External sulfate attack is an important factor causing a decrease in the mechanical properties of cement-based materials. In this paper, a computational prediction model of elastic modulus, considering the characteristics of sulfate corrosion from outside to inside and the influence of the interface transition zone (ITZ), was established to predict the elastic modulus of mortar under the external sulfate attack. Firstly, the backscattered electron (BSE) images of mortar and the algorithm of image threshold segmenting were used to determine a reasonable thickness of corroded ITZ. Secondly, the nanoindentation test was adopted to acquire the microscopic elastic parameters of phases  in corroded mortar. Moreover, the mortar mix proportion and Lu and Torquato’s model were adopted to calculate the volume fractions of phases. Finally, a computational prediction model of elastic modulus of mortar under sulfate attack was proposed with homogenization methods. The results indicate that the thickness of corroded ITZ is 20 mm, and the error values of elastic modulus between the theoretical prediction results and the experimental results are within 8%, indicating that the macroscopic elastic modulus of corroded mortar can be precisely predicted by the computational prediction model of elastic modulus. Elastic modulus has an important influence on the stress distribution and deformation of concrete structures, and, as such, it is a key parameter in the design of such structures . SulfateAs is known, the macroscopic mechanical behavior of material is largely dependent on the compositions, contents, and mechanical properties of phases at the nano/micro-scale . The upsThe interface transition zone (ITZ) is between the matrix and the inclusions. The ITZ is the weakest phase in concrete material, with the characteristics of low elastic modulus, low strength, high porosity, and high permeability ,13,14. TIn this paper, a reasonable thickness of ITZ in corroded mortar was obtained using image processing software with the SEM-BSE image of mortar. Then, the elastic parameters of phases  in mortar were determined based on the nanoindentation test and theoretical calculation. In addition, the volume fraction of each phase in the mortar was determined by calculating the mortar mix ratio and applying Lu and Torquato’s model. Finally, the homogenization method was used to establish the elastic modulus prediction model of mortar under sulfate attack. The theoretical calculation results were compared with the experimental results to verify the effectiveness of the elastic modulus prediction model. The research route is shown in P.I 52.5 Portland cement was selected in this study. The mineral composition of the cement is shown in 2SO4) was selected to prepare the sulfate solution, and the molar concentration of sulfate ion in sulfate solution was 352 mol/m3 (mass concentration was 5%).Sodium sulfate  and humidity (65%) for 24 h, and then demolded. Subsequently, the mortar samples were cured under standard curing conditions (temperature of 20 °C and relative humidity of 95%) for 28 d. Finally, mortar samples were soaked in NaThe uniaxial compression test, the nanoindentation test, and the SEM-BSE test were conducted, and mortar samples were selected with a processing period of 88 d (sulfate corrosion time of 60 d). For the uniaxial compression test, each mix proportion selected five mortar specimens to obtain the constitutive relation curves. For the nanoindentation test, specimens were selected from mortar specimens with dimensions of 5 mm × 5 mm × 10 mm. Owing to the high requirement of flatness for the nanoindentation test, nanoindentation specimens were pretreated ,22. FirsThe uniaxial compression experiments were conducted by a servo-controlled setup (Zwick/Roell Z050) with a loading speed of 0.5 mm/min. Two linear variable differential transformers (LVDT1 and LVDT2) were symmetrically arranged 10 mm away from two sides of the specimen to measure the deformation under loading. Instrumented nanoindentation (G200), produced by Agilent Company, Santa Clara, CA, USA, was adopted to acquire the microstructural mechanical properties of corroded mortar specimens. The elastic modulus and hardness of the specimen varying with the nanoindentation depth were obtained by a continuous stiffness method (CSM). The schematic of the nanoindentation test for the microscopic elastic modulus of the corroded mortar is shown in E is the elastic modulus of tested material, S is the contact stiffness (P is the maximum loading), A is the corresponding contact area at the maximum load, During the loading process, elastic deformation firstly appeared in the specimen. As for the plastic deformation of the specimen, the loading curve was non-linear with the increase in loading. During unloading, only the elastic properties of the indentation point were restored. The nanoindentation test was carried out on ITZ, cement, and sand of the mortar specimens. An 8 × 8 lattices of 64 points on the corroded mortar specimen was measured to obtain the microscopic elastic modulus.In order to obtain a relatively stable elastic modulus value, the indentation depth needs to reach the micrometer scale . MoreoveThe micromorphology of nanoindentation points and corroded mortar was observed by backscattered electron (SEM-BSE) images. The SEM-BSE images were obtained by Quanta 250FEG, produced by FEI Co., Hillsboro, OR, USA. The SEM-BSE images were acquired with a resolution of 3.5 nm under a high vacuum mode, and the distance between the sample and scanning electron microscope lens was 10 mm. The stress–strain curve of the eroded mortar sample was obtained via a uniaxial compression test, so that the macroscopic elastic modulus (E) of the sample could be obtained. The macroscopic elastic moduli of corroded mortars with types of M1 and M2 were 29.88 GPa and 33.33 GPa, respectively.The BSE images of corroded mortar were processed by the algorithm of image threshold segmenting. Each of cubic volume elements (voxels) in the image was assigned a unique grayscale value, then the data were organized as a collection of cubic volume elements (voxels).Next, the data of distinct material phases were segmented into several distinct categories . The matn is the total number of pixels in the processing region.Firstly, the porosity of each region was calculated, and then 10 regions with the same distance to the edge of the sand are selected, and the average porosity of these 10 regions was calculated as the porosity of the region. According to Equation (2), the variation in porosity with the increase in the distance to the edge of the fine aggregate can be obtained, as shown in A lattice of 8 × 8 nanoindentation points, including the sand, ITZ, and paste, was carried out around the fine aggregate in corroded mortar. As known, the macro-elastic modulus of mortar can be measured by the static compressive test, and the macro-mechanical properties are mainly dependent on micromechanical properties and micro-components. The homogenization methods can be used to calculate elastic modulus of mortar from multi-scale for studying the influence of sulfate corrosion on the mortar.The corroded mortar can be divided into two levels according to the multi-scale method, as shown in −6 m to 10−4 m, is composed of unhydrated cement particles , hydrated products H6), sulfate corrosion products (Aft), and pores, etc. The elastic modulus of cement paste can be obtained by the micromechanical model and homogenization method from the micro-elastic modulus of each phase in cement paste. As is known, when the reference medium in composites was hard to determine, the self-consistent (SC) method can be selected as an effective homogenization method [On level I, the cement paste matrix, with a characteristic length range from 10n method ,31,32. IOn level II, two kinds of multi-level homogenization methods were considered, as shown in Using the elastic method and Eshelby equivalent medium method ,33, ChriA, B, C, The effective shear modulus of equivalent particles can be obtained from the following Equations (7)–(14) :(7)A(μ¯sAccording to Equations (5)–(14), the bulk modulus Under the corrosion of an external sulfate environment, sulfate ions enter the mortar through micropore structures and react with cement hydration products to produce expansive products, leading to the change in the mechanical properties of mortar ,38,39,40It is generally believed that sulfate ions will not react with sand in mortar . TherefoIn a sulfate environment, sulfate ions entering into mortar specimens and cause the corrosion of cement-based materials as part of a gradually decreasing process from outside to inside. Therefore, the corrosion degree of mortar was uneven inside and outside. Considering the influence of temporal and spatial distribution for the process of sulfate attack on mortar, the author of this paper coupled the effects of cement hydration and sulfate corrosion and established an X-CT-hydration–deterioration model . On leveAccording to the results of the nanoindentation tests in According to the mix ratio of mortar, the volume fraction of sand in corroded mortar can be obtained by the following Equation (18):Where t, and the overlapping of the ITZ between adjacent aggregates was considered. Thirdly, the remaining part in the mortar was the cement paste matrix.The volume fractions of cement paste matrix (not including ITZ) and ITZ in the mortar were calculated by Lu and Torquato’s model, as shown in ve(t). If the thickness of ITZ is infinitely large, t → ∞, then the area of cement paste matrix tends to zero, ve(t) → 0. Therefore, the volume fraction of ITZ in corroded mortar can be expressed as follows:Based on the above assumptions, the volume fraction of the cement paste matrix was t of ITZ and the particle size distribution of sand are known, ve(t) can be obtained, as follows:t is the thickness of ITZ in corroded mortar, A is a constant value , When the thickness 3), cement (3), and water (3), the volume fraction of sand aggregate was calculated by Equation (18). (2) Substituting the particle size distribution of sand and the thickness of the ITZ (20 µm) into Equation (19), the volume fraction of ITZ was obtained by Lu and Torquato’s model. (3) Finally, the volume fraction of cement paste matrix (not including ITZ) was calculated by Equation (26). The theoretical calculation results of volume fractions the of phases in corroded mortar are shown in Based on Lu and Torquato’s model and the mix proportion of mortar, the volume fractions of phases in the corroded mortar can be calculated as follows: (1) Based on the mix ratio of the mortar and the densities of sand  were 7.7% and 1.9%, respectively, and the error value of the elastic modulus predicted by the theoretical model is less than 8% compared with the actual elastic modulus obtained by the test. The data show that the theoretical calculation model proposed in this paper can well predict the elastic modulus of corroded mortar.The error value between the theoretical and experimental values of the macro-elastic modulus of corroded mortar can be calculated by the following Equation (27):(1)The BSE images of corroded mortar and the algorithm of image threshold segmenting can be used to acquire the thickness of ITZ, and the thickness of ITZ in corroded mortar at 88 d is 20 µm.(2)Combined with the results of nanoindentation test and the characteristics of corroded mortar, the macro-elastic modulus of ITZ in the corroded mortars can be accurately and effectively evaluated. The values of ITZ in corroded mortars (M1 and M2) are 15.2 GPa and 18.9 GPa, respectively.(3)Based on the mortar mix proportion and the thickness of ITZ, the volume fractions of sand, cement paste, and ITZ in corroded mortar can be well evaluated by Lu and Torquato’s model.(4)The computational elastic modulus prediction model, considering the influence of ITZ, can well predict the elastic modulus of mortar under sulfate attack.In this paper, the elastic parameters and the volume fractions of the phases  in mortar were obtained by tests and theoretical calculation. Then, a computational prediction model of the elastic modulus was established with homogenization methods to predict the elastic modulus of mortar under external sulfate attack. The conclusions are shown as follows:


