure of sandstorm activity. Cross validation indicated good model predictive ability (R2 = 0.71), and there were considerable spatial and temporal differences in PM2.5 across the region. Annual average PM2.5 predictions for Iraq and Kuwait were 37 and 41 μg/m3, respectively, which are greater than current U.S. and WHO standards. PM2.5 concentrations in many U.S. bases and large cities  had annual average PM2.5 concentrations above 45 μg/m3 with weekly averages as high as 150 μg/m3 depending on calendar year. The highest annual PM2.5 concentration for both Kuwait and Iraq were observed in 2008, followed by 2009, which was associated with extreme drought in these years. The lowest PM2.5 values were observed in 2014. On average, July had the highest concentrations, and November had the lowest values, consistent with seasonal patterns of air pollution in this region. This is the first study that estimates long-term PM2.5 exposures in Iraq and Kuwait at a high resolution based on measurements data that will allow the study of health effects and contribute to the development of regional environmental policies. The novel approach demonstrated may be used in other parts of the world with limited monitoring networks.Iraq and Kuwait are in a region of the world known to be impacted by high levels of fine particulate matter (PM The satrvations , China  or observational meteorological values to simulate the relationship between AOD and PM2.5 globally  to predict high spatiotemporal resolution visibility, as visibility data throughout the region were available as result of military airport activity. We then derived relationships between daily PM2.5 and visibility using existing monitoring stations in the region to convert visibility to PM2.5. In the absence of extensive air quality monitoring sites, this approach leverages historical visibility data, satellite AOD, and existing regional PM2.5 monitoring stations to understand the intensity and spatiotemporal variability in PM2.5 exposures over Iraq and Kuwait.As part of a Department of Veterans Affairs Cooperative Studies Program health study of land-based U.S. military personnel, we predicted spatially and temporally resolved PM2.2.1.2.1.1.https://www.diva-gis.org/datadown)  satellites . Terra watadown) . The ave2.1.2.https://www.ncdc.noaa.gov/isd). Seventy-five percent of the visibility data in this study was from U.S. Air Force and 25% was from NOAA. We calculated average daily visibility from the available hourly data. The distribution of the 263 visibility monitoring stations available in Iraq and Kuwait during the study period is shown in Hourly visibility data for Kuwait and Iraq were provided by the U.S. Air Force 14th Weather Squadron for all airports and meteorological stations in Kuwait and Iraq. We also obtained additional hourly visibility data from National Oceanic and Atmospheric Administration (NOAA) Integrated Surface Database , PM2.5 concentrations we previously collected in three monitoring sites in Kuwait during 2004–2005 . ERA5 is a widely used global reanalysis, which has been verified by comparisons with observations   and additional particle parameters  (https://disc.gsfc.nasa.gov/datasets/M2TUNXAER_5.12.4/summary). MERRA-2 is an atmospheric reanalysis platform provided by NASA, which is agreed well with the results of other satellite observations and those of ground-based measurements   were obturements . MERRA-22.1.6.https://data.nodc.noaa.gov/cgi-bin/iso?id=gov.noaa.ncdc:C00813). This dataset was acquired by the Advanced Very High Resolution Radiometer (AVHRR) sensor at a spatial resolution of 5 km × 5 km and a temporal resolution of 1 day. Elevation of each grid cell was extracted from ETOPO1 Global Relief Model . ETOPO1 is a 1 arc-minute global relief model of Earth’s surface that integrates land topography and ocean bathymetry. Information regarding industrial areas was provided by the U.S. National Geospatial-Intelligence Agency. Since industrial emissions may influence the relationship between AOD and visibility, we calculated the distance from the center of each grid cell to the nearest industrial area for use as a potential adjustment variable. Road data were obtained through OpenStreetMap (https://www.openstreetmap.org) and traffic density in each 1 km2 grid cell was calculated by the total Highway road length in the grid cell.Land use parameters including Normalized Difference Vegetation Index (NDVI), elevation, distance to the nearest industrial area, and traffic density were included as model predictors . NDVI da2.2.2.5 concentrations in each 1 km × 1 km grid cell, we developed a 4-stage model  were computed.We performed a 10-fold cross-validation by randomly dividing the visibility