Women following the “Beans-vegetables” pattern had significantly lower GWG  and had a 16% lower risk of excessive GWG and 11% higher odd of adequate GWG. The association between the “Beans-vegetables” pattern and birth weight was negatively mediated by GWG. A dietary pattern enriched in beans and vegetables is beneficial for effectively controlling GWG and increasing birth weight. GWG serves.The associations among maternal diet, birth weight, and gestational weight gain are still inconclusive. This study aimed to investigate the associations between maternal dietary patterns and birth weight, and further explore whether GWG mediates these associations. A total of 3,334 pregnant women who completed a validated semi-quantitative food frequency questionnaire from the Tongji Maternal and Child Health Cohort were included. Dietary patterns were extracted by using principal component analysis. Regression models and mediation analyses were performed to explore the associations between dietary patterns and birth weight and the effects of GWG on these associations. Five dietary patterns were identified: “Beans-vegetables,” “Fish-meat-eggs,” “Nuts-whole grains,” “Organ-poultry-seafood” and “Rice-wheat-fruits.” Only women following the “Beans-vegetables” pattern had heavier newborns . Birth weight is an important index to evaluate the health status of newborns. Inappropriate birth weight is related to an increased risk of infant mortality and diseases in adulthood life –4. ThereNutrition plays a crucial role in the growth and development of a fetus. Several studies focusing on specific nutrients and foods have advanced our comprehension of the links between maternal nutrition and birth weight . In pracIt is concordance across many studies that optimal birth weight was associated with what were perceived as healthier diets, such as the Mediterranean diet and the Dietary Approaches to Stop Hypertension diet, which was rich in vegetables, beans, and seafood, among other , 8. The Furthermore, gestational weight gain (GWG) of mothers is associated with the birth weight of newborns . A meta-The Tongji Maternal and Child Health Cohort (TMCHC) is a prospective cohort in Wuhan, Hubei province, central China, to investigate the associations between maternal dietary, lifestyle factors, and the pregnant outcomes of mothers and newborns from January 2013 to May 2016. Pregnant women at 8–16 weeks of gestation were enrolled in this cohort. This was the time when they went to the hospital for their first antenatal visit in one of three public hospitals in Wuhan. At enrollment, all participants completed an interviewer-administrated questionnaire which included some baseline information. Trained investigators conducted lifestyle and dietary intake interviews during each trimester. This study was carried out in accordance with the Declaration of Helsinki and approved by the Ethics Review Committee of Tongji Medical College, Huazhong University of Science and Technology. All participants gave informed written consent upon recruitment.n = 86), multiple gestations (n = 135), an implausible total energy intake  (n = 81), who had abortion or stillbirth (n = 22), or who did not have information about the diagnosis of GDM (n = 364) were excluded in this study. Finally, a total of 3,334 women were available  and been sure to undergo regular prenatal examination and delivery in the above hospitals. Those who reported a previous gestational diabetes mellitus (GDM) or history of diabetes ; the newborns were defined as large for gestational age (LGA) when the body weight was >90th percentile for gestational age and small for gestational age (SGA) when it was <10th percentile for gestational age; excessive GWG, adequate GWG, and insufficient GWG were evaluated based on the recommendation of Institute of Medicine  was calculated as birth weight (kg)/birth lengthMedicine .2. Smoking status was divided into “yes (either smoking or passive smoking)” and “no (none)” according to whether smoking was a habit before pregnancy, or whether participants were frequently exposed to second-hand smoke . Alcohol consumption was divided into “yes” and “no” categories based on whether alcohol was consumed before pregnancy . History of disease included chronic non-communicable diseases, infectious disease, and hereditary disease. History of a family disease considered these diseases of immediate families (parents and siblings). The history of specific diseases was considered as binary variables, divided into “yes” and “no”.The demographic and socioeconomic characteristics, anthropometric parameters, and lifestyle of the participants were recorded using a structured questionnaire at the baseline enrollment. The questionnaire was completed by trained investigators, including the information of maternal age, educational level, average personal income, ethnicity, pre-pregnancy weight, height, history of the disease, history of the family disease, parity, physical activity, consumption of smoke and alcohol, and so on. Maternal age was categorized as ≤24, 25–29, 30–35, and ≥36 years old. Ethnicity was categorized as Han Chinese and others. Education level was divided into ≤9 (junior high school or under), 10–12 , 13–15 (bachelor or college degree), and ≥16 (master or above) years . Per capDietary patterns were extracted by principal component analysis. This analysis was a data-driven technique that reduced the dimensions of the data and grouped correlated variables to identify common factors/components  by varimax rotation. The principle of principal component analysis was to decompose the total variance of the original indicators into the sum of the variances of several independent composite factors. The first principal component contributed the most to explaining total variation. The greater the contribution of variation among the factors, the better their ability to combine the original indicators. So, the factors represented the combinations of food consumed by individual participants. The number of factors retained was based on the eigenvalues, the breakpoints of the scree plot, cumulative variances, and factor interpretability. Thereinto, the factor loading reflected the relevance between the original 16 non-overlapping food group frequency and newly extracted factors (dietary patterns). In this study, the food groups with factor loadings of 0.40 or higher on a factor were considered important to the interpretability of each pattern. As the patterns were extracted, each participant had the dietary pattern score corresponding to each dietary pattern. The score was calculated by summing the mean standardized frequency of food groups weighted by their factor loadings. Higher dietary pattern scores indicated greater adherence to the extracted patterns , 20.t-tests. To explore the associations between dietary pattern scores and pregnant outcomes, we treated dietary pattern scores as quartiles and as continuous variables. Multivariate linear regression models were used to analyze the relations between dietary pattern scores and continuous pregnant outcomes, such as GWG and birth weight. In addition, the changes in pregnancy outcomes and P-value were calculated when the dietary pattern score increased by one unit. For the relations between scores and binary pregnant outcomes, such as excessive GWG, adequate GWG, insufficient GWG, SGA, and LGA, Logistic regression models were used. Stratified analyses were conducted by sex of newborns. Mediation analysis was an approach to assess the importance of various pathways and mechanisms, which had expanded over the past decade . Other social and demographic characteristics of participants were present in Overall, 3,334 participants were included in this analysis. Five dietary patterns were identified , and we Pregnant outcomes of all participants were presented in Ptrend = 0.012). Considering the score as a continuous variable, normal birth weight increased by 17.58 g  with one unit increase in this score. Although “the Fish-meat-eggs” pattern score had no significant relationship with birth weight, the risk of LGA increased 18% as the score increased by one unit . Other associations between dietary pattern scores and birth outcomes were exhibited in Whether unadjusted or adjusted, the relationships between dietary pattern scores and normal birth weight only existed in the “Beans-vegetables” pattern, as displayed in Ptrend = 0.024). One unit increase in dietary pattern score was associated with the increase of 20.41 g of birth weight of boys .After stratified analysis according to the offspring sex, the relation of “Beans-vegetables” only appeared in boys. Compared to the lowest quartile, the birth weight of the highest quartile increased 56.79 g , fiber , total iron  and non-heme iron , which indicated that these nutrients contributed to the association between “Beans-vegetables” pattern score and birth weight . With a unit of “Beans-vegetables” pattern score increased, GWG decreased 0.36 kg . In addition, this pattern reduced the 16% risk of excessive GWG and increased the 11% likelihood of adequate GWG , while it increased 14% risk of insufficient GWG in the meantime . For the other three patterns, the GWG of the highest quartile all showed an increase compared with the lowest quartile. GWG increased 0.18 kg for “Fish-meat-eggs” , 0.23 kg for “Nuts-whole grains”  and 0.26 kg for “Rice-wheat-fruits”  as score increased by one unit. And the risks of insufficient GWG for these three patterns decreased 15, 13, and 17%. Moreover, the “Rice-wheat-fruits” pattern increased the 14% likelihood of GWG in the appropriate range   .βindirect = −4.50; 95% CI: −7.27, −2.04). After adjusted covariates, this mediation was still significant with an estimated mediating proportion of −26% . Besides, this mediating effect also remained in newborns of boys, and the estimated mediating proportion was −21.3% .Mediation analysis was performed to clarify the role of GWG in the relationship between the “Beans-vegetables” pattern and normal birth weight, as displayed in We extracted five dietary patterns to represent the dietary habits of pregnant women in central China, naming them as “Beans-vegetables,” “Fish-meat-eggs,” “Nuts-whole grains,” “Organ-poultry-seafood,” and “Rice-wheat-fruits,” respectively. Women who tended to have higher adherence to the “Beans-vegetables” pattern had infants with relatively high birth weight. Higher adherence to the “Beans-vegetables” pattern was significantly associated with the decrease of GWG, the reduction of excessive GWG risk, and the increase of adequate GWG odd. The association between the “Beans-vegetables” pattern and birth weight was partly mediated by GWG.In this study, we observed that the “Beans-vegetables” pattern contributed to increasing birth weight in the normal range, without generating the risk of LGA. Consistent with our results, some studies came to the conclusion that maternal dietary patterns rich in plant foods were associated with larger birth sizes in multiethnic Asian and Chinese populations , 11. ThePrevious studies have found a positive link between GWG of women and the birth weight of newborns, and GWG above the recommendations was associated with a higher risk of LGA , 26, 27.via GWG.Our further investigation revealed that plant protein, fiber, and iron served as contributors to the association between the “Beans-vegetables” pattern and birth weight, which may explain the potential role of plant-based foods. It is worth mentioning the beneficial effect of plant protein in plant-based foods. There have been few studies that show a direct effect of maternal plant protein intake on birth weight. However, Lai et al. found a positive association between plant protein-enriched foods and optimal GWG . The resThe findings of the beneficial effect of the “Beans-vegetables” pattern only appeared in boys in the present study. This may be explained by a sex-specific growth mechanism during the fetal period. Generally, boys have higher birth weights, possibly due to an interaction between sex hormones, fetal insulin, and genetic factors . AvailabThe predominant advantage of this study is the use of prospective design to clarify the association between maternal dietary patterns and birth weight. Moreover, we took the mediation of GWG into consideration and analyzed direct and indirect effects of dietary patterns, respectively when exploring their relationship with birth weight. However, we acknowledge some limitations of this study. First, the dietary data of this study was obtained from a single FFQ investigation for 24–28 weeks. But as a previous study suggested, a single source of dietary data can provide reliable information throughout pregnancy . Second,In conclusion, this prospective cohort study in pregnant women in central China has shown that adherence to the “Beans-vegetables” pattern, which is characterized by high vegetables and beans intake, is beneficial for effectively controlling GWG in mothers and moderately increasing birth weight in newborns. In addition, GWG serves as a mediator in the association between this dietary pattern and birth weight, helping to control birth weight in a normal range. These novel findings may provide guidance for pregnant women to adhere to a healthy diet for beneficial pregnant outcomes. Well-designed intervention studies are needed to confirm our findings and elucidate the metabolic mechanisms underlying these findings.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Tongji Medical College, Huazhong University of Science and Technology. The patients/participants provided their written informed consent to participate in this study.YL and XZ: conceived and designed the research, acquired the data, performed the statistical analysis, drafted the manuscript, and played an important role in interpreting the results. YZ, CZ, LHu, XC, RC, JW, QL, GS, HY, GX, LHa, and NY: acquired the data. XY: revised the manuscript and further contributed to the discussion. All authors read and approved the final vision of the manuscript.This work was supported by the National Program on Basic Research Project of China (NO.2013FY114200) for NY, and the National Natural Science Foundation of China (81973043) and Chinese Nutrition Society Nutrition Science Foundation—Feihe Tizhi, nutrition and health research fund (CNS-Feihe2019A33) for XY.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.