Childhood adversity can induce maladaptive behaviors and increase risk for affective disorders, post-traumatic stress disorder, personality disorders, and vulnerability to stress in adulthood. Deprivation of maternal care interrupts brain development through the disturbance of various neurotransmitters, however, the details remain unclear. The features of the symptoms of disorders are largely determined by early stress protocol, genetic characteristics (line), and the sex of the animals. The purpose of current study was (1) to assess behavioral changes in adult Wistar rats of both sexes after early life stress; (2) to determine the levels of monoamines in brain structures involved in the motor, emotional, and social reactions in rats aged 1 and 2 months; and (3) to determine the level of monoamines after physical or emotional stress in adult rats. The rat pups were separated from their dams and isolated from siblings in tight boxes at a temperature of 22–23 °C for 6 h during postnatal days 2–18. The data were processed predominantly using two-way analysis of variance and the Newman–Keys test as the post hoc analysis. The adult rats demonstrated an increase in motor activity and aggressiveness and a decrease in levels of anxiety and sociability. Behavioral disturbances were accompanied by region-, sex-, and age-dependent changes in the levels of monoamines and their metabolites. The dopaminergic and noradrenergic systems were found to be sensitive to psycho-emotional stress. Traumatic experiences are not uncommon in life. The term “complex trauma” refers to traumatic events that are chronic, invasive, and primarily interpersonal  . These lThe monoamine systems have a great impact on memory, cognition, and emotions . StudiesSignificant progress in understanding the neurochemical mechanisms involved in the development of long-term impairments in response to traumatic experiences can be achieved through animal modeling. The large list of PTSD symptoms, many of which can be defined only in a subjective manner , make itStudies on rodent models of early stress have revealed behavioral disorders in adult animals and changes in the monoamine levels in several brain regions associated with abnormal behavior. The separation of rat pups from their mother on postnatal day 1–21 (P1–P21) increased DA turnover in the prefrontal cortex and hippocampus of females and 5-HT turnover in males. In addition, an increase in the NE turnover in the striatum was observed in females and a decrease in DA turnover in the hippocampus in males . In anotAccording to modern understandings of the pathogenesis of mental disorders, childhood adversity creates predisposition to psychopathology, which can manifest itself after repeated stressful exposure in adult age (the double-hit hypothesis) . In modeConsidering that the weaning protocol can influence the pattern of behavioral disorders associated, among others, with a change in the functioning of monoamine neurotransmission, the aim of the present study was (1) to assess behavioral changes in adult young rats of both sexes after the early stress of maternal and sibling separation; (2) to determine the levels of monoamines in brain structures presumably involved in the formation of emotional and social disorders in rats aged 1 and 2 months; and (3) to determine the levels of monoamines after repeated stress in adult rats.The study was performed on Wistar rats bred at the vivarium of the Research Institute of General Pathology and Pathophysiology . Pregnant and lactating females and later animals of the experimental and control groups were housed in controlled room temperature and humidity conditions (22 ± 1 C and 55 ± 25%) with natural lighting and free access to water and food . During the experiment, the health status of the animals was monitored by a veterinarian. All manipulations with animals were carried out under supervision of the Ethical Committee of the Research Institute of General Pathology and Pathophysiology  in accordance with Directive 2010/63/EU of the European Parliament and of the Council “On the protection of animals used for scientific purposes”, and Guide for the Care and Use of Laboratory Animals: Eighth Edition, 2011.After mating, pregnant females were kept in individual plastic cages (36 × 20 × 14 cm) until delivery . On P1, the rat pups were separated from their mothers and weighed, their sex was determined, and then the pups were re-distributed to nursing females. For reducing the influence of the genetic factor and the maternal care factor, 6–8 male and female rat pups from different litters born on the same day were left with each dam. These mixed litters were randomly assigned to two groups according to inclusion/exclusion criteria. The groups included male and female Wistar rats from at least six different litters, born on the same day, somatically healthy. Sick animals and some pups from very large litters were excluded so that all lactating females had no more than 6–8 pups. The sample size was determined based on articles published by other authors with a similar study design. One group consisted of the pups that were subjected to the procedure of daily separation from their mother and isolation from siblings for 6 h on P2–P18 . The boxes (6 × 6 × 5 cm) with separated rat pups were placed in a special room with a temperature of 22–23 °C. The other group included pups that were handled and immediately returned to the female daily (Control group). The juvenile rats were weaned on PND24 and housed in new cages (57 × 37 × 19 cm). In each cage, animals of the same sex and nursed by one dam were housed. We performed two experimental series. In Series 1, approximately half of the randomly selected males and females were decapitated at the age of 28 days and brain tissue samples were obtained for monoamine assay. The remaining animals were not disturbed until the behavioral experiments at the age of 56–60 days, after which they were decapitated and the brain samples were obtained. In Series 2, after conducting a battery of behavioral tests at the age of 56–60 days, the animals were subjected to additional stress for 5 days for 10 min per day: some rats were subjected to unavoidable foot-shock stress  and others witnessed this procedure . Before “double stress” and after it, electrocardiograms were recorded to assess the autonomic balance in rats (data under processing). Immediately before decapitation, the animals were placed in the context of the previous stress, but no electric shocks were inflicted. The scheme of the experiment is shown in The tests were carried out during daytime from 9.00 to 17.00; the cages with control and experimental animals were alternated  to reduce the influence of the daytime factor. Behavioral testing included assessment of motor  and exploratory  activity in automated open field (aOF) and classical (cOF) tests. These tests differ in aversive conditions, in particular, by the size and shape of the open space and the intensity of illumination. The cOF is more stressful. It is known that testing in a large open space in a classical situation of OF testing aversive for animals allows for the detection of sex differences in animal reactions . AnxietyIn the aOF test, an arena (48 × 48 × 21 cm) with transparent walls was used . The measurements were carried out in dimmed light (17 lux) for 10 min. Horizontal (motor) activity was evaluated by the traveled distance (in cm) and vertical (exploratory) activity was assessed by the number of rearings using the applied software. In this and all other tests, the experimental chamber was wiped with 70% alcohol and dried with a towel after each animal.In the cOF test, we used a round white arena  divided into squares of 20 × 20 cm. Measurement was carried out for 5 min: 3 min in bright light , 1 min in red light (a 40 W lamp positioned at a height of 80 cm above the center of the OF), and 1 min again in bright light. Behavioral parameters were assessed visually. Motor activity was evaluated by the number of crossed squares, and exploratory activity was assessed by the number of rearing postures. Modulation of lighting conditions was used to evaluate the reaction to novelty, which was calculated as the ratio of the number of crossed squares during the fourth minute in red light to that during the first minute of the session in bright white light. In addition, the total activity over two minutes after switching off and returning to the bright light was taken into account.In cOF, the anxiety–phobic state of the rats was measured in bright light using several stress tests based on situations that could cause anxiety in rats: descent from a platform 10 cm high; exit from a transparent box (11 × 15 × 15 cm); passage through a hole (11 × 7 cm) separating two compartments of the chamber (25 × 23 × 23 cm); and exit from the center of the OF (for a detailed description see ). In eacThe level of anxiety in the EPM was assessed using the equipment and software of the VideoMot2 video system . The maze was located 70 cm above the floor and consisted of two opposite closed arms (CAs) with side and end walls (43 × 14 × 22 cm), two open arms (OAs) of the same size but without walls, and a central platform (14 × 14 cm). The illumination was 29 lux in OA and 1 lux in CA. Rat behavior in the EPM was recorded using a standard approach  accordinIn a SIT, social contacts of a rat with a conspecific are evaluated under the conditions of free behavior of both . TestingThe TST assessed the sociability of rats, i.e., the choice between an unfamiliar social object  and a non-social object (an empty cylinder), as well as the preference for “social novelty” (the choice between familiar and unfamiliar conspecifics). The social testing apparatus was a gray plastic box 120 × 80 cm, divided with partitions into three equal chambers (40 × 80 cm). The partitions had openings allowing access to all chambers. Twenty-four hours before testing, each rat was placed in the empty three-chamber box with open doors and allowed to explore it for 5 min for adaptation to the experimental conditions. Intact rats of the same sex and age as animals of the experimental groups were adapted for 5 min to a cylinder cage  with metal wire walls. The intact rats were kept in a separate room of the vivarium during the study. The testing was performed in two stages.In Stage 1, two cylinders, wire cages, were placed in two side chambers; the intact rat was placed in one cylinder as a social object, while the second cylinder was empty. The experimental rat was placed in the middle chamber with closed doors for 1 min for habituation, then the doors were opened and the rat could explore all three chambers for 10 min, after which the doors were closed again, and the experimental rat was returned to the middle chamber. In Stage 2, another intact rat  was placed into the empty cylinder. Then the doors were opened, and the