stations into 10%−90% splits (10 random groups). For each split, the model was trained with data from 90% of the visibility sites and was used to predict visibility in the 10% of sites not included in the training. This process was repeated 10 times, and the cross-validation R2.2.2.For grid cells with AOD but without visibility measurements, we used the Stage 1 model to estimate daily visibility for each grid.2.2.3.PredVisij is the predicted visibility at grid-cell i on a day j from the stage 2 model; MVisj is the mean value of predicted visibility from stage1 on a day j across the study region; α and β are the fixed intercept and fixed slope, respectively;μi and vi are the random intercept and grid-cell specific random slope, respectively. X is longitude, and Y is latitude; “Smooth” is a thin plate spline function; and k(j) is the two-month period in which day j falls. To estimate the goodness of stage 3 model, we use the same cross-validation validation method as in stage 1.For grid cells missing both AOD and visibility measurements, a generalized additive mixed model (GAMM) was used to predict daily visibility based on the output from Stage 2 model . This me2.2.4.2.5 and visibility Because of the paucity of PM2.5 data in this region, we used data during 2017–2018 to train the model and used data during 2004–2005 for validation. Inverse of visibility and squared form of relative humidity were used as variables in the model based on the comparison of models using different combinations of variables  and R2 value of 0.74 in 2004–2005, indicating good model fit  which is mostly desert had low average PM2.5 concentrations, except for large urban areas in the east . PM2.5 levels in the study region also showed distinct temporal variability. The highest annual PM2.5 concentration for both Kuwait and Iraq were observed in 2008, followed by 2009. The lowest values for both Iraq and Kuwait were observed in 2014. It is possible that the extreme drought in 2008–2009 contributed to the high PM2.5 level in 2008 and heavy rain in the end of 2013 contributed to lower levels in 2014. By regions, the higher PM2.5 levels in 2008 and 2009 were predominantly in the urban areas. The monthly average predictions for Kuwait ranged from 32 to 49 μg/m3, and for Iraq ranged from 29 to 44 μg/m3. July had the highest predicted concentrations, and November had the lowest values. This is consistent with seasonal patterns of PM2.5 observations and indicates that PM2.5 pollution in the region has been a long-term problem.In general, the PM2.5 exposures. Some bases were located in regions with lower PM2.5 concentrations such as in the Syrian Desert, which have average PM2.5 concentrations below 30 μg/m. However, many U.S. bases located in urban area and had very high PM2.5 concentrations. The weekly average PM2.5 at Baghdad International Airport, the US base in Balad, and Kuwait City International Airport are shown in 2.5 concentrations ranged to more than 150 μg/m3 for the bases in Baghdad and Balad in summer of 2008 and 2009.As demonstrated by the considerable spatial and temporal variability over the region, deployers in Iraq and Kuwait experienced variability in PM4.2.5 exposures at 1 km × 1 km resolution for Iraq and Kuwait, where the monitoring system is very limited. Our results indicated that this region had very high PM2.5 levels, which varied by location and year.We developed a model to predict daily PM2.5 concentrations for Kuwait (41 μg/m3) and Iraq (37 μg/m3) from 2001 to 2018 were considerably higher than the U.S. National Air Quality Standard of 12 μg/m3 and the WHO guideline of 10 μg/m3. Very few studies conducted PM2.5 measurements in Kuwait or Iraq. 2.5 samples approximately four times per month at five sampling sites in Iraq and four sampling sites in Kuwait in 2006. 10 for the year 2012 in a suburban area of Kuwait. 2.5 measurements in three monitoring stations in Kuwait from 2014 to 2017. Our estimates have the same spatial distribution with the PM2.5 samples of 10 in 2.5 concentrations predicted in this study are consistent with a previous WHO air pollution database (2.5 concentrations in (3) are generally higher than our results, possibly because they selected hourly PM2.5 values above a minimum background concentration of 8.83 μg/m3 to report.The annual mean predicted PMdatabase . Howevertions in   that was accounted for by modeling, and the local crossing time of the AOD data were relatively fixed between 10:30 am and 1:30 pm. Although these factors could result in systematic bias of the PM2.5 predictions, the model would still be internally consistent.A limitation of our approach is that the stage 4 model regression results was based on PMd Kuwait . It has operties . In supp2.5 concentrations at a high resolution in Iraq and Kuwait that takes advantage of all available measurement data. This high-resolution model makes it possible to assess the health effects of PM2.5 exposures both in the native populations and the military personnel. This study is also helpful to the construction of regional environmental policies in areas with different pollution levels.To the best of our knowledge, this is the first model to estimate historical PMsupplement