In contrast, pharmacological inhibition of large-conductance Ca2+-activated K+ (BKCa) channels and ATP-sensitive (KATP) channels did not abolish vasodilation. Inhibition of the vasodilatory signaling axis involving endothelial nitric oxide (NO), smooth muscle cell soluble guanylyl cyclase (sGC) and protein kinase G (PKG) did not abolish empagliflozin-evoked vasodilation. Inhibition of the endothelium-derived vasodilatory molecule prostacyclin (PGI2) had no effect on the vasodilation. Consistently, empagliflozin-evoked vasodilation remained unaltered by endothelium denudation. Overall, our data suggest that empagliflozin stimulates smooth muscle cell KV channels KV1.5 and KV7, resulting in vasodilation in resistance-size mesenteric arteries. This study demonstrates for the first time a novel mechanism whereby empagliflozin regulates arterial contractility, resulting in vasodilation. Due to known antihypertensive properties, treatment with empagliflozin may complement conventional antihypertensive therapy.The antidiabetic drug empagliflozin is reported to produce a range of cardiovascular effects, including a reduction in systemic blood pressure. However, whether empagliflozin directly modulates the contractility of resistance-size mesenteric arteries remains unclear. Here, we sought to investigate if empagliflozin could relax resistance-size rat mesenteric arteries and the associated underlying molecular mechanisms. We found that acute empagliflozin application produces a concentration-dependent vasodilation in myogenic, depolarized and phenylephrine (PE)-preconstricted mesenteric arteries. Selective inhibition of smooth muscle cell voltage-gated K Type 2 diabetes (T2D) and hypertension are two major and independent risk factors for the development of cardiovascular diseases including heart disease, stroke, peripheral arterial disease and chronic kidney disease . HyperteClinical studies suggest that there is considerable residual risk for cardiovascular events in the hypertensive-diabetic group despite standard pharmacologic treatment and lifestyle modifications . TherefoV1.5 ion channels [V1.5 ion channels [Empagliflozin is a potent and highly selective SGLT2 inhibitor that received U.S. FDA (The United States Food and Drug Administration) clearance for use in T2D in 2014 . A signichannels . In addichannels . In an echannels . This rachannels ,33,34,35+ channels KV1.5 and KV7 to elicit vasodilation in resistance-size mesenteric arteries. This study is significant as it unveils a direct role for empagliflozin in relaxing mesenteric arteries that reduces vascular resistance and may explain blood pressure lowering effects of this drug. This study also lends additional support for the use of this drug in hypertensive-diabetic patients for reducing the risk of adverse cardiovascular events such as heart attack and stroke.Our data demonstrate that empagliflozin stimulates smooth muscle cell voltage-gated K+-PSS (30K), which depolarizes arteries to the same extent as 80 mmHg intraluminal pressure. We found that empagliflozin relaxed both PE and 30K-preconstricted arteries in a concentration-dependent manner , and a cumulative concentration response to empagliflozin was performed. Our myography data show that empagliflozin produced a concentration-dependent vasodilation in mesenteric arteries A,B. Empat manner A–D, simiEndothelial cells (ECs) that line the lumen of all blood vessels are a critical regulator of smooth muscle contractility and vessel diameter. ECs produce vasodilators that act on underlying smooth muscle cells to elicit arterial vasodilation. NO, produced by eNOS, is one of the most prominent vasorelaxant factors that diffuses into arterial smooth muscle cells to stimulate sGC and cGMP production. Elevated cGMP levels activate PKG, which in turn activates myosin light chain phosphatase, resulting in vasodilation .Considering the critical role of the NO-sGC-PKG signaling axis in vasodilation, we examined whether pharmacological inhibitors of this signaling axis could attenuate empagliflozin-mediated arterial vasodilation. We found that L-NNA, an inhibitor of eNOS, ODQ, a sGC inhibitor or KT5823, an inhibitor of PKG did not abolish empagliflozin-induced vasodilation A,B. Thes2 that acts on its smooth muscle cell receptors to produce vasorelaxation [2 production to induce vasodilation. To perform this, we preincubated mesenteric arteries with indomethacin, an inhibitor of COX, and then applied empagliflozin. Our pressure myography data revealed that indomethacin application did not abrogate empagliflozin-induced vasodilation in mesenteric arteries (2 production.Cyclooxygenase (COX) enzymes in the endothelial cells synthesize another major vasodilator PGIlaxation ,38. Therarteries A,B. ThesTo assess the role of endothelium further, we compared empagliflozin-induced vasodilation in endothelium-intact and endothelium-denuded mesenteric arteries. Endothelium denudation was performed by slowly passing air bubbles through the vessel lumen and confirmed as described by others previously ,40,41. B+ channels that control membrane potential and arterial contractility [+ channels (KV) are reported to be expressed in smooth muscle cells, and their activation causes K+ efflux, smooth muscle hyperpolarization and vasodilation [Arterial smooth muscle cells express a wide variety of Kactility . Severaldilation ,44.V channels to cause vasodilation in mesenteric arteries. Indeed, our data showed that the application of 4-aminopyridine (4-AP), a non-selective KV blocker, [V channel stimulation is critical for empagliflozin-mediated vasodilation in mesenteric arteries. We next explored the contribution of major KV channel isoforms present in mesenteric artery smooth muscle cells, including KV1.3, KV1.5 and KV7 [V1.5 inhibitor [V7 [V1.5 and KV7 channels were simultaneously inhibited by the co-application of DPO-1 and linopirdine, empagliflozin-evoked vasodilation was strongly suppressed by ~69%  and ATP-sensitive K+ channels (KATP) which, when stimulated, caused arterial hyperpolarization and vasodilation [Arterial smooth muscle cells also express large-conductance Cadilation .Ca and KATP channels in empagliflozin-induced vasodilation. Myography data showed that the application of paxilline, an inhibitor of BKCa channels [ATP channels [V channels, specifically KV1.5 and KV7 but not BKCa or KATP channels.Here, we analyzed the contribution of BKchannels  and glibchannels , did notV1.5 and KV7 ion channels.In this study, we demonstrated for the first time that empagliflozin stimulates vasodilation in resistance arteries such as the small mesenteric arteries. Our data also demonstrate that empagliflozin-evoked vasodilation does not depend on endothelial signaling; rather, it depends on the activation of arterial smooth muscle cell KAccumulating evidence suggests that empagliflozin treatment is associated with substantial reduction in adverse cardiovascular events, such as heart attack and stroke, in diabetic patients . Cardiov+ PSS opens voltage-gated Ca2+ channel CaV1.2 in arterial smooth muscle cells, resulting in Ca2+ influx, rise of intracellular Ca2+ concentration and vasoconstriction. PE binds to smooth muscle cell α1 adrenergic receptor (Gq11-coupled), which activates phospholipase C (PLC), resulting in the formation of inositol triphosphate (IP3). IP3 binds to ryanodine receptors on sarcoplasmic reticulum (SR) to stimulate the release of stored SR Ca2+ into the cytoplasm, causing smooth muscle contraction [V1.5 channels [V1.5 channels in mesenteric arteries, as it did in the aorta. Furthermore, our study demonstrates that empagliflozin-induced vasodilation in mesenteric arteries is dependent on the activation of two smooth muscle cell KV ion channels, KV1.5 and KV7 (V7 inhibition produces 15% more vasodilation than the inhibition of KV1.5 channels. As KV1.5 channels are the predominant KV isoform (65%) [V7 which has relatively lower abundance  [V7 over KV1.5 channel. This suggests that KV7 channel activation is the major contributor to empagliflozin-elicited vasodilation in mesenteric arteries. In contrast, a lack of involvement of KV7 channels in mediating empagliflozin-induced relaxation of the aorta may be attributed to the difference between aorta and mesenteric artery microenvironment. Unlike the previous finding that suggests a role for PKG in aorta relaxation, our data rule out the contribution of PKG activation and its upstream signaling components involving sGC and endothelium-derived NO in empagliflozin-induced mesenteric artery vasodilation. These findings suggest that, although PKG activation is required for empagliflozin-induced relaxation of aorta, it has no role in mesenteric artery vasodilation or the activation of KV1.5 and KV7 channels. Since empagliflozin relaxed 30K-constricted mesenteric arteries, it is possible that other mechanisms, in addition to KV ion channels, are also activated by this drug to induce vasodilation. Regardless of the mechanisms involved, relaxation of resistance mesenteric arteries upon acute empagliflozin application may be beneficial for reducing blood pressure and the risks of heart attack and stroke that are associated with elevated arterial tone and hypertension.Here, we demonstrated that empagliflozin relaxes myogenic (80 mmHg-constricted), 30K- and PE-constricted resistance-size mesenteric arteries . Intralutraction . Althougchannels . Our fin and KV7 . We demorm (65%) ,44 in me all KV)  may indiSGLT2 inhibitors including empagliflozin have been shown to have a wide range of effects in the cardiovascular system and beyond ,33,34,35Our study has several limitations. Since our findings are based on healthy, young animals, future studies using diabetic animals will be required to validate the vasodilatory action of this drug in a relevant model. We tested the vasodilatory action of empagliflozin in an ex vivo preparation of resistance-size mesenteric arteries. Proof of concept studies such as blood pressure measurement in ambulatory animals as well as in vivo blood flow measurement will be required to understand the full therapeutic potential of this drug in blood pressure and blood flow regulation. Similar experiments in human vessels and the measurement of blood flow and blood pressure will significantly enhance the translational aspect of this work.V1.5 and KV7 ion channels. Empagliflozin-evoked vasodilation does not depend on endothelial signaling or other smooth muscle K+ channels. This finding is significant as it uncovers a direct role for empagliflozin in reducing vascular resistance, which may provide an explanation for its blood pressure lowering action in clinical studies. As diabetes and hypertension coexist, the use of empagliflozin (perhaps other SGLT2 inhibitors) in this high-risk diabetic population may complement conventional antihypertensive therapy to potentially reduce the overall risk of adverse cardiovascular events such as heart attack and stroke.In summary, we demonstrated that empagliflozin relaxes resistance-size mesenteric arteries by stimulating smooth muscle cell KAll animal protocols were approved by the Mercer University Institutional Animal Care and Use Committee  Male Sprague Dawley (SD)  rats, 8–2 gas into a CO2 euthanasia chamber followed by decapitation. An entire mesenteric artery bed was dissected and placed into ice-cold PSS (see composition below). Third and fourth order branches of mesenteric arteries (150–250 µm) were dissected cleaned of adventitial tissue in ice-cold PSS. Cleaned arterial branches were cut into 1–2 mm long segments that were kept in ice-cold PSS until cannulated for pressure myography [Animals were euthanized by passing compressed COyography ,40,41,573 1.18, MgSO4 1.18, KH2PO4 1.18, CaCl2 1.8 and glucose 10. The pH of the PSS was adjusted to and maintained at 7.4 by continuous flow of normal air into the PSS. The amount of 60 mM K+-PSS (60K) was prepared by equimolar replacement of NaCl with KCl in the PSS. Empagliflozin was purchased from Ambeed Inc.  and used in the concentration range of 0.001–100 µM. Phenylephrine (PE) and 4-aminopyridine (4-AP) were purchased from Sigma-Aldrich  and used at a final concentration of 1 µM and 1 mM, respectively. Indomethacin (10 µM), DPO-1 (1 µM), Linopirdine (10 µM), Psora-4 (100 nM), Glibenclamide (10 µM), paxilline (10 µM), ODQ (10 µM), KT 5823 (1 µM), L-NNA (10 µM), SNP (10 µM) and acetylcholine ) were purchased from Tocris . 