experimental rat was allowed to explore all three chambers for 10 min. The parameters recorded at each stage were the time spent by the rat in each chamber with the objects, the number of visits to these chambers, the time spent near each object, and the number of approaches to the objects . Sociability in Stage 1 was assessed by the preference for a social chamber and a social object by the following formulas:[ − (time spent in the chamber with the empty cylinder)]/ × 100%;[ − (time spent near the empty cylinder)]/ × 100% .In Stage 2, the preference for social novelty was assessed by the preference for a chamber with the novel social object (unfamiliar rat) and the new social object itself vs. familiar social object. The animals that did not explore all three chambers during the habituation session and the animals that did not visit one of the side chambers during stage 1 were excluded from the study.Patients with BPD may differ from healthy individuals in the functioning of the stress response system, and the most traumatic situation for them is psycho-emotional stress during social interaction ,54. For After decapitation, the brain was removed, placed on a cold surface, and the frontal cortex, hippocampus, striatum, and hypothalamus were isolated according to Paxinos and Watson  and Chuig.In the brain tissue samples, the concentrations of biogenic amines norepinephrine (NE), dopamine (DA), 5-hydroxytryptamine (5-HT), and their metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (DA metabolites) and 5-hydroxyindoleacetic acid  were measured using High-Performance Liquid Chromatography with Electrochemical Detection (HPLC-ED). The samples were homogenized with a Labsonic M ultrasonic homogenizer  in 0.1 n HClO4  with 250 pmol/mL internal standard 3,4-dihydroxybenzylamine hydrobromide . After that, the samples were centrifuged for 15 min at 2000× HPLC separation was carried out on a reversed-phase column ReproSil-Pur, ODS-3, 4 × 100 mm with a pore diameter of 3 µm  at 26 °C and a mobile phase speed of 1 mL/min coupled with an LC-20ADsp liquid chromatograph . The mobile phase consisted of 0.1 M citrate–phosphate buffer pH 2.58, 0.3 mM sodium octanesulfonate, 0.1 mM EDTA, and 8% acetonitrile . The electrochemical detector Decade II  was equipped with a working glass carbon electrode (+0.80 V) and an Ag/AgCl reference electrode. Peaks of interest and the internal standard were identified by their elution time in the standard solution. The concentrations of amines were calculated by the internal standard method using a calibration curve using LabSolutions software . The samples were normalized to tissue weight. In addition, the 5HIAA/5HT, DOPAC/DA, and HVA/DA ratios were calculated.The data were processed using Statistica for Windows 7.0 software. If the hypothesis on normal data distribution was not rejected after preliminary testing using the Kolmogorov–Smirnov test, parametric methods were used. The effect of factors Sex , Rearing (2 gradations—growing with mother (Control) or maternal separation and isolation from littermates (MSI)), and Stress  and emotional stress (EmS)) were assessed using a multivariate ANOVA. The intergroup differences were evaluated using a Newman–Keuls post hoc analysis. If the hypothesis on normal data distribution was rejected, the nonparametric two-way Mann–Whitney U test for independent variables was applied with adjustment for multiple comparisons using the FDR method . The sigSex and Rearing did not affect the body weight of the animals at the age of 1 and 2 months (Sex factor at the age of 2 (P58) and 3 (P75) months: males had greater body weight than females, but no differences between 2-month-old females and males within the groups (Control and MSI) were found  = 50.053, p < 0.001. In MSI groups, locomotor activity in both males (p < 0.00): in females, the distance traveled was longer than in males in both the Control group  and the MSI group .In both series, the influence of the factor of Rearing on the total (over 10 min) locomotor activity of rats in aOF was revealed: in Series 1 F  = 25.318, th males A,C and fth males B,D was hp < 0.001) (p = 0.003). In both Control and MSI groups, females had higher exploratory activity than males  = 18.945, p < 0.001).In Series 1, males and females of the MSI groups demonstrated higher exploratory activity than animals of the corresponding Control groups  = 17.073, < 0.001) . In Serip = 0.043) and Series 2  = 6.101, p = 0.015). Note that in Series 1, an increase in motor activity was observed in female MCI (pronounced trend), and in Series 2, in male MCI (p = 0.036): in females, activity was higher than in males. Under conditions of changing illumination (fourth and fifth minutes of the test), the influence of the factors Rearing and Sex was revealed in Series 2 only:  = 4.775, p = 0.031 and F  = 11.061, p = 0.001, respectively). In Series 1, the Rearing  = 2.405, p = 0.128) and Sex  = 0.040, p = 0.842) factors did not affect motor activity. The level of exploratory activity of the rats did not differ significantly in all subgroups.In the cOF test under bright illumination (1–3 min), the level of locomotor activity was influenced by the factor of Rearing: motor activity in the MSI group was higher than in the Control group in Series 1  = 4.325, male MCI . The infp = 0.030): in MSI females, the total distance traveled in EPM was greater than in the Control . Only MSI females showed an increase in the duration of hanging from the OA in comparison with the Control  = 4.672,  Control C. The di Control A.p = 0.017 was statistically significant for locomotor activity in OAs: the distance traveled in OAs increased in both males  and females . For total locomotor activity, the Rearing main effect was F  = 12.368, p < 0.001 and Sex main effect was F  = 7.010, p = 0.009. MSI males accumulated longer distances in the EPM than Control males . In females, the differences did not reach the level of statistical significance , The preference for OAs .In Series 2, the Rearing main effect F  = 6.688,  for OAs B and dur for OAs D was greIn cOF, no differences in the indicators of the anxiety–phobic level scale were found in both series.In SIT, MSI males and females did not differ by the number and duration of non-aggressive social contacts from the corresponding Controls in Series 1 and 2 A,B. In bp = 0.035 and F  = 4.587, p = 0.037, respectively; for Series 2 F  = 4.303, p = 0.041 and F  = 2.784, p = 0.099 (trend), respectively. Only in MSI males was the preference for the social chamber reduced in comparison with the corresponding values in control males; in Series 1, the intergroup differences did not reach the level of statistical significance  = 4.681, ificance A,B. A teificance C,D. In Sales see B.At the age of 1 month after weaning, the Rearing main effect was significant for the hippocampus, and the Sex main effect and Sex × Rearing interaction were significant for the hypothalamus . Post hoAt the age of 2 months after a battery of behavioral tests, as well as at the age of 1 month, the Rearing main effect was significant for the hippocampus, and Sex × Rearing interaction was significant for the hypothalamus. However, the NE level was reduced in the MSI group compared with the Control only in the hippocampus of males and in the hypothalamus of females. In the frontal cortex and striatum, no differences in NE levels were revealed at the ages of 1 and 2 months.In adult animals after repeated stress, the Stress main effect was significant for the frontal cortex and for the hippocampus see . After tin the frontal cortex, the Rearing main effect was significant for HVA level and HVA/DA ratio: these parameters in the MSI were lower than in the Control  in females in the frontal cortex was lower, and in the hippocampus, it was higher than in males. In the striatum, the Rearing main effect was significant for the levels of DA, HVA, and the DOPAC/DA and HVA/DA ratios, while the Sex main effect was significant for the levels of DA and HVA. The DA level was higher in females than in males and decreased in MSI females compared to Control. The DOPAC/DA ratio, on the contrary, was lower in females than in males and increased in MSI females compared to the Control. In MSI males, the level of HCV and HVC/DA turnover were reduced in comparison with the Control. In the hypothalamus, the Rearing main effect was revealed for the levels of DOPAC and HVA and the ratios of DOPAC/DA and HVA/DA. The DOPAC level in MSI rats was lower than in the Control; no differences between the subgroups were found. The DOPAC/DA ratio was elevated in Control females in comparison with the males; in MSI females, the DA turnover was decreased compared to the Control. The HVC level and the HVA/DA ratio were higher in females than in males and reduced in both males and females of the MSI group.At the age of 2 months, in the frontal cortex, the DOPAC/DA ratio was lower under EmS conditions than under FS conditions as a result of the Stress factor. In the striatum, the Stress main effect was significant for the DA level and its metabolites, DOPAC and HVC, which were higher after EmS than after FS. In the hypothalamus, DA turnover (DOPAC/DA) was significantly reduced under conditions of EmS in comparison with FS.After repeated stress, in the hippocampus, the influence of the factor Rearing on the level of 5-HT was revealed: 5-HT level was elevated in MSI rats   and on the 5-HT/HIAA ratio . In the hypothalamus, the Rearing main effect was significant for the 5-HT level, while the Sex × Rearing interaction was significant for the HIAA level. Both indicators were reduced in MSI males compared to the Control. In the frontal cortex, no differences between the groups were revealed.At the age of 1 month, ls test) . In the in the frontal cortex, a Sex × Rearing interaction was revealed for the levels of 5-HT and HIAA: they were increased in males and reduced in females in comparison with the corresponding Control subgroups; for the HIAA/5-HT ratio, the Sex main effect was significant: the turnover of 5-HT was lower in females than in males. In the hippocampus, the Sex main effect was revealed for the levels of 5-HT  and for HIAA and the HIAA/5-HT ratio ; no differences between the subgroups were found. In the striatum, a Sex main effect was identified: the 5-HT level and HIAA/5-HT ratio in females were lower than in males. The Rearing main effect was significant for HIAA level: it was higher in MSI rats. In the hypothalamus, the levels of 5-HT and HIAA were lower in MSI males, and the HIAA/5-HT ratio was lower in MSI females than in the corresponding Control.At the age of 2 months, in the hippocampus and hypothalamus was revealed: in males, the level of this metabolite in animals subjected to EmS was higher than in animals subjected to FS.Under conditions of repeated stress in adult rats, the influence of Sex × Stress interaction on the HIAA level Repeated maternal separation is the most widely used animal model for investigating the relationship between early life stress and its psychiatric and physical consequences. Despite non-standardized protocols, the MS paradigm is mainly considered as a tool for modeling anxiety- and depression-like behavior of juvenile and adult rodents ,59,60,61In our experiments, complex developmental trauma was used, including separation of rat pups from the mother and littermates at room temperature with partial limitation of motor activity due to the small size of the boxes. We expected that early-life abuse would lead to attachment disorder in rat pups and to formation in later life of persistent neurobehavioral deficits not