The lowest estimate of disease-free-survival (DFS) was for grade 1 expression, with 50 months, confidence interval (CI): 95% 13.3–23.9 months and the highest for grade 3 expression, 167.9 months . ROC (receiver operating characteristic) curves revealed that the positivity for nestin (+/−) in relation to malignancy, presented a sensitivity of 50.98%, a specificity of 81.08%, with an area under the curve of 0.667 (p = 0.009). Conclusions: Nestin could be a useful marker to detect the presence of stem cells in head and neck tumors that have a role in tumor initiation and progression.Background: The aim was to investigate the clinical significance of nestin immunohistochemical expression in head and neck area lesions and to study its role in patient survival and recurrence. Methods: 39 (44.3%) nasosinus, 37 (42%) major salivary gland (6 submandibular and 31 parotid) and 12 (13.6%) oral cavity lesions of paraffin-embedded samples were retrospectively included. Results: The expression was categorized into grades, negative for 55 (62.5%) cases, grade 1 in 10 cases (11.4%), grade 2 in 12 cases (13.6%), and grade 3 in 11 cases (12.5%); 100% of pleomorphic adenomas were positive for nestin with grade 3 intensity, 100% of polyps and inverted papillomas were negative ( Nestin is a class VI embryonic intermediary filament protein and has been shown to be a marker of immature/stem cell-like tissue, such as the brain , arteriaDuring embryogenesis, nestin is expressed in cellular migration and proliferation. By contrast, in adult tissues nestin is restricted mainly to regenerative areas, being abundant in progenitor cells derived from embryonic stem cells, which have the potential to develop into neuroectodermal, endodermal and mesodermal lineages, indicating regenerative potential . It is iNestin blockade has been shown to inhibit the proliferation of colorectal cancer cell lines , migratiGiven the lack of studies on nestin expression in head and neck conditions, the aim of this work is to investigate the clinical significance of nestin immunohistochemical expression in head and neck area lesions  and to study its role in patient survival and recurrence.Study design: This is a retrospective observational study, designed according to Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) recommendations  and apprSample selection and histological evaluation: a total of 88 paraffin-embedded samples of benign and malignant conditions with a confirmed histopathological diagnosis were included. Five µm of haematoxylin-eosin stained sections from all cases were histopathologically analyzed and reviewed by two of the authors (JMSP and MPS) who adjusted the diagnosis according to the international recommendations for histopathological analysis. The clinical data of the patients and the characterization of the lesions (AJCC American Joint Committee of Cancer 8th Ed. 2017) were collected in a pseudonymized database.Immunohistochemical evaluation: immunohistochemistry was performed using a rabbit monoclonal anti-nestin antibody EPR1301(2)  with an automatized equipment . In brief, epitope retrieval was performed in 10 mM sodium citrate buffer (pH 9.0) using a water bath for 40 min at 95–99 °C. Endogenous peroxidase was blocked using peroxidase-blocking reagent  for 5 min. Incubation with the primary antibody (1/500) was performed at room temperature for 20 min and staining was revealed with EnVision (20 min) and DAB (diaminobenzidine) 10 min) . Finally, slides were counterstained with HE for 15 min.Immunohistochemically, only unequivocal cytoplasmic staining was considered positive. Results were evaluated semi-quantitatively attending to the following criteria: negative (Score 0) when no cells stained for nestin; weak (Score 1) when only scattered cells (less than 10% were positive); moderate (Score 2) when positive cells represented 10 to 50%, and strong (Score 3) when over 50% of the cells stained with the antibody. For statistical purposes, nestin expression was also evaluated as negative (score 0) or positive . Stromal or vascular staining was not considered.p ≤ 0.05.Statistical analysis: SPSS v.24.0  was used for statistical analysis. Results were described by mean and standard deviation