4-AP, SNP and PE were dissolved in distilled water. Empagliflozin, DPO-1, linopirdine, psora-4, indomethacin, glibenclamide, paxilline, ODQ, KT5823, L-NNA and ACh were dissolved in dimethyl sulfoxide (DMSO). The final DMSO concentration (<0.1%) in the myograph chamber had no significant effect on arterial contractility.PSS for vessel dissection, isolation and preparation contained (in mM) the following: KCl 6.0, NaCl 112, NaHCO2, 5% CO2 and 74% N2 to maintain pH 7.4. A 60K-induced constriction was used for testing the viability of the arteries at 10 mmHg before all experiments. Intraluminal pressure gradually increased to 80 mmHg with increments of 20 mmHg at each step to allow the development of myogenic tone. Intraluminal pressure was altered using a pressure servo controller with peristaltic pump  and monitored with a pressure monitor. At 80 mmHg, mesenteric arteries developed 25 ± 3.91% myogenic tone, after which increasing concentrations of empagliflozin (0.001–100 µM) were applied. Diameter changes were recorded at 1 Hz using a charge-coupled device (CCD) camera connected to a Nikon Ts2 microscope and an automatic edge-detection function of IonWizard software . Luminal flow was absent during the experiment. Myogenic tone (%) was calculated as follows: 100 × (1 − Dactive/Dpassive), where Dactive is active arterial diameter in the presence of Ca2+ and Dpassive is the passive arterial diameter determined in Ca2+-free PSS supplemented with 5 mM EGTA [Arterial segments were cannulated in a perfusion chamber , continuously perfused with 37 °C PSS and gassed with a mixture of 21% O mM EGTA ,40,41,57For the mechanistic studies, we used 1 μM PE-preconstricted arteries. Arteries were preincubated for 15 min with different pharmacological modulators prior to the application of empagliflozin in the presence of the modulator.Endothelium was denuded by slow passage of air bubbles through the vessel lumen for 1–2 min. Absence or at least 90% reduction in ACh-induced vasodilation was used as the indicator of successful endothelium denudation ,40,41. St-tests (two-tailed) to test hypotheses. A p value of <0.05 was considered statistically significant [OriginLab software version 9.55 (2019b)  was used for data analysis. The results were expressed as mean ± SEM. Considering the interval-ratio data with normal distribution, we performed unpaired Student’s nificant ,29,39.


Preterm birth (PTB), defined as birth before 37 completed weeks of gestation, is a major cause of infant morbidity and mortality. Inflammation is an important component in the physiopathologic pathway leading to PTB but results from cross-sectional studies on associations between inflammation, as measured by cytokines, and PTB are inconsistent. Timing of cytokine measurement during pregnancy varies between studies and may contribute to inconsistent findings. We investigated the effects of timing on associations between 16 cervico-vaginal cytokines  and PTB among 90 women throughout pregnancy. We used logistic regression to compare associations between concentrations of cervico-vaginal cytokines from periods in pregnancy and PTB. Trimester 1 cytokines had the strongest positive associations with PTB; for example, OR = 1.76  for IL-6. Second and third trimester associations were weaker but largely positive. IL-1α was the only cytokine with a negative association . Strong first trimester associations between cytokines and PTB suggest that measuring cytokines early in pregnancy may hold promise for early identification of PTB risk. Variations in cytokine measurement during pregnancy may contribute to inconsistencies among studies. Preterm birth  is a major public health problem worldwide ,2. The bThe inflammatory response, involving fetal membranes, decidua and cervix, is one of the pathways linked to the development of PTB ,12,13,14Our study addresses these previous limitations and uses a design that includes the same set of participants across the study. We utilized 16 cytokines obtained from monthly cervico-vaginal samples to estimate the associations between cytokines from different points in pregnancy and PTB.We analyzed data from the Pregnancy Research on Inflammation, Nutrition, & City Environment: Systematic Analyses (PRINCESA) cohort, a longitudinal study (enrollment 2009–2014) based in Mexico City . The resStudy participants were pregnant women in Mexico City, Mexico. Inclusion criteria included singleton pregnancies, and resided and/or worked in Mexico City and surrounding areas. In addition, participants had to be 18 years or older, in their first or second trimester of pregnancy, and able to attend approximately monthly prenatal visits. Exclusion criteria included presence of medical or obstetric complications in the current pregnancy. All participants provided written informed consent. Clinical samples, behavioral, and demographic information were collected monthly. The parent study included 935 participants and birth outcomes were available for 838 participants. This analysis was limited to the 90 participants who met all of the following requirements; (i) participants had to have had at least three visits (ii) at least one visit during each trimester (iii) cytokine measures for each corresponding visit, and birth outcome including gestational age had to be available for each participant. There were no fetal abnormalities among participants in the current study. Participants who developed obstetric complications were referred to a tertiary level hospital and did not continue follow up in the study.Fetal gestational age was calculated using the date of visit and the reported first day of the last menstrual period (LMP) and confirmed by ultrasound; gestational age at birth was calculated based on the infant’s date of birth and the first day of the LMP. Both calculations were based on reliable and accurate recall of LMP from data collected during screening. PTB was defined as being born before 37 weeks of gestation. Spontaneous preterm labor and pre-labor rupture of the membranes were included.Sampling occurred by the use of a vaginal mirror rinsed in a sterile saline solution to visualize the cul de sac. Cervico-vaginal samples were obtained using a Dacron swab rotated for 10 s in the cervico-vaginal section of the reproductive tract. Collected samples were placed in a buffering solution and transported to the laboratory. Two additional samples of the exudate were taken using a similar method and were processed for conventional microbiologic cultivation and for confirmatory probes if infection was suspected. Samples were collected during each visit and frozen at −20 °C for later processing. Samples used in this study were collected between 2010 and 2014 and cytokines were quantified between 2011 and 2014.® MAP human cytokine/chemokine magnetic bead panel kit  based on the manufacturer’s published protocol. Analyses were performed on 50 µL of previously frozen duplicate cervico-vaginal exudate samples. Cytokine data included left and right censored observations. Limit of detection (LOD)/√2 (left censored) and a value of 10,010 (right censored) were substituted for these observations.The following cytokines were quantified in picograms/milliliter (pg/mL) from cervico-vaginal samples: Eotaxin, Interferon gamma (IFNγ), Interleukin (IL)-10, IL-12p40, IL-12p70, IL-17, IL-1RA, soluble IL-2 receptor alpha (sIL-2rα), IL-1α, IL-1β, IL-2, IL-4,IL-6, IL-8, Interferon gamma inducible protein (IP-10), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein 1 alpha (MIP-1α), macrophage inflammatory protein-1 beta (MIP-1β), tumor necrosis factor alpha (TNFα), and vascular endothelial growth factor (VEGF). Cytokine quantification was done using the Millipore MILLIPLEXp-values from the logistic regression models were adjusted using Benjamini–Hochberg corrections for multiple testing [Cytokine distributions were evaluated using histograms to assess whether cytokine data needed to be transformed. Fisher’s exact test and T-test were used to evaluate differences between term and preterm births. To evaluate differences in the distribution of cytokine concentrations between groups defined by preterm birth status, we used the non-parametric Kruskal–Wallis test because the normality of residuals assumption required to use analysis of variance was not met. We evaluated the association between each cytokine and PTB on the same group of participants over time. To account for potential cytokine variability within each trimester and avoid having single measures represent cytokine levels during a trimester, we averaged individual cytokine measurements for each exposure period . Comparative analyses were conducted using logistic regression models for individual cytokines for the following averages: i. first trimester; ii. second trimester; iii. third trimester; and iv. overall pregnancy.  testing . All anap < 0.05), we observed no statistically significant associations between demographic characteristics and preterm birth status (A group of ninety participants (78 term and 12 preterm births) was included in study. The median gestational age at enrollment was higher among term births than preterm births (11.2 weeks vs. 10.3 weeks). Other than finding that participants who delivered preterm babies were more likely to be nulliparous , seven , two (IL-1β and IL-6) and nine cytokines from the first, second, third trimesters and overall pregnancy average, respectively. Cytokines IL-6 and IL-1β were the two cytokines that were significantly different across all points in pregnancy between term births and preterm births .Individual cytokines from the first trimester had the strongest positive associations with PTB; 14 cytokines  were either statistically significant or marginally significant after adjusting for multiple testing. Models using entire pregnancy average for each cytokine had 11 estimates that were either statistically or marginally significant compared to eight for second trimester concentrations. First trimester VEGF concentrations had the strongest association with PTB  and remained the strongest statistically significant predictor at other points during pregnancy. In general, statistically significant associations were fewer and the strength of the associations was attenuated as the pregnancy progressed. IL-1α was the only cytokine that exhibited a protective association .We compared the distributions of 16 cervico-vaginal cytokines at each trimester of pregnancy between women who delivered term or preterm babies, and examined the age-adjusted associations between cytokine concentrations and risk of preterm birth. Samples across trimesters were obtained from the same group of women, a key strength of this longitudinal design. Both significant differences in cytokine concentrations and age-adjusted associations were found, with the strength of associations varying across trimesters. Cytokines obtained during the first trimester were the strongest and most frequent predictors of PTB. This finding suggests that processes occurring early on in pregnancy are potentially