associated with the development of depression-like phenotype. It was shown that more pronounced disorders in behavior and neurotransmission were observed in rats subjected to complex aversive influences during brain development ,63.In two series of experiments, maternal separation and sibling isolation in the neonatal period led to similar changes in the behavior of young adult animals. In Series 1, we evaluated the effect of early-life stress on the levels of monoamines and their metabolites in the frontal cortex, hippocampus, striatum, and hypothalamus of juvenile and adult animals . These bBody weight is one of the basic indicators of physical status in animals. Early life experiences have a strong influence on brain programming and can affect the control of eating behavior and body weight later in life. However, there is no consensus about the relationship between neonatal stress and eating behavior. Many researchers reported that rats subjected to early-life maternal separation had lower body weight in the juvenile and mature age ,65,66,67In both series of our study, adult male and female rats subjected to repeated early MSI showed increased locomotor and exploratory activity in OF in comparison with the Control. The MSI-exposed rats demonstrated increased exploratory activity in aOF in dim lighting conditions. Under the bright light in the cOF, the vertical activity in the MSI groups did not differ from the control, which may be due to a decrease in the exploratory behavior of these rats under stressful conditions. We observed a decrease in signs of anxiety in MSI females in EPM: they had an increased preference for open arms and number and duration of hangings from the open arms. In males, the effect of early stress on anxiety-like behavior was less certain and was reduced only in Series 2.The use of different early life stress schemes may have different effects on motor and exploratory activity and on the level of anxiety. So, maternal separation for 4 h on P2–P5, for 8 h on P6–P16, and weaning on P17 decreased locomotor activity, increased anxiety-like behavior and resulted in a passive coping strategy in Sprague–Dawley rats of both sexes . MaternaThe results obtained in this block of behavioral tests indicate that the protocol of neonatal complex aversive exposure applied by us leads to the formation of a hyperactive phenotype in young adult rats. The other authors have shown that a complex aversive effect  interferes with the normal process of cell proliferation, apoptosis, and synaptogenesis in the frontal cortex  and hippIn our study, sex differences in the effects of MSI on the social behavior of animals were revealed. The MSI males demonstrated increased aggressiveness under conditions of free interaction of animals in an unfamiliar environment, reduced preference for socializing, and increased reaction to the presence of social novelty under the conditions of restricted motor activity of conspecifics.A number of studies have demonstrated that MS disrupts the social behavior of rats at a later age, and males are more sensitive to early-life stress. In juvenile Wistar males, daily long-term MS caused an increase in the number of aggressive elements in social play behavior in a social interaction test  and moreWe revealed no changes in sociality (sociability) in rats of both sexes by the results of SIT: the number and duration of non-aggressive interaction did not differ in MS and control groups. These results are at odds with the conclusions of Mintz et al.  that MS Various studies of the effect of MS on social preference and social novelty in adult rats have yielded contradictory results. There is data that the social behavior of adult MS rats does not change . Other dThe design of our experiment  was most close to that of the study of Kambali and colleagues . HoweverThe aversive events during the critical periods of the development of an organism can disrupt the maturation of many neurotransmitter systems, including monoaminergic ones (see review ). The loIn 1-month-old males, the NE level was increased in the hippocampus and hypothalamus; the DA level was decreased in the hypothalamus, and DOPAC and HVC levels were decreased in the striatum; the 5-HIAA level was decreased in the hypothalamus . In femaSince behavioral tests were not performed after weaning at 1 month of age, we are unable to assess the relationship between changes in monoamine levels and behavioral disturbances in adolescent rats. However, according to the literature, the dysfunction of NE and DA neurotransmission in animals is often associated with the development of a hyperactive phenotype . The incIn adult rats, we observed motor and exploratory hyperactivity in both sexes, increased aggressiveness and decreased sociability in males, and signs of decreased anxiety in females. It is believed that all DA systems are involved in the development of the hyperactive phenotype ,96. AccoMost researchers recognize the priority contribution of the DA system to the development of hyperactivity, but not everyone shares the point of view that the disorders are based on the hypofunction of all DA systems. Experimental data indicate that in some cases the symptoms of hyperactivity are associated with an increase in the functional activity of DA ,93,98. TIt is well known that the 5-HT system plays an important role in the pathophysiology of anxiety, depression, and aggression . AlthougIt has been proposed that early life negative events may alter the response of a subject to a subsequent traumatic experience, increasing the likelihood that later trauma will lead to PTSD or affective disorders . WitnessWe expected MSI rats to be more sensitive to emotional impact than control animals. We evaluated the effect of prolonged (5 days) physical and emotional stress on the levels of monoamines and their metabolites in several areas of the brain in male and female rats reared with their mother or under conditions of MSI. Although the influence of the factors Sex, Rearing, and Stress as well as their interaction was identified, we did not obtain a wide range of differences between groups see . StatistEarly studies have demonstrated that physical and psycho-emotional stresses cause different regionally dependent changes in the level of DA  and 5-HTThe first limitation of the current study is related to the determination of monoamine levels. The concentrations of monoamines and their metabolites were measured in relatively large areas of the brain and not in specific nuclei. Given the mediator and functional heterogeneity of the structures chosen for the study, the assessment of the contribution of monoaminergic systems to the development of behavioral disorders can be quite rough. More accurate bioassays of monoamines and their metabolites in these cores should be performed in future studies. In addition, we did not evaluate basal monoamine levels during an experiment in which animals were subjected to additional adult stress, so we cannot conclude the actual effect of physical or emotional stress on monoaminergic neurotransmission. Our data allow us to judge only the differences in response of monoaminergic systems to stressors of different natures. In the present study, we wanted to evaluate the effect of growing conditions on the level of monoamines and determined the content of neurotransmitters at the time of weaning. Therefore, we did not screen the behavior of adolescent animals, which makes it difficult to interpret the observed changes in the state of neurotransmitter systems of the brain. We conducted an additional series of studies in which the level of neurotransmitters in adolescents was determined after assessment of behavior. These results are being processed.Another limitation is related to the complexity of assessing the influence of rat pup rearing factors on the formation of the endophenotype and behavioral profile of adult animals. Early stress, in particular maternal deprivation in rat pups, does not always lead to the development of post-traumatic stress disorder and other stress-related disorders in adulthood. There is no doubt that different outcomes of early trauma are associated with the plasticity of the endocrine, nervous, and immune systems. Plastic changes can be adaptive or maladaptive in nature and be mediated by environmentally dependent changes in active maternal care on the one hand and the rigidity of environmental influences on the other . In our In the present study, we show that the protocol of maternal separation and isolation from siblings that we used led to the formation of a hyperactive phenotype in adult animals and disruption in emotional regulation and social interaction. At the same time, females were more characterized by a decrease in anxiety, while males were characterized by an increase in aggressiveness and a decrease in sociability.Early life stress effects on brain monoamine concentrations were sex-dependent and region-specific. The opposite directions of 5-HT and 5-HIAA shifts in the frontal cortex may be associated with differences in behavior patterns in adult male and female rats.An increase in the levels of DA and DOPAC in the striatum of animals of both sexes under conditions of witness stress may indicate an increased sensitivity to emotional stress in rats reared under conditions of deprivation of maternal care.Thus, maternal deprivation in rodents, as a model of traumatic childhood experiences, is considered promising for studying neurodevelopmental disorders and subsequent vulnerability/resistance to the onset of mental disorders. Although our results show that MSI alters the levels of monoamines in brain structures associated with the development of stress-related diseases in adult rats, it remains unclear how these changes are recruited in the formation of endophenotypes and behavioral features of animals (including state/vulnerability to stress). Changes in the level of neurotransmitters can reflect both the dysfunctional state of monoaminergic systems and their involvement in adaptation processes. To answer this question, an assessment of the content and activity of enzymes of synthesis and degradation and the expression of receptors is needed . In addi


