In 1 (and derivatives), the B⋅⋅⋅B separation is ca. 3 Å, appropriate for one boron centre bonding to N and one to the C7 of indoles and indolines. This suitable B⋅⋅⋅B separation enables double E−H (E=N/C) functionalisation of indoles and indolines. Specifically, the activation of 1 with HNTf2 generates an electrophile that transforms N−H indoles and indolines into N/C7‐diborylated indolines, with N−H borylation directing subsequent C7−H borylation. Indole reduction to indoline occurs before C−H borylation and our studies indicate this proceeds via hydroboration—C3‐protodeboronation to produce an intermediate that then undergoes C7 borylation. The borylated products can be converted in situ into C7‐BPin‐N‐H‐indolines. Overall, this represents a transient directed C−H borylation to form useful C7‐BPin‐indolines.Pyrazabole  borylation of N−H‐indoles/indolines. Due to the robust B Organoboranes are ubiquitous in modern synthesis in part due to the power of the Suzuki–Miyaura reaction.A, Figure 2‐electrophiles that are: i) sufficiently reactive for double electrophilic borylation, and ii) can be transformed into synthetically useful boron species post borylation is challenging. One notable report on addressing the first point comes from Wagner et al. using B , double electrophilic borylation using B has not been combined with further transformations to form organoboranes ubiquitous in synthesis (e.g. pinacol boronate esters (BPin)). Thus, identifying readily accessible ditopic diboron electrophiles able to perform double E−H borylation and then be transformed readily into synthetically useful boron units is an unmet challenge. If realised this would streamline the synthesis of desirable borylated (hetero)arenes.Conceptually, if two electrophilic boron centres present in a single molecule are separated by an appropriate distance then one boron centre can act as a directing group by borylating the most reactive E−H site (E=C or N). This will position the second boron centre appropriately to transform a C−H into a C−B unit at a site otherwise hard to borylate ;2N4 core A amine)1 with commercially available HNTf2. The addition of HNTf2 to 1 resulted in immediate H2 evolution and crystals suitable for X‐ray diffraction studies were formed directly from the reaction mixture. The solid‐state structure of [Tf2N(H)B(pyrazole)]2, 2  Å out of the N4‐plane. The B⋅⋅⋅B separation in 2 is in the expected range at 3.126(2) Å, while the B‐NTf2 distance at 1.609(2) Å is unremarkable for a B‐NTf2 unit,2 could be isolated in good (74 %) yield on multigram scale using two equiv. of HNTf2 with respect to 1. While 2 is poorly soluble  it displays a δ11B=−3 , shifted downfield from that of 1 . Attempts to isolate a mono‐NTf2 derivative, compound 3, by using equimolar HNTf2 and 1 led to broad 1H NMR spectra and precipitation of 2 on standing, with 0.5 equiv of 1 left unreacted in solution. NMR spectra from combining equimolar mixtures of 1 and 2 suggest an exchange process occurs as new broad 1H resonances not corresponding to 1 or 2 are observed  along with new 11B resonances at −1.8 and −4.5 ppm (consistent with a lower symmetry pyrazabole such as 3). Thus, the mono‐NTf2 species 3, appears accessible in solution although only 1 and 2 can be isolated as crystalline solids in our hands.Due to the precedence in C−B bond formation using boron electrophiles derived from L‐BR2 is a ditopic electrophile, for example the addition of excess MeCN to 2 leads to displacement of both NTf2 anions and formation of the salt 22‐(MeCN). This crystallises as the cis‐isomer with both MeCN molecules on one face. Therefore, the formation of 2 does not lock a trans arrangement, important as a cis‐configuration is essential for borylation directed borylation. Note, the displacement of [NTf2]− by MeCN indicates that 2 and 3 will be reactive borenium equivalents.+[3] (Scheme −) using the hydride affinity relative to BEt3 methodology,+[3] is indeed a strong electrophile. In fact, +[3] has a hydride affinity greater than the [(catecholato)B(NEt3)]+ borenium cation ,3 should be sufficiently electrophilic to react with activated heteroarenes.Compound  Scheme  towards 2 electrophiles do indeed effect double borylation of N−H‐indoles, with it selective for N and C7−H borylation. However, this proceeded alongside the reduction of the C2=C3 unit, leading to formation of a C7‐borylated indoline. The initial product was converted in situ to the pinacol boronate ester 4 a  via a simple work up (addition of pinacol/K2CO3(aq)). In contrast, combining 2 and N−H indole led to no C7 borylation (significant 2 remained even after 18 h at 100 °C). However, the combination of a hindered base, 2 and N−H indole did lead (on heating) to C2−C3 reduction and borylation at N and C7 to form an analogous C7‐borylated indoline (see below). C7‐functionalised indolines are of significant interest, for example as anti‐cancer and anti‐inflammatory agents.N‐pivaloyl‐indoline and BBr3 also leads to C7‐borylated indolines,With electrophilic pyrazabole derivatives accessible the functionalisation of indoles was explored targeting double E−H borylation (C3/C4 or N/C7). Work up using pinacol/base would then form mono‐BPin indoles by selective protodeboronation of the more reactive E−B bond (C3−B or N−B).7, 23 3 is more atom efficient than using 2/hindered base only 3 is utilised in the scoping) revealed reduction/C7‐borylation to be compatible with substituents at indole positions C2−C6, although C6 substitution (4 e) slows pinacol installation meaning higher temperatures are required for this step. The transformation was compatible with N−H‐indoles mono‐substituted with methyl (4 b–4 e), halide (4 f–h), OMe (4 i), SMe (4 j), a C2/C3 disubstituted indole (4 k) and even with a tBu group at the C3 position (4 l). Furthermore, the majority of BPin products can be isolated in good purity (>95 %) without column chromatography. From these studies the functional group tolerance is comparable in scope to other electrophilic C−H borylation reactions using borocations , although groups prone to hydroboration (e.g. esters) are not compatible.Scoping studies . The major intermediates formed at room temperature displayed NMR data consistent with hydroboration and binding of the second boron at N . Heating to 100 °C consumed mixtures of 5‐H(Cl)/6‐H(Cl) with formation of one new product in each case. These displayed resonances consistent with 7‐H  and 7‐Cl  and these are the major products formed at the end of the reaction. Based on this data, the precedence from Fontaine and co‐workers and subsequent reactions using N−Me‐indole and N−H‐indoline as substrates (see below) we propose the mechanism shown in Figure 6 proceeding via D (formed by N−B bond formation and tautomerisation) is feasible but is disfavoured by the absence of diagnostic iminium C2‐H resonances (by 1H and 13C{1H} NMR spectroscopy).In situ NMR spectroscopy studies of N−H‐indole borylation using 7‐H . This is consistent with our hypothesis that pyrazaboles are appropriate diboron scaffolds for indole/indoline double borylation at N/C7 (and presumably C3/C4). Electrophilic C−H borylation requires a base to facilitate deprotonation of arenium species (via a step‐wise or concerted SEAr mechanism).