useful in identifying women at risk of delivering before 37 completed weeks of gestation and may inform future studies directed at early prevention of preterm labor. During pregnancy, the feto-maternal tolerance has to be established to allow growth and development of the fetus, while concurrently activating the innate and adaptive mechanisms to promote survival of the developing fetus. The resulting immune mechanisms involved in these processes may be systemic or operate in the local components of the maternal-fetal unit . Feto-maIdentifying individuals who are at high risk of preterm birth during the first trimester may lead clinicians to closely monitor high-risk patients and provide an opportunity to treat risk factors, for example asymptomatic lower reproductive tract infection, associated with increased risks of preterm birth. In addition, in other cases where increased risks are not associated with lower reproductive tract infections, initiation of a low-dose aspirin regimen during pregnancy could play a role in reducing preterm birth risk and should be explored as a prevention for preterm birth. A recently published double-masked, placebo-controlled randomized trial of 11,976 participants conducted in six countries found a statistically significant reduction in preterm birth and other key measures among women taking low-dose aspirin initiated between the 6th week and end of the 13th week of pregnancy compared to women taking placebo . Additio10) and carbon monoxide (CO)) and the same cytokines (N = 7) quantified from cervico-vaginal samples and serum samples [Several previous studies have evaluated associations between PTB and a limited number of cytokines obtained from a single point during pregnancy. Although some of these studies found associations between cytokine levels and PTB, results have been inconsistent, ranging from null, to positive and negative associations ,19,20,34 samples . Using aBased on the limitations of previous studies, the simultaneous evaluation of 16 cervico-vaginal cytokines obtained over the course of gestation among a fixed group of women who delivered at term and preterm is a main strength of this study. Limitations of this work include that almost all participants were enrolled at three months of gestation, so we could not evaluate associations between cytokine levels at month 1 and 2 of gestation and PTB. Second, our sample size was relatively small, limiting our ability to adjust for known confounders other than maternal age. Third, we used substitution methods for censored cytokine observations and limitations have been reported when the percent above the LOD exceeds 25% . The perPositive associations between PTB and cytokines obtained during the first trimester suggest that samples taken early in pregnancy may be useful for risk screening. Consistently weaker associations seen later in pregnancy suggest that sampling from different periods of pregnancy may contribute to inconsistencies found in the PTB literature. Similarly designed studies are needed to further evaluate these findings.


In nine identified cases, 14C analysis of tooth crowns provided an estimate of the true date of birth with an average absolute error of 1.2 ± 0.8 years. Analysis of 14C in tooth roots also showed a good precision with an average absolute error of 2.3 ± 2.5 years. Levels of 14C in bones can determine whether a subject has lived after 1955 or not, but more precise carbon turnover data for bones would be needed to calculate date of birth and date of death. Aspartic acid racemization analysis was performed on samples from four cases; in one of these, the year of birth could be predicted with good precision, whereas the other three cases are still unidentified. The stable isotope 13C was analyzed in tooth crowns to estimate provenance. Levels of 13C indicative of Scandinavian provenance were found in known Scandinavian subjects. Teeth from four Polish subjects all showed higher 13C levels than the average for Scandinavian subjects.The identification of unknown human remains represents an important task in forensic casework. If there are no clues as to the identity of the remains, then the age, sex, and origin are the most important factors to limit the search for a matching person. Here, we present the outcome of application of so-called bomb pulse radiocarbon ( Dead victim identification constitutes an important task for forensic professionals including forensic pathologists, anthropologists, and odontologists in their daily casework, particularly in mass disasters. If there are no clues as to the identity of the deceased, age, sex, and origin represent particularly important information to limit the search for possible matching persons. Whereas the sex often can be determined by morphological characteristics, or by DNA analysis, the age of adult subjects is more difficult to estimate. Since structural parts of teeth and bones are highly resistant to decomposition, chemical degradation, and heat, they may constitute the only material available for forensic analyses. Many different methods for estimating the age of a deceased person have been published, most of them based on examination of teeth and bones. Anthropological analyses based on morphological changes typically provide age estimation errors for adults of ±10 years ,3,4,5,6,In practical casework, the primary focus is to determine whether the find is of human origin, and then whether it is recent or old, warranting a further police investigation or not. One powerful technique that is useful in this respect is the retrospective radiocarbon dating of modern biological material ,21. In f14C) is naturally formed in the atmosphere by cosmic rays’ interactions with nitrogen-14 (15N). Carbon atoms in the atmosphere are quickly oxidized to form CO2, which is then incorporated into plants via photosynthesis. The 14C levels in the atmosphere have stayed fairly stable for several thousands of years. However, atmospheric above-ground detonations of nuclear weapons during the Cold War (1955–1963) almost doubled the concentration of 14C levels in the atmosphere, which was rapidly distributed around the globe [14C levels in the atmosphere have been decreasing exponentially. This decrease is not due to radioactive decay  but is caused by mixing with large marine and terrestrial carbon reservoirs.Carbon-14  analysis. We have taken advantage of this by analyzing 14C in tooth enamel, which does not exchange any carbon atoms with the environment after it has been formed. In their pioneering study, Spalding et al. [14C analysis of tooth enamel. Similar precision has subsequently been reported in several studies [14C incorporation times in tooth enamel, which are more practical to use than radiographic laydown times for the calculation of a person’s date of birth [14C levels. Further, analysis of tissues with a high turnover—virtually all soft tissues except the lens of the eye [The bomb pulse-generated artificial variation in atmospheric g et al.  reported studies ,28,29,30of birth . The lim the eye —will not14C levels will match times in between the birth and the death of the individual. For archeological studies, this is not a critical issue, but when dating post-bomb skeletal remains in criminal investigations, it is certainly important to be aware of the variation in levels that may be found in different types of bones of persons of varying ages [In archeological casework, the collagen fraction of bone  is prefeing ages ,35.2H and 18O vary with latitude and precipitation, which in part are associated with the distance from coastal areas, and will show geographical variation, reflected in the drinking water. Hence, analysis of these isotopes in teeth will give a rough idea of the geographical region where a person was raised. In contrast, 13C levels reflect the diet, which partly depends on geography and partly on cultural habits. So-called C4 plants, which include crops that grow in hot areas , enrich 13C due to a different CO2 assimilation as compared to so-called C3 plants , which thrive in colder climate regions [In addition to radiocarbon dating of tooth crowns, it is also possible to analyze stable isotopes in tooth enamel and dentin to obtain clues about the provenance . Althoug regions ,37. We h regions ,31.14C method, AAR analysis, and 13C analysis could provide helpful information.Today, there are numerous publications on a variety of methods for age estimation of human remains, but there is a paucity of reports regarding the performance of these methods in practice. Hence, we sought to study the panorama of cases in which the police requested investigation regarding age, sex, and provenance, and to what extent the bomb pulse The material studied consisted of teeth and bones from human subjects. The remains were obtained from the six Swedish forensic medicine departments, which in turn had received them from the police during the period 2011–2021. 2O and placed again into the sonicator water bath. This procedure was repeated three times, and the material was then dried at room temperature. Next, the entire crown and root were crushed using Qiagen Tissue-lyser II. A fine powder was obtained using a standard program with a run time of 30 s. The powder was then further pre-treated at the AMS laboratory.The soft tissue and dirt were removed mechanically using an odontological electric drill. The tooth crown was cut away from the root at the level of the cervical line. The crown and root were then incubated in 0.2 N HCl at room temperature in a sonicator water bath .The crown and root were washed three times with ddHSample pre-treatment was performed on all samples prior to AMS analysis. The carbonate  fraction was analyzed for both tooth crown and root samples. In some cases, the collagen fraction was also analyzed in tooth roots to allow for comparison between the isotopic levels in the two fractions. For bone samples, only the collagen fraction was analyzed. Procedures for the extraction of each fraction are described below.2 was purified, split, cryogenically trapped, and finally reduced by graphitization with an iron catalyst [In this step, 10 mL 0.8 M HCl cooled to 5 °C was added to 200–500 mg of the tooth sample in a low vacuum reaction chamber. The subsequent leaching reaction was carried out at room temperature for a duration of at least 1 h until no further gas release was observed. The obtained COcatalyst ,39.2 was split, purified, trapped, and reduced by graphitization with an iron catalyst [Collagen extraction was performed by a standard HCl–gelatin procedure . Sample catalyst ,39.14C AMS analysis was performed on the graphitized samples at the Tandem Laboratory, Uppsala University, using the 5 MV pelletron tandem accelerator or the MICADAS (mini radiocarbon dating system) [2 that was split prior to graphitization was used to perform 13C and 15N stable isotope measurement using isotope ratio mass spectrometers, .The  system) ,42. Cali system) . A small14C incorporation times [14C levels to matching atmospheric levels, we used the CALIbomb Levin data set [The average age at which the formation of tooth crowns and roots is completed for each specific tooth has been determined previously and is dependent on the tooth number and gender. We used the formation times for tooth roots reported by Nolla , but foron times . For thedata set  with 1.014C analysis, and conversely, to estimate the year of birth in one case with a known date of death.AAR analyses were performed on nine teeth from four cases to provide an estimate of the person’s age at death. These results were used to estimate the year of death in three cases, in which the year of birth had been estimated by D- and L-forms of aspartic acid were measured