Índice Brasileiro de Privação – IBP).To estimate the prevalence of adult smokers in the 26 capitals and the Federal District according to the Brazilian Deprivation Index (Dataset on smoking were obtained from the Surveillance of Risk and Protective Factors for Noncommunicable Diseases by Survey (Vigitel) system for the 26 capitals and the Federal District, in the period from 2010 to 2013. The IBP classifies the census sectors according to indicators such as: income less than ½ minimum wage, illiterate population and without sanitary sewage. In the North and Northeast regions, the census sectors were grouped into four categories  and in the South, Southeast and Midwest regions into three . Prevalence estimates of adult smokers were obtained using the indirect estimation method in small areas. To calculate the prevalence ratios, Poisson models are used.The positive association between prevalence and deprivation of census sector categories was found in 16 (59.3%) of the 27 cities. In nine (33.3%) cities, the sectors with the greatest deprivation had a higher prevalence of smokers when compared to those with the least deprivation, and in two (7.4%) there were no differences. In Aracaju, Belém, Fortaleza, João Pessoa, Macapá and Salvador, the prevalence of adult smokers was three times higher in the group of sectors with greater deprivation compared to those with less deprivation.Sectors with greater social deprivation had a higher prevalence of smoking, compared with less deprivation, pointing to social inequalities. Tobacco use is associated with variables such as low income, low education, and living in places with high vulnerabilities.According to the World Health Organization (WHO), tobacco is the main risk factor for preventable causes of death and the second largest attributable factor of mortality in the world. To this end, in addition to considering social aspects, epidemiological research makes use of spatial analysis to identify the influence of spaces related to exposure differentials and inequalities, expanding the understanding of the occurrence of health-related events in populations and in the processes of morbidity and mortality.The place of residence is presented, among the social determinants, as a component strongly shaped by the social position in which it is allocated, showing that the aspects of the physical surroundings of the neighborhood can be important factors for the perpetuation of inequities in health.Acting through research in these intra-urban relationships allows identifying where and how interventions should be carried out, and one of the tools used to understand the relationships between social determinants and health outcomes is geoprocessing, an important strategy in identifying areas of vulnerabilityIt is noteworthy that most states lack health information on their population in small areas for formulating local public policy programs, given the high cost of surveys of this nature.. The indirect estimation method for small areas based on models has been widely used in several areas. This method uses survey data and auxiliary information extracted from the last census, at the lowest level, as predictor variables of the model for estimating the variable of interest in smaller areas.In this sense, the area of statistics has contributed with methods for obtaining reliable estimates for smaller areas, such as regional health, districts or sub-regions, not initially contemplated in the research sampling plansCentro de Integração de Dados e Conhecimentos para Saúde – CIDACS) in partnership with the University of Glasgow built the deprivation index for Brazil, called the Brazilian Deprivation Index (Índice Brasileiro de Privação – IBP), using data from the 2010 demographic census. This index allows to highlight the inequalities of different social groups and the comparison between municipalities and Brazilian regions. The index was built to measure inequalities in the country using a single cutoff point for all of Brazil. This index is presented by quartile, quintile, and vigintile of deprivation.In 2019, the Center for Integration of Data and Knowledge for Health  in smaller areas and, thus, support policies to promote equity. The present study aimed to produce estimates of prevalence of adult smokers, according to the IBP, in the 26 capitals and in the Federal District.The use of composite indicatorsVigilância de Fatores de Risco e Proteção para Doenças Crônicas por Inquérito – Vigitel) system, in the 26 capitals and the Federal District, from 2010 to 2013.This is an ecological study using data from the Surveillance of Risk and Protective Factors for Chronic Diseases by Telephone Survey (Vigitel uses probability sampling of the adult population (≥18 years old) residing in the 26 state capitals and the Federal District. The system uses the data frame of residential telephone available annually by the main telephone companies to draw the samples. The sampling process is carried out in two steps:draw of 5,000 telephone lines per city, divided into subsamples of 200 lines;selection of a resident over 18 years of age to be interviewed.The Vigitel weighting process consists of multiplying two factors: the inverse of the number of landline telephones and the number of adults in each household. Post-stratification weights were used so that the system results are representative for the entire adult population of each city. This weighting aims to match the estimated socio-demographic composition of the population of adults with a telephone based on the Vigitel sample in each city to the socio-demographic composition estimated for the total adult population of the same city, in the same year the survey was carried out.The study used the question “Do you smoke?”, regardless of the number of cigarettes, frequency and duration of smoking, to estimate the prevalence of adult smokers according to the IBP in the period from 2010 to 2013.Cadastro Nacional de Endereços para Fins Estatísticos – CNEFE) of the 2010 census. At the end of processing the database, IBP information by census sector was added.Using the Vigitel samples with telephone and complete address information and the interview databases with telephone number information, it was possible to include the census tract by performing a linkage with the National Register of Addresses for Statistical Purposes , given the high concentration of sectors in the low deprivation category and few occurrences in the high and very high deprivation categories .i) is dichotomous, with 1 being a smoker (success) and 0 (failure) otherwise (Table S4). The covariables by census sector were taken from the 2010 census, such as the percentage of households by type of water supply, percentage of households by type of sanitary sewage, percentage of households with no male members, percentage of households with female heads of household, percentage of households with grandchildren, great-grandchildren, son-in-law or daughter-in-law, parents or stepfathers or stepmothers, percentage of households with siblings over 50 years of age, and percentage of households with one or more residents.This study used data from Vigitel and the indirect estimation method to estimate the prevalence of adult smokers by IBP in the 26 state capitals and the Federal District. This method consists of using statistical models to obtain estimates of proportions of adult smokers observed in capitals for smaller areas, such as the IBP. The logistic regression model was used to impute the smoking response variable (Y), yes (1) or no (0), in the set of census sectors without any Vigitel interview. In the construction of the model, the set of sectors with a single interview in the period from 2006 to 2013 was used. This criterion was adopted due to the similarity in the distribution of sectors without an interview in Vigitel according to the IBP . The response variable  represents the vector of covariates;x) is the probability that the respondent self-declares a smoker (success) given the characteristic of x;π  is the vector of model parameters.β =  curve.Multiple logistic regression models were run in Rstudio version 3.6.3 using the Tidyverse packageTo assess the adjusted the model, a two-by-two classification matrix was used with four possible results: true positive (TP) denotes a response of smoking being correctly classified by the model; true negative (TN) denotes a response of non-smoking being correctly classified as non-smoking. False negative (FN) responses were classified as non-smoking, and false positive (FP) responses were classified as no smoking. The sensitivity of the model is defined by. These weights were calculated in the STATA program using the SURVWGT package, requiring sample weight information to run the package. In this study, data from population N1 and N2 extracted from the 2010 census of each region were considered to calculate the weight of the group of sectors with Vigitel interviews 1 is the total number of adults in sectors with Vigitel interviews, N2 is the total number of adults in sectors without Vigitel interviews, n1 is the number of Vigitel interviews and n2 is the number of sectors without interviews.In the joint analysis of the sectors with and without interviews, the post-stratification weight adjusted for the 2010 census population by IBP was calculated using the rake methodThe prevalence ratio of adult smokers due to IBP was calculated with the aim of comparing the groups. This ratio was estimated using the Poisson model, considering the first category as a reference. These estimates were calculated using post-stratification weights.The 65,684 census sectors in the 26 Brazilian capitals and the Federal District correspond to a population of 45,980,581 people. This corresponds to 22% of the total census sectors and 24% of the Brazilian population. Of this total of census sectors, 38,867 (58.2%) sectors had at least one Vigitel interview in the period from 2010 to 2013. Analyzing by region, the North, Northeast, and South regions had 83.1%, 81.3%, and 82.0% sectors with interviews, with a median equal to five, three, and three interviews, respectively. This shows the good spread of the Vigitel samples. While the Center-West Region presented 69.9% (median=3) and 39.2% in the Southeast. In the Southeast Region, the capitals São Paulo and Rio de Janeiro have 18,182 and 10,158 sectors respectively, both with a median equal to one interview per sector, which explains the low percentage of sectors with Vigitel interviews .In general, Vigitel's samples of residential telephones are scattered throughout the capitals, with the exception of São Paulo and Rio de Janeiro.To illustrate the IBP, Figure 1a (Supplementary) shows the census sectors of Salvador  and Figure 1b (Supplementary) the sectors grouped by IBP: low (29.7%), medium (32.0%), high (35.0%), and very high deprivation (3.3%).In the construction of the logistic regression models, the census sectors were selected with an interview in the period from 2006 to 2013 . This number of sectors varied between 7 (Boa Vista) and 4,231 (São Paulo). Due to the high variability in the number of sectors per region, the number of models was reduced from 27, one for each capital, to 5 models: North, Northeast, Southeast, South, and Center-West regions .The adjusted models for the North, Northeast, Southeast, South, and Center-West regions are available in the supplementary material – Tables S6 to 10. The measures of accuracy, sensitivity, and specificity of the models obtained in the two samples showed good adequacy of the models. However, the ability of the model to classify the individual as a non-smoker, given that he is a non-smoker, was greater when compared to its specificity .The trend of increasing prevalence as deprivation increases was found in 16 59.3%) of the 27 cities, indicating a positive gradient. In nine (33.3%) cities, the most deprived sectors had a higher prevalence of smokers when compared to those with less deprivation and, in the other two (7.4%), there were no differences  to 3.