[7‐H shows the indoline‐N has acted as the Brønsted base and is thus chiral (leading to diasterotopic resonances in 7‐H). Thus addition of an exogenous base deprotonates the N−H unit of 7‐H simplifying 1H NMR spectra .Regarding the solid‐state structure of H Figure  this conchanism).22, 282/N−H‐indole in the presence of the hindered base, 2,6‐tBu‐4‐Me‐pyridine , we propose this proceeds via a different sequence, starting with rapid indoleN−B bond formation. This was indicated by isolation of 8 . The formation of 8 indicates that pyrazabole‐NTf2 electrophiles actually react with N−H‐indoles at N in preference to C3 as no C3 borylation was observed under these conditions. However, in the absence of an exogenous base −Bpyrazabole interaction is reversible and this ultimately leads to formation of 5 from combining N−H‐indoles and 3. Thus when using an exogenous base/2, reduction/C7‐borylation still proceeds , but it is via a different sequence of steps to that using 3/no exogenous base.Regarding the mechanism for forming C7‐borylated indolines from 8 Figure  from a r3, if the mechanism in Figure 2/hindered base. The treatment of N−H‐indoline with one equiv. 2 and DBP rapidly forms the N‐borylation product 6‐H, as the major species and then 7‐H . Heating leads to faster consumption of 6‐H with 7‐H formed as the major product  or N−H‐indoline (with 2/base) starting materials. Note, when using N−H indoles in the absence of exogenous base the C3‐B unit (formed by indole hydroboration) can be viewed as acting as the Brønsted base for the N−H borylation step . It should be noted that the unactivated pyrazabole 1 does not react with N−H‐indole or N−Me‐indole (even on heating). In addition, attempts to hydroborate N−Me‐indole with (IMe)BH2NTf2 also led to no reaction at room temperature, suggesting that the facile hydroboration observed on combining 3 and N−Me‐indole maybe due to the bis‐hydroborane structure of electrophilic 3.To further support the initial hydroboration step 3 (B−N 1.650(2) Å).indoline dative bond occurs 9 was heated with or without a hindered base present (targeting borylation at C4). This was unsuccessful leading to extremely slow reactivity at 100 °C , indeed 9 remains the major product even after 14 days at 100 °C) while forming multiple intractable products at higher temperatures (160 °C). Finally, N‐SiR3‐indoles were explored as the larger R3Si groups may disfavour boron coordination at N, e.g. in the R3Si analogues of 9. However, combining 3 with N‐TMS‐indole led to the hydroborated product 10 . Heating of mixtures containing the hydroborated species 10 led to the formation of the C7‐borylated indoline product 7‐H as the major product . Using N‐TIPS‐indole (TIPS=triisopropylsilyl) also led on heating to the formation of 7‐H, however in this case no hydroboration intermediate (e.g. 11) was observed. The major silane by‐product observed by NMR spectroscopy was R3SiH in both cases, presumably formed by abstraction of a hydride from a four coordinate at boron B−H unit of a pyrazabole species by a silicon electrophile, with related processes well‐documented to occur.3Si‐indole/3 to 7‐H, the fact that reduction/C7 borylation proceeds even using the large TIPS protecting group has to date frustrated our attempts to direct reactivity towards C3/C4 diborylation.While the B⋅⋅⋅B separation in 2N4 pyrazabole core is sufficiently robust to persist during electrophilic borylation (enabling borylation directed borylation), the B2N4 pyrazabole core is reactive enough that it can be converted into synthetically ubiquitous organoboron reagents (pinacol boronate esters) via a simple work‐up. This balance between stability of the diboron core during E−H borylation and reactivity during work‐up is essential to access the synthetic desirable organoboron products. Work is ongoing in our laboratory using pyrazabole derived electrophiles and other ditopic diboron compounds to expand the borylation directed borylation approach.In conclusion, pyrazabole derived electrophiles are readily accessible reagents for converting N−H‐indoles into C7‐borylated indolines. The diboron core of pyrazabole, with a B⋅⋅⋅B separation of ca. 3 Å, is key for enabling the formation of two bonds from one pyrazabole unit to one indole/indoline. Importantly, while the BThe authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. 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Previous studies have arguably associated poor embryo morphology with low birth weight in singletons following single embryo transfer. However, the association between birth weight and specific morphological features in the cleavage stage remains less known. The purpose of the study was to investigate whether morphological features of embryos at the cleavage stage affect birth weight following blastocyst transfer.The single-center retrospective cohort study included 4,226 singletons derived from fresh single cleavage-stage embryo transfer , fresh single blastocyst transfer , or frozen-thawed single blastocyst transfer  between 2016 and 2019. Morphological parameters including early cleavage, day-3 fragmentation, symmetry, blastomere number, and blastocyst morphology were associated with neonatal birth weight and birth weight z-score in multivariate regression models. The models were adjusted for maternal age, body mass index (BMI), parity, peak estradiol level, endometrial thickness, insemination protocol, female etiologies, order of transfer, mode of delivery, and year of treatment.Adjusted for confounders, day-3 fragmentation was the only morphological feature associated with birth weight and birth weight z-score, while early cleavage, symmetry, blastomere number, and blastocyst morphology were not. Day-3 fragmentation