and frequency with percentages. The data were subjected to the chi-square and Kruskal–Wallis test to study the relationships between the categorical variables. Kaplan–Meier curves were used to study survival and Cox regression with hazard ratio to determine the role of nestin expression. ROC curves were made to estimate the diagnostic efficacy of nestin positivity in malignancy. The significance level was established for A total 88 patients were studied, 40 (45.5 %) women and 48 (54.5 %) men, with an average age of 54.7 ± 18.9 years. The final distribution of the sample was 39 (44.3%) nasosinus, 37 (42%) major salivary gland (6 submandibular and 31 parotid) and 12 (13.6%) oral cavity lesions .Thirty-seven benign (42%) and 51 malignant (58%) lesions were included. The most frequent histological type was carcinoma, with 36 cases (40.9%), followed by inverted papilloma with 14 cases (15.9%) and nasosinus polyps with 13 cases (14.8%). A complete description of different tumor subtypes can be found in Nestin staining in normal salivary glands was found to be limited to endothelium and nerve fibers, but parenchymal cells, acinar or ductal, were completely negative except for some myoepithelial cells, which were occasionally stained (p = 0.001). Benign lesions were negative in 81.1% of the cases, against 49% of the malignant ones (p < 0.001) (p < 0.001) and the rest of the histological varieties were positive in a variable pattern  cases, grade 1 in 10 cases (11.4%), grade 2 in 12 cases (13.6%), and grade 3 in 11 cases (12.5%). There were no differences in terms of gender. Regarding origin, 84.6% nasosinus lesions were negative compared to those from the salivary gland and oral origin, which were positive (in different degrees) respectively, in 51.4% and 66.7% ( pattern .The average follow-up period was 70.5 ± 70.6 months with a range of 1.4 to 280.4 months. 28.4% of the patients died (14.8% due to the tumor), with an overall survival of 43.7 ± 39.9 months with a range of 1.4 to 119.2 months, and the disease specific survival of 38.1 ± 41.6 months, with a minimum of 1.4 months and a maximum of 113.6 months. During the follow-up period, 33% of the lesions recurred, with a mean of 0.6 ± 1.3 recurrences per patient, with a maximum of 9 per patient (adenocarcinoma being the most recurrent variety). The disease-free survival was 39.8 ± 51.6 months, with a minimum of 2.2 months and a maximum of 235.3 months. An average time until the last recurrence of 87.7 ± 74.2 months and an average period of recurrences of 38.8 ± 36.2 months were recorded .p < 0.001). In relation to recurrence, the majority of cases are negative (51.7%) compared to cases of intense grade 3 expression in which 6.9% (p = 0.038). Grade 1 expression is significantly associated with older patients : 95% 69.5–83 years, p = 0.001). Disease-free survival was significantly lower for lesions with grade 1 expression  than for grade 2  (p = 0.001)  .p = 0.049) (p = 0.002) , and nestin immunohistochemical expression was not graded and only considered as positive vs. negative, survival was worse for positive nestin cases, 30.41 ± 4.27 months  95% 22.04–38.77 months) vs. negative cases with 44.48 ± 4.04 months (IC 95% 36.56–52.40 months)  A. For re= 0.002) B.p = 0.009) (The study using ROC (receiver operating characteristic) curves revealed that the positivity for nestin (+/−) in relation to malignancy (yes/no), presented a sensitivity of 50.98%, a specificity of 81.08%, with an area under the curve of 0.667 (= 0.009) .We have found a positivity for nestin in head and neck region lesions of 37.5% globally, remaining negative in all inflammatory lesions (polyps) and inverted papillomas) and in 51% of malignant tumour lesions. The malignant lesions in the oral cavity reached a level of positivity of 66.7% significantly, although in relation to intensity, 100% of the pleomorphic adenomas of the salivary gland were grade 3. Mortality and recurrence were higher for negative cases and similarly, the DFS was significantly lower in the cases of grade 1 nestin in relation to the most intense grades. The hazard ratio for recurrence was 4.577 for cases with mild nestin expression (grade 1) and 8918 when we adjusted the model by location.The intermediate filament protein nestin, initially considered a marker for neural stem cells, is extensively expressed in progenitor cells derived from embryonic stem cells, which have the ability to become multi-linear . Since iIt has been reported that expression of nestin in the soft tissues of patients with uni- or bilateral cleft lip may indicate a potential increase of tissue regeneration . Also, nMascolo et al.  studied Regarding salivary gland conditions, Yanai et al.  describep = 