% of the In the North Region, Belém and Macapá presented a positive gradient between the prevalence of adult smokers and IBP, whose prevalence estimates were three times higher in sectors with greater deprivation when compared to those with less deprivation. Followed by Boa Vista, Porto Velho, and Palmas with 2.62 (95%CI 1.69–4.05), 2.76 (95%CI 1.61–4.72), and 1.38 (95%CI 1.02– 1.88), respectively. In Manaus and Rio Branco, no differences were detected between IBP prevalence estimates .In Aracaju, Fortaleza, João Pessoa, and Salvador, the prevalence of adult smokers in the most deprived sectors was three times higher than in the low ones. While in Natal, Recife, and Teresina, the prevalence ratio of adult smokers ranged between 2.15 (95%CI 1.51–3.05) and 2.72 (95%CI 2.25–4.59). In Maceió and São Luís, the prevalence ratio was 1.67 (95%CI 1.25–2.23) and 1.79 (95%CI 1.30–2.46), respectively .In the Southeast, South, and Center-West regions, the prevalence ratios of adult smokers ranged from 1.33 (95%CI 1.10–1.60) in Campo Grande to 2.76 (95%CI 1.38–4.02) in Florianopolis. In Curitiba, Florianópolis, and Porto Alegre, the prevalence ratios of adult smokers were twice as high in the sectors with the greatest deprivation when compared to those with the least .This study used the IBP to measure intra-urban inequalities in the prevalence of adult smokers, in Brazilian capitals and the Federal District, using Vigitel data from 2010 to 2013 and the indirect method for estimation in small areas. and in other countries.The study takes an ecological approach to measuring health inequalities, pointing out that the areas of greatest deprivation also had the highest prevalence of adult smokers. In Aracaju, Fortaleza, João Pessoa, and Salvador, the prevalence of smokers in very high deprivation sectors is three times higher than in low deprivation ones. The results found in the study are consistent with the literature, which points to an association between the highest prevalence of tobacco and the population with low income and education in Brazil showed the external validity of the estimate of the prevalence of adult smokers calculated using the indirect estimation method on Vigitel Belo Horizonte data. This study used the Health Vulnerability Indicators (HVI) grouped into four categories to estimate the prevalence of adult smokers in each group. Similarities were found between the estimates calculated in Vigitel and in the household survey, corroborating the results found here.Bernal et al.The work has some limitations. First, in 14% of the Vigitel interviews, the census sectors were not identified in the linkage process. The second is related to the lack of Vigitel interviews in some sectors, mainly in those with high or very high deprivation, requiring the use of statistical models to impute missing data in these sectors. In this sense, the covariates of the model may have underestimated or overestimated the probability of the adult being classified as a smoker or not in the sector. The capitals São Paulo and Rio de Janeiro have 28 and 43% of the sectors with interviews; in these capitals, the model may have underestimated the proportion of adult smokers. Third, the use of data from the 2010 census for the construction of post-stratification weights by IBP to minimize the selection bias of Vigitel in the period from 2010 to 2013 and of the covariates of the models. Due to the long-time span of the last census, these covariates may change over time. Fourth, the joining of the Vigitel databases from 2006 to 2013 given the annual variation in prevalence .Brazil produces a lot of research data in the health area with national coverage, large regions, federation unit, metropolitan region, and capitals. However, most of these states lack health information on their population in small areas, due to the high cost of surveys of this nature. In this sense, the IBP can be used to measure intra-urban inequalities in the country.This study contributes in the methodological aspect to the production of indicators in smaller areas and, thus, subsidize the states with this information for the formulation, monitoring, and evaluation of programs and public policies for the adequate promotion of health to combat smoking.


Endometrial cancer represents an ideal target to evaluate the impact of COVID-19 being the most frequent gynecological malignancy in Italy, generally detected at early stages and correlated with favorable oncological outcomes. The present comparative retrospective study carried out at Campus Bio-medico University Foundation in Rome aims to evaluate the impact of the COVID-19 pandemic on the presentation, diagnosis and treatment of EC. All women with a histological diagnosis of non-endometrioid and endometrioid endometrial cancer between 1 March 2018 and 31 October 2022 were included. The number of cases was higher in period 2 (95 vs. 64 cases). Time to diagnosis did not show statistically significant differences but in period 2, 92.06% of the diagnoses were made following abnormal uterine bleeding, while in period 1, only 67.02% were. The waiting time for the intervention was significantly shorter in period 2. Definitive histology, FIGO staging, surgical technique and adjuvant therapy did not show significant differences between the two periods. The study demonstrates that the impact of the COVID-19 pandemic did not have a direct effect on the diagnostic delay, tumor staging and type of therapy but rather on the presentation pattern of endometrial cancer. Endometrial cancer represents one of the most common gynecological neoplasms in industrialized countries; it is estimated that over 55,000 new cases are diagnosed each year in the United States ,2,3. OneThe present study aims to evaluate the impact of the COVID-19 pandemic on the pattern of presentation, diagnosis and treatment of endometrial cancer to verify whether diagnostic and treatment strategies have changed across these two periods in an oncological center of reference at a territorial level. The primary endpoint aims to evaluate whether the diagnosis of endometrial cancer has been delayed due to the COVID-19 pandemic. The secondary endpoint aims to evaluate the variation of post-intervention therapeutic assistance in terms of time and type of therapy.This is a single-center retrospective two-arm comparative study carried out at Campus Bio-Medico Foundation of Rome. All the patients diagnosed with endometrial cancer undergoing surgery between 1 March 2018 and 31 October 2022 were collected. Inclusion criteria: All women ≥18 years of age, diagnosed at primary or secondary discharge with malignant endometrial cancer, whose histologic diagnosis was endometrioid and non-endometrioid endometrial cancer . All patients underwent the following preoperative work-up: clinical and gynecological examination, transvaginal ultrasound and magnetic resonance imaging (MRI) for preoperative assessment of myometrial and cervical stromal invasion depth. EC was diagnosed after histopathological examination of samples from operative hysteroscopy helpful for having a representative biopsy or for removal of the target lesion. An abdominal and thoracic computed tomography (CT) was considered for investigating the presence of extrapelvic disease in high-risk patients. All patients included have been collected into a database. On the basis of the date of surgery, the population was then split into two periods: period 1 includes patients undergoing surgery between 1 March 2018 and 29 February 2020, corresponding to the pre-pandemic era, while period 2 includes patients undergoing surgery between 1 March 2020 and 31 October 2022, corresponding to the pandemic era. The following data were collected into a database: medical history , surgical history , histological classification , clinical management (start date and type of adjuvant therapy if undertaken), time of diagnosis  vs. date of suspect endometrial thickness at the ultrasound examination and the date of the histological diagnosis obtained with hysteroscopy), waiting time for surgery , initiation time of adjuvant therapy (days elapsed between the date of surgery and of the first day of any adjuvant therapy performed). The taxonomy proposed by WHO was used to classify the histological subtypes of endometrial cancer , while tThe general population is composed of 159 women aged between 33 and 87 years who underwent surgery for endometrial cancer at Campus Bio-medico Foundation between 1 March 2018 and 31 October 2022. In period 1, pre-pandemic, a total of (64) cases were subjected to surgery, while in period 2 (95). The demographic characteristics of the population sample under analysis are reported in p-value = 0.62414). However, these data are attributable only to those cases who underwent hysteroscopy following abnormal vaginal bleeding, 43 cases (67.2%) in period 1 and 88 cases (92.6%) in period 2, for a total of 131 cases (82.4%) in the general population. In parallel, 28 patients (17.6%) in the general population, distributed in 21 patients (13.2%) in period 1 and 7 patients (4.4%) in period 2, achieved histological diagnosis of endometrial cancer despite not having symptoms but only due to periodic specialist visits. The mean waiting time for the intervention from the general population was 62 (59) days. In period 1 it was 79 (83) days, while in period 2, it was 49 (31) days . The waiting time for the intervention was therefore significantly different in period 2 compared to period 1.The average time to diagnosis is 139 days. In period 1, the mean time to diagnosis was 131 days, while in period 2 it was 142 . The mean time to adjuvant therapy initiation was therefore significantly different in period 2 compared to period 1. All the data reported are summarized in The time to adjuvant therapy initiation of the general population averaged 89 (59) days, specifically in period 1 it was 79 (48) days, and in period 2 it was 96 days (66) . As reported in Data regarding the FIGO classification show a clear prevalence of endometrial cancer in FIGO stage I: 124 cases (77.99%) among the general population, divided into 49 cases (76.57%) in period 1 and 75 cases (78.95%) in period 2, as reported in The surgical technique most commonly used in the general population was the laparotomy (LPTM) with 101 (63.52%) total laparotomic interventions, while the laparoscopic technique (LPS) was preferred in 36.48% (58 cases) of interventions. In period 1, the total laparoscopic operations were 19 (29.68%) while the laparotomic ones were 45 (70.32%). In period 2, laparoscopic operations were 39 (41.05%) while laparotomic operations were 56 (58.95%). During surgery, the sentinel lymph node technique was used in 23.90% (38 cases) of cases of the general population, especially in 21.87% (14 cases) of the surgical interventions of period 1 and in 25.26% (24 cases) of period 2 operations. Systemic lymphadenectomy was preferred in 82 (51.57%) operations of the general population, divided into 29 (45.31%) cases in period 1 and 53 (55.79%) cases in period 2. As reported in As reported in Flow chart of the study is shown in The present study aimed to investigate the management of patients with endometrial cancer before and during the COVID-19 pandemic. Compared to other studies in the literature ,40,41,42Considering the BMI and age at diagnosis, no statistically significant differences were found between the two periods, i.e., period 1 (pre-pandemic), and period 2 (during the pandemic), while there is sufficient statistical evidence to state that there is a real difference in the ages of menopause onset among the two groups. This may be attributable to the physiological variation of EC incidence over time.Beginning with the total number of patients gathered at the Campus Bio-Medico Foundation of Rome, we discovered that period 2 had more patients than period 1 (95 vs. 64 cases). These data are justifiable by the fact that our foundation became a point of reference during the pandemic for all patients who were not able to be treated in other facilities reorganized in favor of COVID-19 management, with limited admissions and assistance in other departments. These data are in contrast with what has been reported by Bogani et al. , where iAll three intervals evaluated  show positive asymmetry in distribution, moreover, the mean values  are greater than the median values; this suggests that in the general population, as well as within the two groups, long waiting periods are less frequent than short waiting periods.As the principal theme of evaluation, being the main endpoint of the study, time