increased the birth weight in both the ET  and BT groups  but not in the FBT group . The associations between birth weight and these morphological parameters were confirmed through birth weight z-score analyses. The adjusted odds of large for gestational age (LGA) and high birth weight were also significantly greater in singletons following the transfer of fragmented embryos in the BT group . The presence of fragmentation at the cleavage stage also affected the association between the blastocyst morphology and birth weight. Inner cell mass grades were negatively associated with birth weight in blastocysts with day-3 fragmentation but not in blastocysts without.The birth weight following blastocyst transfer was found to be positively associated with fragmentation at the cleavage stage. The data did not support the argument that transferring a poor-looking embryo may increase the risks of low birth weight. However, concerns for LGA infants remain. Despite the rapid evolution of embryo assessment technologies that has occurred within the past decade, the conventional embryo morphology assessment based on microscopic observation remains a routine procedure for many embryo laboratories and continues to play a key role in quality control systems . MorpholDespite this lower chance, achieving a live birth is still possible following the transferring of embryos with poor morphology. This raises the question of whether children derived from poor-looking embryos differ from those with “standard” morphology at their early stage of life. Several investigators have evaluated the association between embryo quality according to morphology and neonatal outcomes, showing that transferring poor-quality embryos at either the cleavage or blastocyst stage is associated with low birth weight in offspring , 4. It sin vitro fertilization (IVF), even though the morphological grades of the transferred blastocysts were similar. The present study aimed to evaluate the association between day-3 morphological features and birth weight in both the cleavage stage and blastocyst transfer, in addition to examining whether day-3 morphology modifies the association between birth weight and blastocyst morphology.Recent data suggest that unfavorable morphological features at the cleavage stage may impair implantation and live birth following blastocyst transfer, even when high-quality blastocysts were transferred –14. SuchInstitutional review board approval for this retrospective study was obtained from the ethical committee of the Xiamen University Affiliated Chenggong Hospital.Data of all patients who underwent single embryo transfer in either fresh or frozen-thawed cycles in the affiliated Chenggong Hospital of Xiamen University between January 2016 and December 2019 were accessed for potential inclusion. Only cycles resulting in singleton live birth were included. The exclusion criteria were a vanishing twin, very preterm birth , patients reporting maternal gestational hypertension or diabetes, uterine abnormalities, and unknown perinatal outcomes due to incomplete records. To minimize the maternal confounding, we also excluded maternal metabolism-related conditions, including Polycystic ovary syndrome (PCOS) and glucose intolerance. Female smokers were excluded from the study rather than being considered confounders because of the small number of events (n = 28). The present study did not analyze the embryos with early compaction on day 3 because our grading system did not record the cell number or fragmentation if early compaction had occurred. A previous study has suggested that the effects of early compaction may be modified by other morphological parameters .2, and 5% O2.Patients received ovarian stimulation as previously described . BrieflyMorphological evaluations were performed on days 1, 3, and 5 of the culture. In this study, all of the morphological evaluations at the cleavage and blastocyst stages were conducted by two of the authors (XJ and ZL). Each has over a decade of experience as an embryologist.On day 1, the occurrence of early cleavage was observed. On day 3, the embryo quality was assessed through a combination of cell number, blastomere size, and degree of fragmentation. Grade I embryos  were defined as eight-cell embryos with evenly sized blastomeres and no more than 5% fragmentation. Grade II embryos  were deemed 7–10-cell embryos with minor defects in blastomere size or moderate fragmentation (10%–15%). Grade III embryos  were embryos with any two of the following defects: cell number <7 or >10, abnormally sized blastomeres, and a fragmentation rate of ≥10%. Embryos with severe fragmentation (≥50%) or those with a combination of more than two major defects were not considered for transfer. Day-3 fragmentation here was defined as small anuclear extracellular cytoplasmic structures. Due to the subjectivity of the fragmentation evaluation, representative images for day-3 embryos with different fragmentation degrees in our clinic are shown .Embryo transfers were performed using a Cook catheter  under the guidance of abdominal ultrasonography. Assisted hatching was not performed during the period of the study. Luteal support continued until 10 weeks of pregnancy.The main outcomes measured were absolute birth weight and birth weight z-score adjusted for gestational age and gender. The birth weight z-score was calculated as the weight of the individual child minus the median weight of a reference population of children born at the same gestational age and of the same gender divided by the standard deviation from the same reference population. For the calculation, we used a birth weight reference for the Chinese population generated by the national population-based Birth Defects Surveillance System . The smaThe association between the embryo morphological parameters and birth weight outcomes was analyzed using generalized linear models (GLMs). The models were adjusted for a set of covariates that may affect the birth weight outcomes following embryo transfer; these included maternal age, BMI, parity, peak estradiol level, and endometrial thickness. Also incorporated were potential confounders, including the insemination protocol (IVF or ICSI), female etiologies , order of transfer (1 or >1), mode of delivery , and year of treatment. To analyze the absolute birth weight, the models were also adjusted for gestational age and gender. The same set of covariates was also used in the multivariate analyses for LGA, SGA, high