0.049). In head and neck tumors, the vast majority of the stem cells are found within a 100 μm radius of a blood vessel, suggesting the existence of a perivascular niche [In our series we have not been able to demonstrate immunohistochemical expression in any benign neoplasm, except for pleomorphic adenomas, in which it seems to be related with the epithelial-mesenchymal transition and not with the benign or malignant character of the lesion. Instead, malignant tumors have shown different grades of nestin immune expression and, independent of its degree, it could be helpful to assess the malignant character of a lesion in small biopsies. Although overall nestin expression does not seem to be correlated with survival , tumor relapsing is associated with nestin staining. Moreover, when pleomorphic adenomas were excluded and nestin immunohistochemical expression was not graded and only considered as positive vs. negative, survival was worse for cases positive for nestin (ar niche . It coular niche  but it i


Objectives: To estimate the prevalence of musculoskeletal pain of hotel housekeepers (HHs) and to describe the work conditions and perception of health in this occupational group in the Balearic Islands, Spain. Methods: Cross-sectional descriptive study with HHs of the Balearic Islands, performed in primary care. Random sample of HHs who worked during the 2018 season. We collected information on sociodemographic variables, job characteristics, workload, pain, perceived health, and physical activity. After participants signed the informed consent form, we conducted a face-to-face interview in the primary care centre and accessed the participants’ electronic health records. Results: 1043 HHs aged 43.3 ± 10 years and with 10.7 ± 9.1 years worked as HHs were included. 51% (95% CI: 48–54%) reported chronic pain, mainly in the lower back 28.7% (95% CI: 25.9–31.5%), hands/wrists 23.7% (95% CI: 21.1–26.4%), neck 21.6% (95% CI: 19.1–24.3%), shoulders 19.9% (95% CI: 17.4–22.4%), and back 17.8% (95% CI: 15.4–20.2%). Pain was associated with older age, more years worked, more beds made/day and difficulty in pushing the housekeeping cart. More than half HHs reported that they did not incorporate occupational risk prevention measures (ORPMs) into their routine; 17.3% (95% CI: 15.1–19.7%) HHs considered their health as poor or very poor. Perception of health was worse in HHs with chronic pain. Conclusions: A high percentage of HHs of the Balearic Islands reported chronic pain, a low compliance with ORPMs and compared to women of the same sociodemographic profile they perceive a worse health status. According to the World Health Organization, musculoskeletal pain is the main occupational disease . Within It has been suggested that HHs are at high risk for MSD , due to Occupational risk factors can be physical, chemical, biological, and psychosocial . Most stAssessment of the physical burden of HHs is complex, since it varies in each workplace based on the number and type of rooms/day, the number of checkout rooms/day, the number and type of beds/day, and other aspects. While there is no standardized instrument to measure all dimensions of physical risks in HHs , it is cTourism is the main economic sector of the Balearic Islands. In 2018, it constituted 44% of the annual GDP of the region . A totalHotel Housekeepers and Health (ITS’17-096). This project follows the structured phases proposed by the British Medical Council (BMC) regarding trials for complex interventions [The framework for this study is the project ventions  to promoThe main objective of this study is to estimate the prevalence of musculoskeletal pain in HHs of the Balearic Islands and to describe their sociodemographic characteristics, work conditions and perception of health.A descriptive cross-sectional study was conducted between November 2018 and February 2019 in 39 of the 58 primary healthcare centres (PHCs) of the Balearic Islands. Participants were contacted by telephone and asked for inclusion criteria, and set up a date for the interview, which was carried out in the Primary Care Health Centre, where each HH had her general practitioner. We conducted face-to-face interviews during the winter months, because most HHs were not working and thus more available.The study population was comprised of HHs who had worked during the 2018 summer season and had a public health services card for the Balearic Islands. The 39 centres with the highest proportion of HHs were selected. A simple random sampling of HHs attending these 39 PHCs was carried out. Informed consent was obtained from all subjects involved in the study.Exclusion criteria included: HHs under 18 years of age; HHs unable to go to the PHC; HHs