to diagnosis did not show statistically significant differences between the two intervals. This is in contrast to what we would have expected given the reduction in hospital and routine outpatient visits, as reported in the literature, in order to avoid crowding and therefore the spread of the infection ,45,46,47Compared to the multicenter study conducted by Bogani et al. , and in The timing of initiation of adjuvant therapy does not statistically differ between the two groups, for this reason, there was no evidence of longer waiting times to access adjuvant therapies during the pandemic compared to the previous period. All this is in agreement with what was reported by Bogani et al.  and is pAs reported in the guidelines ,51,52,53In parallel, in the present study, the type of lymphadenectomy (systematic vs. sentinel) did not show significant differences between the two groups. These data are in contrast to what was reported by Bogani et al. , who poiFinally, in relation to our secondary endpoint, focused on the investigation of the modifications in terms of post-surgical treatments, there are no statistically significant differences between period 1 and period 2 in terms of types of adjuvant therapy undertaken. In contrast to the evidence of the study by Bogani et al. , there wA 2018 Israeli Gynecologic Oncology Group retrospective study evaluated the role of EC diagnosis in asymptomatic patients . The autThe study demonstrates that the impact of the COVID-19 pandemic did not have a direct effect on the delay in diagnosis but rather on the pattern of presentation of endometrial cancer. Therefore, in pursuing our primary endpoint, which consisted of the evaluation of whether a diagnostic delay had occurred in the pandemic phase compared to the pre-pandemic one, we verified that no statistically significant delay was recorded. However, a change in the presentation pattern of the disease and in the investigation method through which the initial clinical suspicion was raised were highlighted. In fact, patients came to our attention more frequently with endometrial cancer symptoms, primarily abnormal vaginal bleeding, as a result of the decreased access to regular gynecological checkups, rather than after an ultrasound examination evidencing an increased endometrial thickness.The main limitations of the present study, which could be otherwise interesting starting points for future investigations are the following: the limited number of samples in the exam and, due to the absence of long-term follow-up, the lack of evaluation of the impact of the COVID-19 pandemic on the oncologic outcomes of EC patients involved. However, the aim of this research was to assess how the pandemic impacted patients’ access to care and endometrial cancer pattern of presentation rather than to determine the impact the pandemic produced on survival and oncological outcomes. Further studies may have to assess how waiting time for diagnosis and treatment and different patterns of presentation may have impacted survival outcomes, but a more prolonged follow-up is certainly needed to determine aspects of this relevance.The main strengths of this paper consist of a data analysis that includes a very long time span (4 years) for the first time in the literature on this topic and carrying out the study in one of the few centers in Italy that has not undergone substantial reconditioning for the management of the pandemic crisis, becoming a first-level oncological center of reference at a territorial level. A further point of strength is related to the large number of patients for whom it was possible to reconstruct the entire process (from diagnosis to adjuvant treatment completed in our facility) and therefore precisely calculate the various timescales in the exam.In conclusion, the present study shows that the characteristics and patterns of care are changing during the COVID-19 pandemic, highlighting that the diagnosis was made mainly as a result of alarm symptoms, rather than for evidence during routine gynecological visits, and despite this, no diagnostic delay was detected. Conversely, waiting times for surgery were reduced and no impacts on FIGO staging, tumor grading, need for and type of adjuvant therapy were found compared to the pre-pandemic era.The Campus Bio-Medico Foundation Hospital has therefore been able to maintain excellent therapeutic assistance for patients with endometrial cancer during the COVID-19 pandemic, reducing waiting times for diagnosis and surgery and guaranteeing the start of adjuvant therapy comparable to previous years. These aspects are related to the fact that the center is a first-level oncological center of reference at a territorial level.Further studies, on a larger sample, will be needed to translate what has been highlighted in terms of survival and oncological outcomes in order to demonstrate if and how the pandemic has changed long-term outcomes.


Background: Maternal vaccination is safe and provides protection against COVID-19 in mothers and neonates, and it is necessary to evaluate its effect on the induction of immune responses through the levels of neutralizing antibodies present in maternal and neonatal blood. Methods: An observational study with transversal analysis was carried out. Included in the research were neonates (<1 month) whose mothers had been immunized whilst pregnant with at least one dose of the vaccine BNT16b and had not shown any symptoms of COVID-19. The blood of the mothers and newborns was collected during the Guthrie test and sent to the laboratory for the detection of neutralizing antibodies against SARS-CoV-2. Results: A total of 162 pairs of mothers and neonates were analyzed with an average age of 26.3 ± 5.97 years and 13.4 ± 6.90 days, respectively. All samples collected present neutralizing antibodies with an average percentage of 91% in the mothers and 92% in the neonates. The most satisfactory immune response was observed in neonates and mothers vaccinated during the second trimester of gestation. Conclusions: The vaccination of expectant mothers with the immunizer BNT162b2 has promoted a robust immunological response in both the mothers and the neonates. During 2020, the World Health Organization (WHO) decreed the coronavirus disease as a pandemic COVID-19) caused by the coronavirus-2 associated with acute respiratory distress syndrome (SARS-CoV-2) 9 caused .With the rapid spread of the pandemic, global efforts were made with the intent to develop efficient vaccines in a short time span . Among tCurrently, the evidence shows that the immunization of expectant mothers with mRNA vaccines such as BNT162b2 is safe, and in addition to protecting mothers, they are associated with the development of a neonatal immunological response through the possibility of passive transmission, supported by the presence of specific antibodies against SARS-CoV-2 present in breast milk, maternal serum, the placenta, and the umbilical cord’s blood . Data frHowever, there are still questions to be answered due to the lack of literature on various topics, for example, concerning the gestational time point when vaccination will promote a stronger immunological response in the mother as well as in the neonate  and aid This study comprises an observational study, with transversal analysis, classified as field research of an applied quantitative nature.The study was conducted in the town of Garanhuns, a town with 141, 347 inhabitants situated in the region of Meridional Agreste, 232 km away from the capital, Recife. The collection of data and biological samples was carried out in the children’s hospital Palmira Sales, a health center and philanthropic organization. Included in the research were neonates (<1 month) whose mothers had been immunized while pregnant with at least one dose of the vaccine BNT16b2 . Mothers who had shown symptoms of COVID-19 were not suitable for this research, as were preterm neonates and children that had shown symptoms of the disease.Biological material (blood) from the neonates was obtained during the Guthrie’s test procedure, which was carried out during the 28 days after birth, where 2 mL of blood was collected with a needle attached to a 3 mL syringe. Maternal blood was obtained following the same specifications. The samples were then placed inside a specific cooling unit for transportation, with the temperature being monitored by a thermometer in order to maintain a temperature of between 2 and 8 °C, and were later sent to the laboratory headquarters, ALFALAB, where the neonates’ test registration and the processing of samples for analysis took place.The detection of neutralizing antibodies was carried out through fluorescent immunoassays (FIA) for the quantification of NABs against SARS-CoV-2 using the Boditech Med Incorporated kit. The results were classified as positive or negative according to the cut-off index (COI), which is found by analyzing the interference of the protein spike RBD from SARS-CoV-2 with the receptor ECA-2 through the presence of NABs. If the result is equal or superior to 30%, the test is considered positive regarding the presence of NABs. .p > 0.05. To investigate at which gestational time point maternal vaccination is able to induce a robust immunological response in the neonate, Tukey’s test was carried out, comparing all average pairs based on the minimum substantial difference (D.M.S.). All statistical analyses were carried out using the software RStudio 2022.02, E..U.A. After triage and the collection and processing of biological material, the data obtained were analyzed descriptively through absolute distribution and percentages using the software Microsoft Excel 2013, E.U.A For the correlation of the dependent and independent variables, a Pearson’s test with a posterior construction of the graph through multiple linear regressions was carried out. The F test was utilized to verify the independent variables, and the T test was used to investigate the correlation between the average maternal and neonatal antibodies in relation to the gestational month of vaccination; results were considered significant when The study was approved by the Ethics Council of Research Multicampi Garanhuns University of Pernambuco under statement nº 5.281.016. The collection of data and biological material only took place after the signing of the Terms of Free Informed Consent (Termo de Consentimento Livre Esclarecido TCLE) by the mother/individual responsible for the child.Between April and October of 2022, 172 samples were collected from women who were vaccinated with BNT162b2 during the gestational period. A total of 172 samples were also collected from the newborns, resulting in a total of 172 pairs. Of these, ten were removed as they did not meet the selection criteria , resulting in the analysis of 162 pairs in this study. Respectively, the average ages of the mothers and neonates were 26.3 ± 5.97 years and 13.4 ± 6.90 days, respectively, with an average gestational age of 39.19 ± 1.16 weeks. The mother and neonate’s data are given in Women under 30 years of age and neonates under 14 days during sample collection were analyzed in higher numbers. The women vaccinated during their first gestational trimester were the majority (105/162), representing 64.81% of the total. Regarding the first dose of the vaccine during pregnancy, in 87 (53.70%) of the analyzed mothers, the first dose received during pregnancy was also their first dose of BNT162b2; in 49 (30.2%) mothers, the first dose during pregnancy was their second dose of the vaccine; and in 26 (16.05%) mothers, the first dose during pregnancy was the