birth weight  and low birth weight .The morphological parameters evaluated were early cleavage on day 1 (yes or no), fragmentation (<10% or ≥10%), cell number , and symmetry (even or uneven) on day 3, as well as ICM/TE  morphology on day 5. We used two methods to evaluate the combinational effects of the cleavage stage and blastocyst morphology on birth weight. First, the morphological parameters were assumed to be independent and to serve as confounders to each other in the multivariate models. Second, interactions between morphological parameters were introduced into the multivariate models. To simplify the interpretation of the data, only two-way interactions were considered. The postulated interaction patterns were first checked in a main-effect-only model and then confirmed in a multivariate model.All of the calculations were performed using SPSS .A total of 5,228 singleton live births following single embryo transfer were reviewed for potential inclusion. After excluding incomplete records (n = 20), very preterm births (n = 20), vanishing twin (n = 27), reported gestational diabetes (n = 26) and hypertension (n = 59) cases, uterine abnormalities (n = 28), glucose intolerance or diagnosed diabetes (n = 21), PCOS (n = 470), female smokers (n = 28), and early compaction (n = 176), 4,353 singletons were included. Of these, 1,185 were following fresh cleavage-stage transfer, 127 were following frozen-thawed cleavage-stage transfer, 787 were following fresh blastocyst transfer, and 2,254 were following frozen-thawed blastocyst transfer. Due to the limited sample size, we did not further analyze singletons following frozen-thawed cleavage-stage transfer; the characteristics, outcomes, and multivariate analyses for these patients are detailed in the supplementary material  g, -0.11 (-0.74 to 0.55), and 39 (38–40) weeks, respectively.For singletons following fresh single cleavage-stage embryo transfer (ET), fresh single blastocyst transfer (BT), and frozen-thawed single blastocyst transfer (FBT), the patient characteristics, embryo parameter distribution, and neonatal outcomes are provided in The associations between the birth weight z-score and each morphological feature of the transferred embryos are provided in We also evaluated the association between the birth weight z-score and embryo/blastocyst quality  on the association between morphology and birth weight. The size of association of fragmentation with the birth weight and birth weight z-score in the FBT group decreased by 142.36 (95% CI: 15.86–268.87) and 0.34 (95% CI: 0.01–0.66), respectively, in comparison with that of the BT group.To examine whether birth weight increased with the severity of fragmentation, we further categorized the degree of fragmentation into four groups . In fresh transfer cycles (cleavage and blastocyst stage), multivariate analyses suggested that in comparison with the no fragmentation group, the low fragmentation, moderate fragmentation, and high fragmentation groups experienced birth weight increases of 2.04 (95% CI: -59.28 to 63.36) g, 114.7 (95% CI: 36.38–193.1) g, and 214.8 (95% CI: 62.54–367.08) g, respectively. A similar trend was also observed with the birth weight z-score  may increase the risks of low birth weight , as we oCompared with previous studies –11, our However, our study is also limited by similar methodological defects to those affecting previous studies, including the subjectivity of embryo assessment and the retrospective nature of the study. The most profound bias may lie in the fact that patients receive a poor-looking embryo only when they have no choice. Therefore, singletons following transfer of a poor-looking embryo may be more likely to be delivered by mothers with a poor prognosis. In addition, because the proportion of live births resulting from day-3 fragmented embryos remained small in the birth cohort, the association between fragmentation and birth weight should be verified in a larger cohort.Cleavage-stage embryo fragmentation has been defined as the presence of anuclear membrane-bound extracellular cytoplasmic structures and used as a marker for embryos with poor prognosis. Embryo fragmentation has been associated with reduced blastocyst formation, decreased implantation, and increased early abortion. However, its effect on birth weight is less widely known. Ebner et al.  demonstrThe pathogenesis and biological significance of fragmentation in cleavage-stage embryos are not fully understood. A transmission electron microscopy study revealed that the most abundant organelles in the fragment were mitochondria . The detAmong the studies investigating the association between embryo quality and birth weight, Zhang et al.  suggesteSeveral studies have associated singleton birth weight with specific morphological features rather than the overall embryo score, yielding conflicting results –11. LiccThe association between blastocyst morphology and developmental potential might also be affected by their cleavage-stage origin. Recent studies have suggested that the developmental potential of blastocysts might differ according to their different morphological features in the cleavage stage, even though a similar blastocyst grade may be given –14. In aOur study failed to associate any morphological features with birth weight in frozen-thawed cycles, and the data suggested a significant modification of frozen-thawed on our estimate. This was not the first time that data have suggested that technologies may modify the association between embryo morphology and neonatal outcomes. Lieberman et al.  showed tIn conclusion, our data suggested that fragmentation in the cleavage stage is associated with increased birth weight following either cleavage- or blastocyst-stage transfers in fresh cycles but not in frozen-thawed cycles. Transferring day-3-fragmented embryos appeared to be safe for offspring, as infants with a higher birth weight may show positive long-term health, development, and educational outcomes . HoweverThe original contributions presented in the study are included in the article/This study was reviewed and approved by Ethical Committee of the Xiamen University Affiliated Chenggong Hospital. The patients/participants provided their written informed consent to participate in this study.JC, LL and JR contribute to conception and design. JHC, ZL and XJ contribute to the acquisition of data. JLC and LL contribute to the analysis and interpretation of data. All authors contributed to drafting the article or revising it critically for important intellectual content. All authors read and approved the final manuscript.This work was supported by the National Natural Science Foundation of China [grant number 22176159]; the Xiamen medical advantage subspecialty construction project [grant number 2018296]; the Special Fund for Clinical and Scientific Research of Chinese Medical Association [grant number 18010360765]; Xiamen Science and Technology Benefit Plan [grant number 3502Z20194053].We thank Xinli Wang for her assistance in the data processing.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