by gas chromatography using a glass capillary  after hydrolysis and derivatization. The column of the glass capillary was 30 m in length, 0.3 mm in internal diameter, and coated with Chirasil-Val . The concentration of the D-form was related to the concentration of the L-form, and the racemization ratio was expressed as ln((1 + D/L)/(1−D/L)). Plotting the chronological age on the x-axis and the racemization ratio on the y-axis, we derived the following linear regression equation by the least squares method:t is the chronological age, and k is the racemization rate constant. To estimate the chronological age, we plotted the age against the racemization ratio and derived the following linear regression equation by the least squares method.In Case 2, the AAR analysis was performed according to a previously described protocol . BrieflyThe estimated age was obtained by substituting the D/L ratio in this linear regression equation with that of the specimen to be estimated. For a detailed flowchart illustrating the method of racemization analysis, see .Cases 14 and 16 were analyzed at the forensic toxicology laboratory in Linköping, Sweden. The analysis was performed on 1 mm-thick median longitudinal sections, prepared, cleaned, and pulverized as described above. The aspartic acid enantiomers were measured by  with mass spectrometric detection, after hydrolysis and derivatization. The chiral column Astec Chirobiotic T 2, 1 × 250 mm, 5 μm was used. The concentrations of the D- and L-forms were determined, and the racemization ratio was expressed as above. Case 28 was analyzed at the Department of Legal Medicine, University Hospital, Düsseldorf, Germany, by gas chromatography with flame ionization detector according to a previously described protocol . BrieflyTM and GlobalFilerTM STR kits  were used according to the manufacturer’s instructions.DNA analysis was performed at the Department of Forensic Genetics, Swedish National Board of Forensic Medicine, Linköping, Sweden. The IdentiFilerAll statistical analysis was carried out using SPSS version 25 . Results are expressed as mean ± SD. Linear regression analysis was used to calculate the correlation between the estimated and true date of birth.14C bomb pulse dating. Nine of these 18 cases were eventually identified, and a high correlation was found between the estimated and true year of birth for these individuals with an average absolute error of 1.18 ± 0.83 years . Even if the person was born before the onset of the bomb curve, there is a possibility of observing positive 14C readings in the teeth: For the third molar in the upper jaw, the crown and the root are formed on average 11.7 and 17.0 years, respectively, after birth. For instance, in Case 33, tooth 43 showed a pre-bomb 14C level in the crown, but the root displayed a post-bomb value and allowed for an estimate of the person’s year of birth. This particular case is still under investigation and the identity not yet established. We also found a high correlation between the estimated age based on 14C levels in tooth roots and the actual age of the subject with an average absolute error of 2.3 ± 2.5 years , both trabecular and cortical bone was analyzed. In Case 41, the trabecular bone sample showed a higher  he died . When ad14C analysis on the femur and found levels matching 2008.3, hence supporting the mother’s account. This implied that the woman was not prosecuted, given that she was underaged at the time of the event.Case 42 regards a newborn baby who was found severely decomposed in a plastic bag in a garden in 2018. The police suspected a 24-year-old woman to be the mother, and this was soon confirmed by DNA analysis. According to the woman, she gave birth to the child in 2009 (when she was 15 years old), yet this statement was questioned since the body had been lying outdoors and soft tissues remained. Since the baby lacked teeth, we carried out 13C levels in the carbonate fraction of all 114 tooth samples (crowns and roots) from 34 individuals were analyzed with IRMS. All subject information and raw data are listed in 13C levels were higher than expected for Scandinavian subjects [13C levels of −12.7 ± 1.8 compared with −15.9 ± 1.8 for tooth samples from Swedish subjects in this study and −14.9 ± 0.3 for all Scandinavian subjects that we have analyzed previously reported [13C levels averaged −12.6 ± 4.3 and −20.5 ± 4.2 in the carbonate and collagen fraction, respectively.The subjects ,31. All reported ,29,31. I13C levels were also measured in the collagen fraction of 24 bones from 21 individuals  averaged 3.3 ± 0.1, supporting a good quality of the stable isotope samples; the C/N ratio should be in the range of 2.9–3.6 [The ividuals . The lev 2.9–3.6 .14C analysis, and this information helped the police to identity the person. The identities of the other three subjects have not as yet been determined. However, since we were able to estimate the year of birth for Cases 2 and 28, the AAR analysis allowed us to calculate the year of death by adding the estimated age at death, which was of particular interest to the police. However, both investigations are still ongoing.AAR was performed on root dentin samples from four cases . In Case14C analysis on the crowns of three teeth, but all showed pre-bomb values, including tooth 48, which has an average 14C incorporation time of 11 years after birth [Case 16, named the Isdal woman, has attracted wide interest since her partly burnt body was found in 1970 in a remote spot, Ice Valley , near Bergen in Norway . We obtaer birth . Hence, TM or GlobalFilerTM PCR amplification kits, by which the gender can also be determined. Comparative analysis with these kits was performed in all nine cases that were eventually identified. In a further 12 cases, DNA profiling failed due to poor quality of the DNA. In the remaining cases, a few well-preserved bodies could be visually identified, and in other cases DNA profiling was not considered meaningful since the remains were too old to justify an investigation.In 13 cases, a successful DNA profile could be obtained using the Identifiler14C analysis of teeth is a technique that can be used in forensic practical casework to estimate the year of birth with a precision that is just as good as that observed for analysis of teeth extracted from living patients [14C levels since the carbon turnover in the samples is unknown and hence it is not possible to tell which values are to be expected if contamination is absent. Regarding tooth enamel and dentin, however, we found that bomb pulse 14C analysis could predict the true year of birth with excellent precision even though several cases had been exposed to soil, air, and water for extended times, hence excluding any major degree of contamination of the teeth.We show here that radiocarbon bomb pulse patients ,30,31,50patients . For theBy cutting off the crown, removing dentin remnants, and then pulverizing the enamel mechanically with a bead mill, we could dramatically expedite the standard extraction procedure previously reported  while maWe also show that radiocarbon dating of tooth roots provides estimates of year of birth nearly as good as enamel, with no systematic deviation in the prediction of an individual’s date of birth. This suggests that dentin, even though it is considered a “living tissue”, has a very low turnover of carbon, otherwise we would have obtained estimates indicating a more recent year of birth for the subjects studied. This is in agreement with the results of AAR analysis of dentin, which requires a low turnover in order for the D-aspartic acid to be formed . Roots a14C analysis in bone difficult. There are up to 220 different types of bones in the body, and their carbon turnover rates are not well characterized. Ubelaker et al. [14C value suggests that the person was alive after 1955, whilst a pre-bomb value suggests that the person either was not alive after 1955 or died soon after that. If the 14C values are very high, it may be deduced that the person died within a decade or so after the bomb peak. In Case 47 (14C analysis of these can provide an estimate of date of death with a good precision as they will show contemporary values [14C dating since they will all have been formed within a short time window.Compared with tooth enamel and dentin, bone tissue is continuously remodeled, implying a turnover of carbon at a pace that makes dating the birth or death of a person from r et al. ,54,55. T Case 47 , the levy values ,56,57. W14C analyses can be performed on teeth to provide information about both the year of birth and year of death of an individual [14C birth dating or an estimation of the postmortem interval. One advantage of AAR analysis is that it is independent of the bomb peak and hence can be used for age determination of subjects born long before the beginning of above-ground nuclear weapon testing. Several factors, however, will affect the precision of this method; as the racemization process is effectively a function of temperature and time, the precision varies depending on the tooth type analyzed, most likely due to their exposure to different ambient temperatures [We have previously reported that combined AAR and AMS dividual . AAR anaeratures . In Case13C levels in teeth extracted from patients in many different countries [13C levels in teeth from subjects raised in Poland all had higher levels. When assessing people on the American continent, the high 13C levels result from a traditional dietary intake of products from C4 plants, such as corn and sugar cane, but for people living in continental Europe, no such reference 13C data or suggested explanation for the elevated concentrations exist. Case 18 was born in Vietnam but raised in Sweden and showed, on average, a 13C concentration of 14.6 [13C levels in tooth crowns are shown in Studies on hair samples from subjects from different countries  have shoountries ,31. In t of 14.6 ,60. The 13C levels are different in the carbonate and collagen fractions due to variable fractionation in biological tissue structures [13C in the collagen fraction will be approximately −20 while the levels in the carbonate fraction will show a higher average, around −15, irrespective of whether the collagen is extracted from teeth or bone. Whilst considerable data detailing carbonate δ13C in teeth exist from different countries [Finally, it should be noted that ructures . For Scaountries ,31, no s13C levels, we also measured the 15N concentrations and calculated the C:N ratio, which should range between 2.9 and 3.6 in collagen samples from bone [In order to exclude the possibility that external contamination had affected the rom bone . For all14C levels still remain elevated in the teeth of the population of interest, which implies two or three decades to come.We show that radiocarbon dating of tooth enamel provides a very precise estimation of an individual’s date of birth and that very small amounts are sufficient for analysis. AMS determines the date of tooth formation, whilst AAR determines age of death. The combination of these methodologies offers considerable power to forensic pathologists and police authorities to help determine the identity and time of death of unidentified individuals. We expect an increased use in this strategy for as long as the bomb pulse