booster dose. Regarding the feeding of the neonate, 69.13% were exclusively breastfed until the point of data collection.The average amount of antibodies in neonates whose mothers received the first dose during gestation (87 mothers) corresponds to 90%; of those, four did not receive their second dose, with an average of 94% of antibodies in the mothers and 88% in the neonates. A total of 66 mothers received two doses, with an antibody average of 89% in mothers and 90% in neonates; eleven received three doses—including the booster dose—with an antibody average of 95% in mothers and 96% in neonates; and six provided no information. From the information above, we can say that in our data, mothers who were vaccinated with a single dose during pregnancy passed on a lower amount of antibodies to their babies than mothers who received two doses, and the same can be seen if we compare mothers who received two doses with those who received three doses.In mothers whose first dose during pregnancy was their second (49 mothers), newborns had an antibody average of 94%, and of these mothers, 24 received the booster dose during pregnancy , and 25 did not. Of the 25 who did not receive the booster, both newborns and mothers had an average of 94% neutralizing antibodies, and the same occurred with the 24 mothers who received the second dose of the vaccine. Finally, in mothers whose first dose during gestation was a booster dose and who therefore received only one dose during gestation (26 mothers), the mothers’ antibody average was 92%, and the neonates’ was 94%.Specific neutralizing antibodies against COVID-19 were detected in all maternal and neonatal blood samples, with an average percentage of 91% (10 to 99%) in mothers and 92% in neonates (8–99%). A total of 138 mothers (85.19%) and 141 neonates (87.04%) presented a level of neutralizing antibodies equal or superior to 90% by means of the multiple linear regression. It was possible to correlate the antibody average in neonates and mothers to the month of vaccination during the gestational period, as illustrated in The existing relationship between the percentage of neutralizing maternal and neonatal antibodies was observed, resulting in a Pearson’s correlation coefficient equal to 0.95 and demonstrating that as the average maternal antibody level increases, the average antibody level of the neonates also tends to increase .p = 0.201, which means that, according to the results of this test, there is no way to predict/relate the best month for the vaccination of mothers to ensure that the neonate obtains a higher percentage of antibodies. However, the p-value of the average maternal antibody variable was 0.000106 < 0.05, which means that, for every 1% of the mother’s antibody level percentage, the percentage of the neonate increases by an average of 0.78%.In the multiple linear regression model, all assumptions of reliability were met, indicating that it can be used to predict the average level of antibodies in the neonate from the maternal antibodies and gestational month of the first dose during pregnancy. The calculated R2 (coefficient of determination) was equal to 0.935, and the adjusted R2 was equal to 0.9133 (indicating a high explanation of the model in the dependent variables); thus, the month in which the mother was vaccinated and her level of neutralizing antibodies explain 93.5% of the variability of the neonate antibodies. However, there was no statistically significant relevance regarding the month in which the mother received the vaccination, at p = 0.0000283). Beyond that, the value of R2 = 0.9133 indicates that the month in which the mother was vaccinated and her level of neutralizing antibodies explains 93.5% of the variability of antibodies in the neonate. That is to say, a neonate whose mother was vaccinated during the first 6 months of gestation and presents 95% neutralizing antibodies will have, on average, a rate of antibodies equal to 74.3%.Using an ANOVA test, it was possible to demonstrate that there is no difference in the production of antibodies in the mother when compared to the neonate; however, a difference was noticed in the production of antibodies in the neonate according to the month in which the mother was vaccinated (p = 0.0000283).In order to obtain answers regarding the best period for vaccination during pregnancy with the aim of achieving higher percentages of neutralizing antibodies in neonates, an ANOVA test was also performed. Through this test, it was identified that there is no difference in the antibody production of the mother when compared to the neonate; however, there is a difference in antibody production by the neonate, depending on the month in which the mother was vaccinated , the presence of anti-RBD was detected in the maternal blood and in the neonate’s umbilical cord in all 91 evaluated pairs . In the In our study, it was not possible to assess whether the antibodies in the neonates come from the transfer at delivery or from breastfeeding; however, we saw that even in cases where the collection was carried out in the first days of the child’s life, antibody levels were high, pointing to a transfer of antibodies from the mother to the baby. Recent research confirms both possibilities of the passive transmission of immunity against COVID-19. Kugelman et al. (2022) detected the presence of neutralizing antibodies in maternal and neonatal blood collected at the time of delivery from 102 patients vaccinated with the BNT162b2 booster dose and in 93 newborns .p < 0.05) [Kashani-Ligumsky et al. (2021) compared the antibody levels in three groups: 1. neonates and mothers infected by COVID-19; 2. neonates of mothers vaccinated with two doses of BNT162b2 during the third trimester of gestation; and 3. neonates and mothers who were not vaccinated or infected. It was observed that the average specific antibody level in the samples of blood and umbilical cords was significantly higher in the newborns of vaccinated women than in those of mothers who contracted SARS-CoV-2 during pregnancy—224.7 ± 64.3 u/mL vs. 83.7 ± 91.6 u/mL ( < 0.05) . Our stuRegarding breastfeeding, several studies have already shown the presence of immunoglobulins against SARS-CoV-2 in breast milk. In the study by Ricciardi et al. (2022), involving 18 infants vaccinated with at least one dose of BNT162b2, breast milk samples were collected at different times after vaccination and analyzed at points before the first dose, two weeks after the second dose and six months after the first dose. The presence of antibodies was detected in all samples analyzed, with a decrease after 6 months post-vaccination [Regarding this issue, in the present study, we observed that 146 of the 162 newborns evaluated were breastfeeding until the time of collection, 112 of them exclusively. It is known that breastfeeding, aside from providing the necessary nutrients for the child’s healthy development and the strengthening of the bond between mother and child . Even soThe recent results confirm that the vaccination against SARS-CoV-2 during the gestational period is not only safe but also provides protection for pregnant women and their children, with detectable maternal antibodies found in the blood of the umbilical cord, breast milk, and serum samples obtained from the infants. Findings indicate the transference of maternal antibodies, whether through the transplacental route or breastfeeding ,11. In gIn our study, we saw that the existing relationship between maternal antibodies and neonate antibodies is high, and we observed that the values are visually very similar, which is numerically confirmed through the Pearson’s correlation coefficient between the two variables, corresponding to 0.95; thus, as the mean maternal antibodies grow, the mean neonate antibodies also tend to grow, and furthermore, maternal antibodies account for about 95% of the variation in neonate antibodies.p = 0.0001) [Such correlation was also observed in our research, corroborating results previously described in the literature. In this comparative study of vaccination effects and maternal infection by COVID-19 in terms of the immunological response, Nir et al. (2022) reported the efficient transmission of specific anti-SARS-CoV-2 antibodies in the 64 women that received the Pfizer mRNA vaccine during pregnancy, with a positive correlation between the maternal serum concentration and antibodies in the blood from the umbilical cord  .It is noteworthy that, in our study, the data were collected over 7 months, which included the dose of the vaccine that the mother received during pregnancy. As time passed, the women became pregnant after having received one or more doses, but all received at least one dose during pregnancy, whether it was the first, second, booster, or even two or three of these. One of the major dilemmas regarding maternal vaccination is the ideal time point, that is to say, which week/month/trimester will provide a stronger neonatal immunological response. In our study, it was observed that during the first and third trimesters of gestation, vaccinated mothers and their neonates presented an average rate of antibodies (91% in both) that was a little inferior when compared to newborns immunized during the second trimester (94%). Such results go against the majority of findings described in the literature.Yang et al. (2022) examined 1359 women that received at least one dose of both the mRNA  and of the viral vector (Jansen) and their 1374 neonates, and it was generally observed that immunization during any period of pregnancy is associated with detectable levels of maternal antibodies at birth; however, higher levels of maternal antibodies and, consequently, antibodies in the blood from the umbilical cord, occur from vaccination in the third trimester .Our study has provided data that differ from some of the research described in the literature in regard to the most suitable time point for vaccination during gestation. This could be a consequence of the higher rate of women vaccinated in the first trimester (64.1%) and significant disproportionality in relation to the second and third trimesters (only 6.79% were vaccinated in the third trimester). This was due to the long period over which the collection was carried out (7 months) to achieve a good number of pairs (mother/infant) for analysis. Furthermore, a larger number of pairs is essential for analysis and is better able to more precisely define the best gestational time point that will provide a stronger immunological response against SARS-CoV-2 in the neonate.However, it is important to emphasize that studies show that mothers vaccinated at any point during gestation present neutralizing antibody levels that are satisfactory and can guarantee the protection of the neonates through transmission via the placenta or breastfeeding ,18. FurtCurrent data suggest that neonatal protection generated by maternal immunization by two immunizing doses of an mRNA vaccine, such as BNT162b2, is also efficient in decreasing the hospitalizations caused by COVID-19 in infants aged under six months infected with SARS-CoV-2 . Thus, tHigh rates of neutralizing antibodies are detected in the maternal and neonatal blood independently from the gestation trimester when immunization occurs, the benefits of which can be transmitted passively, either transplacentally or through breastfeeding, thus contributing to the protection of the neonate. More studies are necessary to further understand the durability and kinetics of these antibodies in the long term, as well as their effectiveness against new variants of SARS-CoV-2.