There is lack of research on body composition and physical fitness in individuals born small for gestational age (SGA) at term entering mid-adulthood. We aimed to investigate these outcomes in adults born SGA at term. This population-based cohort study included 46 adults born SGA with birth weight < 10th percentile at term   and 61 adults born at term with birth weight ≥ 10th percentile  at 32 years. Body composition was examined anthropometrically and by 8-polar bioelectrical impedance analysis (Seca® mBCA 515). Fitness was measured by maximal isometric grip strength by a Jamar hand dynamometer, 40-s modified push-up test and 4-min submaximal step test. Participants born SGA were shorter than controls, but other anthropometric measures did not differ between the groups. Men born SGA had 4.8 kg lower grip strength in both dominant (95% CI 0.6 to 9.0) and non-dominant (95% CI 0.4 to 9.2) hand compared with controls. Grip strength differences were partly mediated by height. In conclusion, body composition and physical fitness were similar in adults born SGA and non-SGA at term. Our finding of reduced grip strength in men born SGA may warrant further investigation. SGA is the most frequently used indicator of intrauterine growth restriction, which is a state where the foetus does not reach its genetic growth potential2. Males may be more susceptible to growth restriction in utero than females3. Being born SGA involves an extra vulnerability for later diseases, such as metabolic syndrome and cardiovascular diseases5. The prevalence of SGA depends on the birth weight standards used. When standards that are based on actual birth weights in the given population are used, 10% of term-born infants are by definition born SGA. In low- and middle-income countries the prevalence is around 20% according to Intergrowth standards6. Hence, the high prevalence represents a major concern for public health.Individuals born small for gestational age (SGA) are often defined as having a birth weight below the 10th percentile for their gestational age4. During childhood, studies have reported that those born SGA remain shorter and thinner with lower body mass index (BMI) than controls9, while there are reports of both less body fat7 and higher central adiposity8. In adulthood, studies have found individuals born SGA to be shorter12 and lighter12 than controls, but their BMI did not differ at 1811 or 22 years of age12. At 26 years of age, we did not find group differences in body composition, but women born SGA displayed lower lean mass than controls13. Another study indicated progression of adiposity from 22 to 30 years, as adults born SGA had more body fat, and their waist circumference increased, but there was no interaction effect with sex12. Also, low birth weight has been linked to reduced muscle mass and reduced muscle strength in childhood and adulthood14. A recent meta-analysis found a positive association between SGA and overweight and/or obesity15, whereas there were inconsistent findings of this association by sex15.Most infants born SGA show spontaneous catch-up growth by two years of age, however approximately 10% do not, and persistent short stature is therefore one of the most common complications after being born SGA16, that both have been reported to be associated with all-cause mortality, as well as non-communicable diseases18. Grip strength, a simple and widely used measure of muscular strength, has proven to be particularly relevant as it is a strong predictor of future physical function, morbidity, and mortality20. Several studies have found associations of birth weight with muscular and cardiorespiratory fitness in adulthood23. However, these studies have either included adults born preterm or not specified the gestational age of their participants, making it difficult to distinguish if the result is due to factors related to preterm birth or low birth weight. Three relatively small studies found no difference in cardiorespiratory fitness, measured by maximal oxygen uptake (VO2max), between term-born young adults with a birth weight at or below the 10th percentile and a control group25. On the other hand, two recent Swedish registry studies that included a large number of 18-year-old men born at term, found strong associations of birth weight with grip strength and cardiorespiratory fitness27.Muscular and cardiorespiratory fitness are two important health-related components of physical fitness28. The aim of this study was to examine whether body composition and physical fitness differed between adults born SGA and non-SGA at term. We hypothesised that adults born SGA at term would display a less favourable body composition and lower level of fitness than the term-born control group.There is lack of research on body composition and physical fitness in individuals born SGA entering mid-adulthood, an age when the prevalence of many non-communicable diseases starts to increaseThis study is a part of the NTNU Low Birth Weight in a Lifetime Perspective study. The present study included two groups of adults born in 1986–1988; one group born SGA at term, and one group born non-SGA at term with birth weight ≥ 10th percentile, which serves as a control group. The participants took part in a larger data collection at 32 years of age. In addition to physical fitness tests, examinations included anthropometric measurements, examination of lung function, visual function as well as fine and gross motor function. Assessments were carried out from September 2019 to October 2020.30. Pregnant women living in the Trondheim region were enrolled before week 20 of pregnancy based on referral from general practitioners and obstetricians. Women were eligible if they had a singleton pregnancy and had been pregnant one or two times before (n = 1249). A 10% random sample of these women were selected to serve as a control group (n = 