who did not have a telephone; and HHs with a language barrier.p = 1 − p). In our study, the prevalence of chronic pain was 51%; then, we can estimate this prevalence with a 2.9% precision. Estimating a 30% drop-out rate, we selected 3 additional HHs of similar age of the same PHC per participant.Information of HHs was provided by the Balearic Occupational Service to the Balearic Public Health Service. This information allowed us to identify to which primary care health centre they belong and their contact data. We obtained 13,000 potential eligible participants. Initially, we calculated that a sample size of 987 HHs was needed, with a 3% precision, 95% confidence and a maximum indeterminacy . In addition, we collected information on the use and maintenance of cleaning equipment and knowledge about occupational risk prevention measures (ORPMs), as stated in the Manual for the Prevention of Ergonomic and Psychosocial Risks of the National Institute for Occupational Safety and Hygiene .Variables related to pain. We used the Nordic Standardized Questionnaire for Musculoskeletal Symptoms in the working population . We askePhysical activity (PA) was collected using the Spanish version of the Brief Physical Activity Assessment Tool . This quPerceived health during the previous year was recorded on a Likert-5 scale from “very bad” to “very good” according to the National Health Survey , and on Finally, we collected willingness to participate in prevention activities during the months with less workload.The HHs were contacted by telephone and invited to participate in the study. If they consented to participate, an appointment was scheduled at their own PHC to conduct an individual interview lasting approximately one hour. Interviews were conducted by previously trained staff with extensive experience in health research studies. The pharmacological treatment was later verified in the electronic health records of the HHs.2) for contrasts between qualitative variables. Statistical significance was set at 0.05. ORs were calculated for the personal variables and job characteristics in relation to the chronic pain variable. We used binary logistic regression with the backward method for the independent variables with a significance level p < 0.05. Missing data were treated as not valid. We used SPSS software  for analysis.We provide absolute and relative frequencies and 95% confidence intervals (95% CI) for categorical variables, and mean, standard deviation (SD) and 95% CI for numerical variables. We used the Chi-square test  HHs explained having suffered from chronic pain during the previous season in at least one body region. In contrast, 7.6% (95% CI 6–9.2%) declared that they just had felt pain “sometimes” (6.4%) or “never” (1.2%) during the previous season. The five body regions most affected by chronic pain were the low back (28.8%), wrists/hands (23.7%), neck (21.7%), shoulders (19.7%) and back (17.8%). p < 0.001) was found between years worked as HH and proportion of chronic pain. A positive correlation (p < 0.001), greater number of beds/day (p < 0.001), more years worked as HH (p < 0.001), type of contract (p < 0.05) and greater number of hours worked per week (p < 0.05) positively correlated with chronic pain. Similarly, chronic pain was associated with a worse perception of health (p < 0.001). In contrast, physical activity and BMI were not statistically associated with pain during the previous season. Older age  HHs reported having felt pain in at least one body region. Notably, 92.6% HHs who suffered from chronic pain in at least one body region during the previous season also expressed having felt pain during the week prior to the interview. Up to 54.7% (95% CI 51.7–57.7%) of HHs explained that they had taken painkillers during the previous year and 17.7% (95% CI 15.5–20.2%) had taken alternative or complementary medication.Although 83.6% HHs reported that they were aware of occupational risk prevention measures (ORPMs) and 83.3% stated that they “always” attended the training courses, only 61.5% considered the information and training received as “always” suitable for the job. Additionally, 81% explained that they never stretched or warmed up before work. Regarding ORPMs, HHs reported that they were “never implemented”: by 55.5% HHs when awkward postures were required ; by 55.7% HHs when performing repetitive movements ; and by 52% HHs when handling loads . Moreover, 53.3% stated that they did not have the assistance of a male room attendant/brace to lift heavy loads. In addition, 41.5% answered that taking breaks was possible “only once” (14.1%) or “never” (27.4%), whereas 33.7% reported that they could “always” take a break.Regarding workload, HHs