Background: Temperament involves individual variations in behavioural tendencies of emotional responses and reactions to stimuli after birth. Because ‘foetal programming' is a strong hypothesis in developing temperament, prenatal and intrapartum factors may be significant determinants of infant temperament. This systematic literature review aims to elucidate the evidence of prenatal and intrapartum predictors, including genetic, biological, environmental, socio-demographic, psychological, and obstetric factors of parents and their child.Methods: Relevant articles were searched using MEDLINE, PubMed, and SCOPUS. The inclusion criteria were (a) original research article, (b) written in English, (c) assessed the temperament of infants 12 months old or younger as an outcome variable, and (d) investigated prenatal and intrapartum factorial variables of infant temperament. Following the PRISMA guideline, the articles found in the three databases were screened and selected according to the inclusion and exclusion criteria before the final review.Results: Finally, 35 articles were reviewed. This systematic review identified a variety of prenatal and intrapartum factors that were significantly associated with infant temperament: (1) genetic and biological factors: certain genotypes, maternal cortisol and ACTH, and CRHs, (2) environmental factors: substance use such as tobacco, alcohol, and illegal drugs, (3) socio-demographic factor: lower-income, (4) psychological factors: depression or anxiety, eating disorders, personality types of mothers, and domestic violence, and (5) obstetric factors: foetal growth (birth weight), hypertension in mothers, nausea (emesis), and preterm birth.Conclusion: The findings support gene-environment interaction and biological mechanisms for developing infant temperament, suggesting the importance of ensuring a safe and comfortable environment for pregnant mothers, unborn infants, and families during pregnancy and delivery. We limited our search to infancy because it is believed that the expression of temperament is relatively stable in infancy (st author). Full-text articles of relevant records were carefully reviewed by M.T. (1st author) and A.M. (2nd author) according to the inclusion and exclusion criteria and the quality of each article was assessed. After a discussion, 35 articles were selected for the review. infancy . FollowiNineteen papers (3.4%) were excluded because they did not meet the inclusion criteria: (a) 13 studies observed the temperament of infants older than 12 months of age, (b) two studies were identified as literature reviews, (c) four studies assessed infants' neuro-motor development but not temperament, and (d) two articles referred to the same study as one of the included articles and one did not assess predictors of a child's temperament. n = 15) or North America (n = 17), whereas only a few were conducted in Oceania (n = 2) and Asia (n = 1). It was also evident that the number of articles published in this field had increased dramatically after 2006, although this research theme has been emphasised since 1978 (n = 10) or assessed infant temperament during the neonatal period (n = 2). Regarding the methodologies of assessing infant temperament, all the studies used parent-administrative questionnaires; however, two studies adopted the observational assessment of child behaviour by clinical specialist (n = 3). The most frequently used questionnaire was the Infant Behaviour Questionnaire (IBQ), its revised version (n =13), followed by the Infant Characteristic Questionnaire (ICQ) (n = 6), the Carey Infant Temperament Questionnaire (CITQ) (n = 4) (n = 2) (ad-hoc methods.nce 1978 . Regardi version  or a ver version  (n =13),re (ICQ)  (n = 6), (n = 4) , and the (n = 2) , among o (n = 2) , as wellIA polymorphism, which is related to the D2 dopamine receptor, and prenatal exposure to tobacco were observed for higher ‘attention' and ‘irritable reactivity' among 4-week old neonates , hypertension among mothers, nausea (emesis), and preterm birth. Further studies should confirm if low or high birth weight , 67 or iSome limitations should be noted. First, most of the reviewed studies adopted parent-administered questionnaires rather than objective measures to assess infant temperament. Although we excluded cross-sectional studies assessing psychological distress of parents and infant temperament simultaneously, perceived infant temperament may be biased due to parental mood at the time of observation (observer bias). Mothers with mental distress may have more negative views of parenting than others, such as a negligent attitude with decreased visual and physical contact with their infants , 85. SinFor future studies, study designs such as a prospective cohort study and adopting both subjective and objective measures may be recommended to avoid bias. Additionally, because most of the studies were conducted in Western societies targeting Caucasian children, more evidence needs to be accumulated in other ethnic groups because temperament is believed to be formed by genetic and biological functions. Furthermore, more evidence regarding pregnancy complications such as hypertensive disorders, nutrition status, delivery complications, and exposure to chemical factors  is needed to confirm the link with temperament .Regardless of these limitations, this systematic review helps us understand prenatal and intrapartum factors of temperament, including genetic, biological, socio-demographic, psychological, and obstetric aspects in the complex mechanism of development. The findings from this systematic review imply the necessity of ensuring and promoting a safe and comfortable pregnancy and childbirth by health care professionals. Raising awareness of the risks of substance use and providing health education for a healthy pregnancy and childbirth to mothers and family members are recommended along with psychological support by nurses and midwives. However, it should be noted that infant temperament would, directly and indirectly, be associated with parent-child interactions as well as other confounders in childhood, subsequently helping to form their personality that would lead to positive or negative consequences later in their life . Hence, This systematic review identified a variety of prenatal and intrapartum factors that are significantly associated with the temperament of infants: (1) genetic and biological factors: certain genotypes, maternal cortisol and ACTH, and CRHs, (2) environmental factors: substance use such as tobacco, alcohol, and illegal drugs, (3) socio-demographic factor: lower-income, (4) psychological factors: depression or anxiety, eating disorders, personality types of mothers, and domestic violence, and (5) obstetric factors: foetal growth (birth weight), hypertension in mothers, nausea (emesis), and preterm birth. These findings support the gene-environment interaction and biological mechanisms for developing infant temperament, suggesting the importance of ensuring a safe and comfortable environment for pregnant mothers, the unborn child, and families during pregnancy and delivery in society.The original contributions presented in the study are included in the article/MTa contributed to the conception and designs of work, selection and review of articles, drafted and revised the manuscript. AM contributed to the selection of reviewed articles, drafted the manuscript. YO and TK contributed to the conception and designs of work, interpretation of data, supervision for drafting and revising the manuscript. MTo and LY contributed to supervision for drafting and revising the 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.


Electrostatic insect exclusion is a physical approach to pest control in which an apparatus forming an electric field (EF) is applied to capture pests. Previous studies have clarified the insect-capture mechanisms of such tools and evaluated their practicality. In the present study, we investigated the biological impact of an EF-forming apparatus on adult houseflies captured by the device. We observed the release of substantial levels of negative electricity from the captured flies, detectable as a specific and transient electric current. The electric current was detected at the time of physical attraction, and during subsequent confinement of the fly to the apparatus. The magnitude of electric current from the fly was voltage-dependent, and detrimental effects caused by electricity release became more apparent as the applied voltage increased. Bioelectrical measurements showed that electric current caused acute damage and delayed the death of captured flies. These findings demonstrate the insecticidal function of the insect-capturing apparatus and provide an experimental basis for establishing a tool for trap-and-kill pest management.Musca domestica) were used as the model insect. The EF producer consisted of a negatively charged polyvinyl chloride membrane-insulated iron plate (N-PIP) and a non-insulated grounded iron plate (GIP) paralleled with the N-PIP. An EF was formed in the space between the plates. A housefly placed on the GIP was physically attracted to the N-PIP, and electricity released from the fly was detected as a specific transient electric current at the time of attraction and during subsequent confinement of the fly to the N-PIP. The magnitude of the insect-derived electric current became larger as the voltage applied to the N-PIP increased. We determined the total amount of electric current and confinement time within the apparatus necessary to kill all captured flies. These results demonstrate the insecticidal function and insect-capturing ability of the EF-producing apparatus.In the present study, we analyzed negative electricity released from insects captured by an electric field (EF)-producing apparatus. Adult houseflies ( Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) [Drosophila melanogaster Meigen  [Scatella stagnalis  (Diptera: Ephydridae) [Megaseria sclaris (Loew) (Diptera: Phoridae) [Lasioderma serricorne (Fabricius)  [Aedes albopictus (Skuse) (Diptera: Culicidae) [Liriomyza sativae (Blanchard) (Diptera: Agromyzidae), bathroom fly, Clogmia albipunctatus (Williston) (Diptera: Psychodidae), green peach aphid, Myzus persicae (Sulzer) (Hemiptera: Aphididae), green rice leafhopper, Nephotettix cincticeps (Uhler) , red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenbrionidae), Azuki bean weevil, Callosobruchus chinensis Linnaeus (Coleoptera: Bruchidae), oriental termite, Coptotermes formosanus Shiraki (Isoptera: Rhinotermitidae), confused flour beetle, Tribolium confusum  (Coleoptera: Tenebrionidae), German cockroach, Blattella germanica (Linnaeus) (Orthoptera: Blattellidae), common clothes moth, Clogmia albipunctatus Williston , western flower thrip, Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae) [Sitophilus oryzae (Linnaeus) (Coleoptera: Curculionidae) [Electrostatic insect exclusion is a physical pest-control approach in which an apparatus forming an electric field (EF) is applied to capture pests. Previous studies have clarified the insect-capture mechanisms of such tools and evaluated their practicality. Some EF-producing pest-capture systems consist of a negatively charged insulated conductor  paralleled with a grounded non-insulated conductor; an EF is generated in the space between them . The insrodidae) ,3, vineghilidae) ,5,6,7, sobiidae) ,8, Asianlicidae) , vegetabionidae) . This apionidae) ,11. The ionidae) , a portaionidae) , and an ionidae) . In thisionidae) . This neionidae) . These oCharged poles within the EF generate an attractive or repulsive force to other charges in the field ; these fMusca domestica (Linnaeus) (Diptera: Muscidae), as a model insect because larger insects produce larger amounts of transient electric current within the EF [Despite active research on the insect-capturing mechanism of EF-producing insect pest-control devices and their practical applications in pest control, no studies have reported the biological impact of these devices on insects. Thus, the main objective of the present study was to determine whether electrostatic insect-capturing