132), using a sealed envelope method. A group of women at high risk of giving birth to an SGA infant were selected for follow-up if they had one or more defined risk criteria for SGA birth; a previous low birth weight child, low pre-pregnancy weight (< 50 kg), previous perinatal death, presence of chronic maternal disease , or maternal cigarette smoking at conception (n = 390). Women in the control group and the high-risk group were thoroughly followed during pregnancy and their infants were examined at birth. The rest of the women (n = 727) were not followed during pregnancy 30.At birth, all SGA infants born to mothers in either group were included in the SGA group Fig. . An infaThe total sample included 104 participants born SGA and 120 controls  in either group (data not shown). From the 26-year follow-up data were available on height, weight, BMI, waist and hip circumference, skinfold thickness and body composition measured by dual-energy x-ray absorptiometry (DXA). In the SGA group there were no differences, but in the control group, participants weighed 8.9 (95% CI 1.0 to 16.8) kg less than those who did not consent or were not assessed clinically.30. Ponderal index (g/cm3) was calculated based on these measurements.At birth, the infants in both groups were weighed to the nearest 10 g on a standard scale, and crown-heel length was measured with both legs extended to the nearest mm31, based on the parents’ education and occupation. This gives a social class rating from 1 (lowest) to 5 (highest).Parental socioeconomic status (SES) was calculated when participants attended the 14-year follow-up, supplemented for two participants at the 19-year follow-up, according to Hollingshead’s Two Factor Index of Social PositionEducational attainment at the 32-year follow-up was collected by self-report and classified according to the International Standard Classification of Education (ISCED) levels 1 through 8. These were recoded into three categories: Lower secondary education or lower (ISCED levels 1–2) as no more than 10th class level, intermediate education (ISCED levels 3–5) as 11th–14th class level, and lower tertiary education or higher (ISCED levels 6–8) as a bachelor’s degree or higher.Assessments were carried out at NTNU/St. Olavs Hospital in Trondheim, Norway. A brief medical interview was conducted prior to examination, including whether the participant was pregnant, had a musculoskeletal diagnosis or other conditions affecting physical functioning. If the participant had a condition that made them unable to perform a physical test or that could be worsened by testing, they did not perform that particular test. All examinations were carried out by experienced and specially trained examiners, blinded to birth weight group. Anthropometric measurements were performed by a nurse and physical fitness tests by two physiotherapists and a medical research student. The examinations were carried out in the same order for each participant.2) and waist-to-hip ratio (waist circumference/hip circumference) was calculated. Bioelectrical impedance analysis measures included percent body fat, fat mass, fat free mass, skeletal muscle mass, total body water and extracellular water using the Seca 115 analytics software .At follow-up, the participants’ height, waist and hip circumference were measured to the nearest mm. Waist circumference was measured at the mid-point between the lowest rib and the crista iliaca, and hip circumference at the maximal circumference over the buttocks. Weight was measured by bioelectric impedance analysis using a Seca medical Body Composition Analyzer (Seca® mBCA 515) with a 100 g accuracy. Body mass index  hand dynamometer was used. The dynamometer has 5 handle positions; position 3 and 4 were used for women and men, respectively. The participants were seated during the test, with shoulder abducted, a 90° angle in the elbow and a neutral position in the wrist, without support of the forearm33 and is modified to improve standardisation. The participants started laying prone on a mat with their hands close to the shoulders and feet hip-width apart with their toes on the mat33. Before every push-up they had to clasp hands behind their back before pushing themselves to a straight leg push-up. In the top position they had to touch either of their hands with the other hand before returning to the push-up position and returning to the down-position. The number of correctly performed push-ups in 40 s were registered. One participant in the control group could not perform the push-up test due to a hand fracture.The 40-s modified push-up test measures the muscular strength and endurance capacity of the upper body34. The participants stepped on and off the step for four minutes paced by a metronome set to 46 beats per minute . The height of the step was adapted to sex: 33 cm for women and 40 cm for men. Heart rate was observed during the test using a heart rate monitor (Firstbeat Technologies Oy) and recorded after 4 min of stepping and after being seated for 2 min. Two participants born SGA were not able to complete the test and were excluded from the analysis.The Åstrand-Ryhming step test is a 4-min submaximal step-test that measures cardiorespiratory fitness36, we performed separate analyses by sex. Differences in physical fitness between groups were adjusted for height as a potential mediating factor in a univariate general linear model, since height has been consistently correlated with both being born SGA37 and physical fitness in previous literature22.The analyses were conducted in SPSS version 27 (IBM Statistics). A p-value of less than 0.05 was considered statistically significant. Background characteristics were examined using Student’s t-test for continuous data, Exact Mann–Whitney U test for ordinal data and Pearson’s Chi square test for dichotomous variables. Group differences in outcome measures were analysed using independent samples t-test. The assumption of normally distributed variables was checked by visual inspection of histogram, boxplot, and Q–Q-plots of standardised residuals. As physical fitness differs between women and menTo investigate whether physical conditions affected the results, sensitivity analyses were performed by excluding participants who were pregnant, had a musculoskeletal diagnosis or other conditions affecting physical functioning, as reported by the participants in the brief medical interview.38, that we would have the power to detect differences of 0.48 SD units