had worked an average of 7 months during the 2018 season, cleaning an average of 18 (±6.5) stay-over rooms/day, 5.4 (±2.8) checkout rooms/day and making 44.6 (±20.7) beds/day.Finally, when asked to assess the adequacy of the equipment, 59.9% and 50.5% considered the housekeeping cart too heavy and too cumbersome, respectively.p < 0.05) with a reduction of chronic pain in the spine and in the wrists/hands when handling loads and performing repetitive movements.Following ORPMs was significantly associated (n = 1040) were considered insufficiently active, since they did not reach the minimum recommended PA. Up to 27.9% and 80% stated that they never performed moderate  or intense PA, respectively.The BMI mean was 25.7 (SD = 4.9) and 49.1% (95% CI 45.9–52.2%) of the HHs were overweight or obese.The average perceived health status on the day of the interview was 72.4 points (SD ± 18.9). Regarding the perceived health status in the previous year, 58.9% (95% CI 55.8–61.9%) evaluated their health as “poor” .n = 1037) answered that they would be willing to attend these sessions, and 70% of the respondents (n = 932) would choose to have them in their PHC. Only 3.4% of HHs chose their workplace for an intervention aimed at improving their quality of life.Finally, and with the objective of designing a future intervention, HHs were asked if they would attend sessions with the goal to improve their quality of life, and where they would like to have these sessions. A total of 90.3%  and the neck (21.7%). In women of the same sociodemographic profile (sociodemographic status VI-unskilled workers), the Spanish National Health Survey estimates prevalence of chronic pain of 26.7% and 23.3% in the low back and neck , respectWe believe that the differences in the prevalence of pain compared to other studies are due to the sociodemographic characteristics of the population and not to improvements in ergonomics or workload, which have been singled out for targeted intervention in various studies ,29,30.Regarding the protective effect of PA, 57% of HHs in our study were considered insufficiently active. In contrast, the proportion of HHs who did not perform PA reached 94.1% in other studies . We beliWith regard to ORPMs, results indicate that most HHs consider that the information provided in the training sessions, which most of them attend, is not transferable to their work routine. We hypothesise that the high workload prevents the implementation of the recommendations when adopting awkward postures. However, future research needs to address the exact reasons behind these behaviours. The most important exposure factors associated with pain are reaching out, overstretching and repetitive movements ,25,26.It is estimated that, while cleaning a room, HHs might change posture every 3 s, which amounts to approximately 8000 posture changes per shift . The higUp to 53.3% of HHs reported that no male room attendant/braces were available to assist in lifting heavy loads. It should be noted that improving ergonomics during lifting can prevent MSD . InteresWork breaks can also reduce the harmful effects of the HHs’ duties . In our study, the OR of chronic pain was 1.56 in participants who could not take any breaks. We emphasize that incorporating breaks during work hours has proven effective and should be integrated in the ORPMs of all hotel businesses .Regarding perceived health status, 17.3% evaluated their health as “bad” or “very bad”, and 41.6% as “fair”. In contrast, the Spanish National Health Survey of the same year for women with the same sociodemographic profile shows a perceived “good” health status in 44.95%, and a perceived poor health status in a lower 10.88%, with “bad” and “very bad” in 7.87% and 3.01%, respectively . RegardiThe prevalence of pain in the HHs of the Balearic Islands agreed with other studies and settings. We believe that interventions aimed to raise the quality of life, reduce pain, and improve the perceived health status of HHs should be urgently implemented. Solutions should promote physical activity, reduce stress, improve work–life balance, decrease workload and encourage the uptake of ORPMs ,33,35. IThis study has some limitations. We should consider the possibility of memory bias, since self-reports were used. Although we administrated a validated pain questionnaire, no criteria for chronicity were explained, and the severity of pain was completely based on subjective criteria . The intHHs of the Balearic Islands present a high prevalence of chronic pain and a low compliance with ORPMs. Compared to women of the same sociodemographic profile, they perceive a worse health status; therefore, interventions to improve the health of HHs are urgently needed.