devices are insecticidal. As the intensity of an EF  is determined as a transient electric current from the insect ,6,10, itn the EF . Moreoven the EF . Using tM. domestica) were purchased from Sumika Technoservice  and reared on a certified diet  [M. domestica [Adult houseflies   in a cloomestica , as show2, 2 mm thickness) were used to construct the EFP; one was coated with a soft polyvinyl chloride (PVC) membrane   for insulation and linked to a negative voltage generator , while the other was non-insulated and linked to a grounded line. The plates were arranged in parallel at a distance of 10 mm. A transformer and Cockcroft circuit [The structure of the EF producer (EFP) is shown in  circuit  were int circuit . Negativ circuit . The oppWe applied voltages between −1 and −15 kV to generate an electric current via the N-PIP to evaluate insect capture, and voltages between −8 and −15 kV to evaluate current generation by the fly and fly mortality. All experiments were conducted in a room controlled at 25 ± 0.5 °C and 60% relative humidity.Within a certain range of the applied voltage, the insulation of the charged iron plate prevents the transfer of the negative charge from the N-PIP to the opposite pole (GIP), i.e., discharge from the PIP to the ground via the GIP A. HoweveFirst, we examined the range of voltages that caused silent discharge by gradually increasing the voltage applied to the PIP. The resulting electric current profiles were recorded using a current detector  integrated into the galvanometer. We also determined the voltage range that caused arc (spark) discharge by further increasing the applied voltage.12 Ω cm) into the space between the PIP and GIP to determine the degree to which electricity released from the N-PIP was intercepted , and single male and female houseflies of various ages  were then transferred onto the P-GIP to determine the voltage range within which the flies were physically attracted to the N-PIP. Flies that were attracted to the N-PIP were continuously observed for 1 h to determine whether they could escape the N-PIP. When flies were confined to the N-PIP during the observation period, capture was considered successful. Thus, we precisely determined the voltage range at which 100% of male and female houseflies of various ages were captured.In this study, we used the EFP shown in Using the same set of houseflies described in In this experiment, we used the modified EFP C chargedIn all experiments, 20 insects were used for each sex, age, and applied voltage. All experiments were repeated five times, and data are presented as means ± standard deviation. Tukey’s test and linear regression analysis were performed using EZR software v1.54  to identify significant differences among conditions and any correlations among the factors.The primary objective of the present study was to suppress the flow of the electric current generated by the mechanical discharge of the N-PIP, which involved both silent and arc discharges. The amount of negative charge that accumulated on the surface of the PIP increased in direct proportion to the applied voltage. The voltage generator used in this study supplied voltages ranging from −1 and −20 kV. However, at >−15.1 kV, the insulator membrane that covered the PIP to generate arc discharge from the N-PIP broke down. Ultimately, we decided not to use this voltage range due to direct exposure of the insect to the arc discharge. In fact, flies exposed to arc discharge tend to be expelled from the EF due to its strong impact ,26. At <12 Ω cm) of the acrylic plate indicated high insulative properties [2, which was used for subsequent insect capture and insect-derived current flow experiments.Silent discharge of the N-PIP can occur along the entire surface facing the P-GIP. To intercept the electricity released from the N-PIP, we partially closed the surface of the N-PIP using an acrylic plate C. High roperties , which sOne of the most important events for pest control by an EFP is the attraction of the insect to the negatively charged insulated conductor within the EF . In the Flies placed in the EF were exposed to a repulsive force from the N-PIP, such that free electrons in the insect body were pushed out of the fly toward the ground via the GIP. This electricity movement was recorded as a transient electric current from the insect ,6,10. EvWe measured the electric current from houseflies using a modified EFP D chargedOur findings indicated that insect-derived electric-current generation caused both an attraction and confinement of the fly to the N-PIP within an applied voltage range of −8 to −15 kV. Houseflies captured by the N-PIP remained positively electrified until the voltage supply to the N-PIP ended. We evaluated the effect of this electrification on the fly in terms of the total amount of negative electricity released therefrom, calculated as the product of the magnitude of electric current and the duration of current generation, which was equivalent to the area bounded by the x-axis and the current generation curve . Figure One fly was released from the N-PIP before it was killed. In the final experiment, after electric-current generation by the fly had ceased and before its death, we switched off the voltage generator to determine whether it was alive or dead. Switching off the voltage generator implies the removal of a repulsive force used to push negative electricity out of the insect body. The positively polarized body likely attracted free electrons from the air , restoriIn the present study, we demonstrated that houseflies in an EF produced by the N-PIP and P-GIP of the EFP were captured by the N-PIP. The present bioelectrical method was useful to confirm the release of negative electricity from the fly, in the form of the electric-current flow from the insect body. This electric-current generation was successfully shown to correspond to the attraction and subsequent confinement of a fly to the N-PIP. These results strongly support our hypothesis that the negative charge of the PIP generates a repulsive force that pushes negative electricity (free electrons) out of the insect body; insects that lost their negative electricity became positively charged and were attracted to the N-PIP . The maiThe next stage in the design of the EFP apparatus is to develop a new contraceptive technique for insect pests. In a preliminary experiment, we determined that some flies laid no eggs during the entire experimental period following intermittent exposure to the attractive force of the N-PIP at an applied voltage of approximately −6 kV. In a future study, we will further analyze the biological impact of the attractive force of the apparatus on the fecundity of houseflies placed within an EF. The findings of this study may facilitate the application of electrostatic-based pest-control methods to many insect species.In the present study, an EF was formed in the space between a negatively charged PIP and GIP within an EF producer. The release of negative electricity from houseflies was detected as they were first attracted, and subsequently confined, to the negatively charged iron plate. This electricity release was detected as the electric current from the fly to the ground via the GIP. The release of electricity from the insect body was dependent upon the applied voltage, and its detrimental effect became increasingly noticeable as the voltage increased. The present study clarified the lethality of the electric current to the housefly, regardless of sex or age.


To evaluate the costs and non‐inferiority of a strategy starting with the levonorgestrel intrauterine system (LNG‐IUS) compared with endometrial ablation (EA) in the treatment of heavy menstrual bleeding (HMB).Cost‐effectiveness analysis from a societal perspective alongside a multicentre randomised non‐inferiority trial.General practices and gynaecology departments in the Netherlands.In all, 270 women with HMB, aged ≥34 years old, without intracavitary pathology or wish for a future child.n = 132) or EA (n = 138). The incremental cost‐effectiveness ratio was estimated.Randomisation to a strategy starting with the LNG‐IUS direct non‐medical costs were calculated. The primary outcome was menstrual blood loss after 24 months, measured with the mean Pictorial Blood Assessment Chart (PBAC)‐score (non‐inferiority margin 25 points). A secondary outcome was successful blood loss reduction (PBAC‐score ≤75 points).n = 115) and 14.2 in the EA group (n = 132); difference 50.5 points (95% CI 4.3–96.7). In the LNG‐IUS group, 87% of women had a PBAC‐score ≤75 points versus 94% in the EA group . The ICER was €23 (95% CI €5–111) per PBAC‐point.Total costs per patient were €2,285 in the LNG‐IUS strategy and €3,465 in the EA strategy . At 24 months, mean PBAC‐scores were 64.8 in the LNG‐IUS group (A strategy starting with the LNG‐IUS was cheaper than starting with EA, but non‐inferiority could not be demonstrated. The LNG‐IUS is reversible and less invasive and can be a cost‐effective treatment option, depending on the success rate women are willing to accept.Treatment of heavy menstrual bleeding starting with LNG‐IUS is cheaper but slightly less effective than endometrial ablation. Treatment of heavy menstrual bleeding starting with LNG‐IUS is cheaper but slightly less effective than endometrial ablation. Heavy menstrual bleeding (HMB) is a common problem, affecting more than a quarter of women of reproductive age.Three decades ago, 60% of women with HMB who were referred to a gynaecologist had a hysterectomy as treatment.Compared with oral medical treatment, the LNG‐IUS shows a greater improvement of quality of lifen = 197) or outpatient gynaecology departments (n = 26) in the Netherlands. The study was approved by the ethics committee of the Academic Medical Centre Amsterdam, the Netherlands (registration number 2011_372), and by the board of directors of each of the participating hospitals. A more detailed description of the design of the MIRA trial has been published elsewhere.A cost‐effectiveness analysis from a societal perspective with a 24‐month time horizon was performed alongside a multicentre randomised controlled non‐inferiority trial . Women with HMB, aged 34 or older, were randomised to either a strategy starting with the LNG‐IUS or a strategy starting with EA after written informed consent. Participants were allowed to undergo a re‐intervention. Women were excluded if they had a wish for pregnancy, abnormal cervix cytology in the past 5 years, intracavitary fibroids, polyps or large intramural fibroids. Women were recruited at participating general practices (®), a second‐generation ablation technique.The 52‐mg levonorgestrel IUS (Mirena) was inserted by either a general practitioner in his/her practice or by a gynaecologist at the outpatient department without anesthesia.P‐value for non‐inferiority were calculated using bootstrapping (10 000 replications). To test the robustness of our result, we added a secondary analysis with a zero‐inflated negative‐binomial (ZINB) model.A core outcome set for HMB has not yet been developed.Both direct medical and direct‐ and indirect non‐medical costs were included in the analyses. Relevant direct cost components were costs of the LNG‐IUS and EA treatment, medication, re‐interventions, GP and specialist consultations, hospital admission, diagnostic tests and home care, consisting of both professional care as well as informal care. Data on healthcare use and re‐interventions were collected from patient questionnaires and medical records. Costs of the treatment procedures LNG‐IUS and EA were calculated according to the bottom‐up principle following the Dutch guidelines for cost studies and using the Dutch tariff in the year of