with an alpha-level of 0.05 and a power of 80%, and 0.67 SD units with an alpha-level of 0.01 and desired power of 90%.A priori power calculations suggested, based on previous follow-up numbers in the SGA (n = 64) and control group (n = 81)The study was approved by the Regional Committee for Medical and Health Research Ethics in Central Norway (23879). Written informed consent was obtained from all participants. All methods were performed in accordance with the relevant guidelines and regulations. The data was pseudonymised and stored securely on a remote server with a two-step identifier. All methods were non-invasive and entailed low risk for injury or adverse events. An appointed doctor was medically responsible during data collection. Participants in need of health services were referred as appropriate.Participants’ background characteristics are shown in Table At follow-up, mean height was significantly lower in the SGA group compared with the control group. The other anthropometric measurements and bioelectrical impedance analysis measures did not differ between the groups Table .Table 2AThe results of the physical fitness tests are shown in Table r = 0.722, p < 0.001 dominant hand, r = 0.711, p < 0.001 non-dominant hand). When we adjusted for height, the difference in maximal grip strength among men decreased to − 2.8 kg .Adults born SGA were shorter than controls Table . Height Results were unchanged regarding anthropometric measures and body composition when we performed sensitivity analyses by excluding eight participants born SGA and five controls who were pregnant, had musculoskeletal diagnoses or other conditions affecting physical functioning. However, the SGA group performed 2.4 (95% CI 0.7 to 4.1) more push-ups than the control group.In this study we found no differences in body composition or physical fitness between adults born SGA and the control group, measured by grip strength, a 40-s modified push-up test and a 4-min submaximal step test. However, men in the SGA group had significantly lower grip strength in both the dominant and non-dominant hand compared with men in the control group.6. At the 32-year follow-up, 56.3% of the invited were assessed clinically. This low participation rate can partly be explained by the data collection being carried out during the Covid-19 pandemic. Even though follow-up rates of 50–80% participation have been suggested to be acceptable in cohort studies39, individuals performing worse may have a stronger tendency to drop out40. This could have led to a selection bias toward physically fit participants. However, there were few differences in background variables between participants and non-participants, and assessment of physical fitness was only a part of a larger follow-up examination. Thus, it seems unlikely that the results were affected by selection bias. Nevertheless, the loss to follow-up limits the sample size and hence gave wider confidence intervals than would be expected with a larger sample size.A strength of this study includes the prospective population-based design, where participants were recruited and followed from mid-pregnancy. At birth, SGA was defined as birth weight below the 10th percentile. This may also comprise individuals who are genetically small and not necessarily growth restricted. Additionally, the control group may comprise individuals who are growth restricted, but still have a birth weight above the 10th percentile. This could possibly contribute to smaller differences between the groups in this study. Nevertheless, the 10th percentile is a common cut-off used to identify SGA individuals41. Assessments were carried out in the same order for all participants by trained examiners blinded to birth weight groups. Bioelectrical impedance analysis by the Seca® mBCA 515 has shown to agree well with the accurate and precise DXA method44, which is considered the reference measurement for differentiating lean and fat tissues. Both grip strength measured by a dynamometer and the modified push-up test are reported to be valid instruments for assessing muscular fitness45. A limitation of the study was the measurement of cardiorespiratory fitness by a submaximal test with heart rate as the outcome, as heart rate is largely individual46, and a maximal exercise test measuring maximal oxygen uptake would evaluate cardiorespiratory fitness more accurately16. However, a submaximal test was considered more feasible in this study, as it is less time consuming and more comfortable for the participants.Another strength is the use of objective measurement tools to assess body composition and physical fitness, as self-reports may be biased by over- or underestimation47. These findings are in line with other studies of children, adolescents, and adults49. However, evidence regarding overweight and adiposity is conflicting. We did not find differences in BMI, waist circumference or waist-to-hip ratio between the groups, consistent with our previous report of similar body composition of participants born SGA and controls at 26 years of age, measured by DXA13. Other studies of term-born adults with a birth weight < 10th percentile have reported both similar BMI and waist-to-hip ratio50, lower weight and reduced lean body mass51, and a higher percentage body fat12 and total abdominal fat mass50 compared with a control group. However, these studies used different birth weight percentiles to define the control group, which may explain some of the discrepancy.In this study, participants born SGA were shorter than controls, which we have previously documented in adolescence and young adulthood27. However, in that study a one SD lower birth weight was associated with 1.8 kg lower grip strength. In the present study, mean difference in birth weight SD score was 1.44, corresponding to a 2.6 kg difference. This is consistent with what we observed in men and would also be included in the CI we observed among women. Further, our finding is also in accordance with other studies that have found strong associations between lower birth weight and reduced grip strength in adulthood, regardless of gestational age at birth21. The reduced grip strength found for men born SGA in this study could indicate increased risk of negative health outcomes, as increased hazard ratio of all-cause mortality ranging from 1.0820 to 1.1619, and for cardiovascular mortality of 1.1719, have been reported for every 5 kg reduction in grip strength.Our hypothesis that adults born SGA