analysis (2018).Missing data on the primary and secondary effect outcomes were not imputed. For missing sick leave data, no productivity loss was assumed if the following data on treatment effect were present at that moment of follow‐up: amenorrhoea (PBAC‐score = 0) or the patient was satisfied with the treatment effect (secondary outcome) or menstrual blood loss was not influencing daily activities .Menstrual blood loss (mean PBAC‐score after 24 months) as well as the average costs per patient over a 24‐month time horizon were measured for each treatment strategy to calculate the incremental cost‐effectiveness ratio (ICER). The ICER expressed the incremental costs per one point difference in PBAC‐score (LNG‐IUS compared with EA). Bootstrap analyses (5000 replications) were performed to create alternative 95% confidence intervals surrounding the point estimate of the ICER, and to create cost‐effectiveness planes.We planned to perform a cost–utility analysis expressing the incremental costs per Quality Adjusted Life Year (QALY) based on the SF‐6D score, derived from the SF‐36 questionnaire. However, the proportion of patients that completed the SF‐36 questionnaire at all follow‐up time points  was 44.7% in the LNG‐IUS group and 62.3% in the EA group. The proportion of missing data was too large (>20%) to perform multiple imputation and therefore reliable utilities could not be calculated and a meaningful cost–utility analysis could not be performed.We performed two secondary analyses: (1) a cost‐effectivity analysis with the proportion of women with a PBAC‐score ≤75 points as a measure of effect and (2) a cost‐effectivity analysis with the proportion of women that were satisfied as a measure of effect.We performed the following sensitivity analyses to explore the robustness of the results: (1) calculation of costs under the assumption that all LNG‐IUS insertions were performed in primary care and (2) calculation of costs under the assumption that all EAs were performed at the outpatient department under local anaesthesia .The funding source (ZonMw) has involved stakeholders, including patients as the target group, in the assessment of the relevance of our study. We have incorporated their suggestions into our study protocol. Furthermore, patients were actively involved in the development of the patient information letter. Patients were not involved in other stages of the development of the research.n = 132) or EA (n = 138) . In the remaining cases, LNG‐IUS insertion was performed by the gynaecologist at the outpatient department (89.3%) or the operating room (3.3%). The majority of LNG‐IUS were inserted without anaesthesia, in four women the procedure was performed under general anaesthesia Table .n = 27) received a re‐intervention compared with 35% of the women in the LNG‐IUS group (n = 44). Additionally, 11 women discontinued the LNG‐IUS without a re‐intervention (discontinuation rate: 43%). Hysterectomy rates were comparable between the two strategies   Table .P = 0.87). Menstrual blood loss decreased to a PBAC‐score ≤75 points in 100/115 women (87%) in the LNG‐IUS group and in 124/132 women (94%) in the EA group . In the LNG‐IUS group, 74/100 women (74%) were satisfied with the treatment effect compared with 98/116 women (84%) in the EA group after 24 months . Longitudinal data analysis of disease‐specific quality of life did not show a significant difference between the two treatment strategies .A complete case analysis was performed with all participants for whom the primary outcome (PBAC‐score at 24 months) was available: 115/132 (87%) in the LNG‐IUS group and 132/138 (96%) in the EA group. The mean PBAC‐score at 24 months was 64.8 in the LNG‐IUS group versus 14.2 in the EA group . Non‐inferiority of the LNG‐IUS (based on a pre‐set margin of 25 points) could not be demonstrated  and less costly  compared with the strategy starting with EA after 24 months. EA costs €23 per additional PBAC‐point reduction of menstrual blood loss   of the LNG‐IUS strategy was 7% lower compared with the success rate of the EA strategy. Our primary analysis showed a cost difference of €1,180 between the two strategies. EA costs €169 per additional 1% of women who are successfully treated Table .Patient satisfaction at 24 months was 10% lower in the LNG‐IUS strategy compared with the EA strategy, with a cost difference of €932 in favour of the LNG‐IUS. There is a 92.5% probability that the LNG‐IUS is less costly with lower patient satisfaction and 4.4% probability that the LNG‐IUS is less costly with a higher patient satisfaction Figure . The EA In the LNG‐IUS strategy, the mean costs of the primary intervention were reduced to €347  but resulted in less reduction of blood loss (mean difference PBAC‐score at 24 months: 50.5 points) compared with a strategy starting with second‐generation EA (Novasure®) over a 24‐month time horizon. The incremental cost‐effectiveness ratio (ICER) for EA compared with LNG‐IUS was €23 per additional PBAC‐point blood loss reduction. This ICER increased to €28 under the assumption that all LNG‐IUS insertions were performed in primary care, and decreased to €22 under the assumption that all EAs were performed at the outpatient department with local anaesthesia. Expressed as the percentage of women with successful treatment (PBAC ≤75), the ICER was €169 per 1% additional success rate for EA compared with LNG‐IUS. Expressed as the percentage of women satisfied with the treatment strategy, the ICER was €89 per additional 1% satisfaction for EA compared with the LNG‐IUS.Strengths of our study are the long follow‐up of 2 years and the bottom‐up calculation of direct medical and indirect costs from clinical trial data, which means calculating each individual cost item. This gives a more accurate estimate of the total costs of a strategy than a top‐down calculation. We believe our study provides a realistic estimate of the number of re‐interventions after LNG‐IUS and EA in the long‐term. Previous studies had a shorter follow‐up of 1 yearTo test the robustness of our results, we performed a sensitivity analysis under the assumption that all LNG‐IUS insertions were performed in primary care, and a sensitivity analysis under the assumption that all EAs were performed at the outpatient department under local anaesthesia. Accommodation costs were included in a fixed overhead percentage of 44%, according to the Cost Guide for Economic Evaluations in Healthcare.To our knowledge, this is the first study comparing the LNG‐IUS with bipolar radiofrequency EA in terms of cost‐effectiveness. Evidence so far is based on the comparison of the LNG‐IUS with other second‐generation ablation techniques . Our study showed that a treatment strategy starting with the LNG‐IUS is cheaper than a strategy starting with EA after 2 years of follow‐up, even if taking into consideration the 43% discontinuation rate for LNG‐IUS. This result is consistent with previous studies comparing the LNG‐IUS with other second‐generation ablation techniques.In our study, the mean PBAC‐score at 24 months was 50 points higher (95% CI 4.3–96.7) in the LNG‐IUS group than in the EA group. We could not demonstrate non‐inferiority nor inferiority of a strategy starting with the LNG‐IUS compared with a strategy with EA because the 95% confidence interval exceeded the predetermined non‐inferiority margin of 25 points. This makes it difficult to interpret when the LNG‐IUS strategy would be cost‐effective. The cost‐effectiveness plane indicates that there is a 14% chance that the strategy starting with the LNG‐IUS is non‐inferior and less costly compared with a strategy starting with EA. However, there is a substantial chance that the strategy with the LNG‐IUS, although cheaper, results in a  higher PBAC‐score after 24 months compared with a strategy with EA. The National Institute for Health and Clinical Excellence (NICE) in the UK generally accepts a cost of £20,000 to £30,000  per additional QALY gained. We calculated the costs per 1 point difference in PBAC‐score, but the price society is willing to pay for an additional decrease in PBAC‐score has not been established.The majority of women in our trial had successful treatment after 24 months . If an 87% chance of success is found to be acceptable, starting with the LNG‐IUS may be a cost‐effective treatment strategy. It is important to discuss the chance of successful blood loss reduction with women who are offered both treatment options, together with other outcomes such as comparable satisfaction rates and quality of life scores. The results of our study are based on a trial in which most LNG‐IUS were inserted in secondary care. In the Netherlands, almost all general practitioners are competent to insert intrauterine devices. Although LNG‐IUS insertions for contraceptive purposes are increasing in general practice, the number of LNG‐IUS insertions for HMB is still low.We performed a cost‐effectiveness analysis with a time horizon of 2 years. As the LNG‐IUS has to be replaced after 5 years and some of the randomised women might be postmenopausal after 5 years of follow‐up, it would be interesting to perform a cost‐effectiveness analysis with a follow‐up of 5 years. However, it has been shown that most  re‐interventions due to treatment failure occur within 2 years of the initial treatment.In conclusion, a treatment strategy starting with the LNG‐IUS is less costly for society than a strategy starting with EA in women with HMB. Although the amount of blood loss reduction after 24 months is less compared with EA, and the total re‐intervention rate is higher, both strategies lead to successful menstrual blood loss reduction after 24 months in the majority of women  and high patient satisfaction. Depending on the success rate women are willing to accept, starting with the LNG‐IUS can be a cost‐effective treatment. Compared with EA, the LNG‐IUS is a reversible and less invasive treatment option for HMB, with a contraceptive effect. It is important to counsel women about the different characteristics of the two interventions and about the expected treatment results in terms of menstrual blood loss reduction, satisfaction, improvement of daily life activities and risk of a  re‐intervention.MJ van den Brink and P Beelen report a grant from ZonMw, the Netherlands Organisation for Health Research and Development, to conduct the study. MY Bongers reports financial relationships with Gynesonics , outside the submitted work. MY Bongers is a member of the communication team of Bayer and the advisory board of Hologic. The remaining authors report no conflicts.MvdB, MH, PG, JD, MBo and MBe were involved in the conception and design of the study. MvdB, MH and PB were involved in data collection for the study. MvdB, PB and KV performed the statistical analyses. MvdB and PB wrote the first draft of the report. All authors participated in manuscript drafting and approved the final draft.The MIRA trial was approved by the ethics committee of the Academic Medical Centre Amsterdam, the Netherlands .ZonMw, the Netherlands Organisation for Health Research and Development, grant number 171202001.Figure S1. Flow diagram.Click here for additional data file.Figure S2. Cost‐effectiveness plane patient satisfaction.Click here for additional data file.Table S1. Intervention—endometrial ablation and LNG‐IUS.Table S2. Effects and costs LNG‐IUS and endometrial ablation—secondary analysis.Table S3. Effects and costs LNG‐IUS and endometrial ablation—secondary analysis.Table S4. Effects and costs LNG‐IUS and endometrial ablation—sensitivity analysis.Table S5. Effects and costs LNG‐IUS and endometrial ablation—sensitivity analysis.Click here for additional data file.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.