would display a lower fitness level than their peers was not confirmed in this study. However, men born SGA had approximately 5 kg lower grip strength than men in the control group in the unadjusted analyses. This result must be interpreted with caution, as the 95% CI was rather wide. The difference in grip strength is consistent with the recent Swedish study that found strong associations between birth weight in men born at term and grip strength at 18 years of age22. Growing up, boys in the general population are reported to have motor problems more often than girls52. In the SGA population, several studies also show that boys are more vulnerable to growth restriction in utero than girls, possibly because of a higher growth velocity3. Thus, boys and men born SGA may be more susceptible to unfavourable development outcomes. In support of this, we have previously reported reduced manual dexterity at 14 years of age in boys born SGA, and not girls47. This may be related to the findings of reduced grip strength in the present study. When we adjusted for height, the difference in grip strength was reduced and no longer significant, indicating that the difference was partly mediated through a lower height in men born SGA. This is in accordance with previous research reporting that height is associated with grip strength22. However, when grip strength is used as a predictor of mortality and functional capacity it is not adjusted for height53. Additionally, even when adjusted for height, the grip strength of men born SGA was lower than the 59 kg normative value for men of the same age in Norway54. This underlines the relevance of the lower grip strength finding in our study.The differences in grip strength among men only may be related to motor development, as associations between motor development and grip strength in adulthood have been documented26 reported that among term-born men, each unit decrease in birth weight z-score was associated with reduced cardiorespiratory fitness of 7.9 W in maximal workload, corresponding to approximately 0.2 SD in that population. That study did not compare men born SGA with men born non-SGA and used a different proxy for cardiorespiratory fitness than we did. However, our confidence intervals among men ranged from less than − 0.5 SD to more than + 0.5 SD, and we may not have had adequate power to observe an association that was observed in the paper of Ahlquist et al.26. Ridgway et al.22 reported a weak association between lower birth weight and lower aerobic fitness, however, the sample also included late preterm born individuals. Our results are consistent with our previous findings at 18 years of age11 and with two other small studies of Danish men that found no differences in VO2max between those with birth weight ≤ 10th percentile and a control group at 19 and 24 years of age25. Thus, it seems unlikely that adults born SGA have any moderate or large deficit in cardiorespiratory fitness, but we cannot exclude a weak association with lower birth weight.Contrary to our hypothesis, we did not find any differences between the groups in the push-up test or step test. In a sensitivity analysis excluding participants with conditions affecting physical functioning, the adults born SGA even performed more push-ups than the controls. In a study of 287,000 male military conscripts Ahlquist et al.53, and is shown to track through life53. It is therefore worrying that men born SGA already at 32 years of age had reduced grip strength compared with men in the control group. Consequently, promoting a physically active lifestyle in men born SGA may be advantageous. A physically active lifestyle has been shown to form early in life55, indicating that promotion of physical activity, especially concerning activities that enhance muscular strength, should be a focus from childhood.Overall, the lack of differences between adults born SGA and non-SGA controls in this study is promising with regards to future health. However, reduced grip strength is an established predictor of future physical function, morbidity and mortalityOverall, we found no differences in body composition or physical fitness between adults born SGA and non-SGA at term. However, men born SGA had lower grip strength than men born non-SGA. There are few studies concerning physical fitness in individuals born SGA at term entering mid-adulthood. Further research is therefore needed to determine whether adults born SGA have lower physical fitness than their non-SGA peers.


Air pollution exposure has been associated with critical neonatal morbidities, including low birth weight (LBW). However, little is known on short-term exposure to wildfire smoke and LBW. In this study, we estimated the association between birth weight following pregnancy and wildfire smoke exposure in more than 1.5 million newborns in Brazil (considered as a very fire-prone region worldwide).We applied a logistic regression model to estimate the percent variation in newborns with low birth weight when exposed to wildfire in different trimesters of the pregnancy.After adjusting the model with relevant covariates, we found that an increase of 100 wildfire records in Brazil was associated with an increase in low birth weight in the Midwest region [0.98% ] and in the South region [18.55% ] when the exposure occurred in the first trimester of pregnancy.Wildfires were associated with LBW and this should be of public health concern for policymakers.Brazilian Agencies National Council for Scientific and Technological Development (CNPq); Ministry of Science, Technology and Innovation in Brazil (MCTI); and Novo Nordisk Foundation Challenge Programme. To our knowledge, the first study on the relationship between air pollution and birth weight was performed by Alderman et al.Our findings add strength to the evidence that mother's exposure to air pollution during pregnancy may cause low birth weight. In our study, we found this evidence accounting for wildfire-related air pollution, which, to our knowledge, was not explored to date in South America. Also, our sample was relatively large, including more than 1.5 million records of birth nationwide over 18 years. To our best knowledge, this is the study with the largest sample size and the largest study period in Brazil.This study adds significantly to the growing evidence that wildfire-related air pollution might be harmful to fetus development and increase the risk of low birth weight. Given that wildfire is a growing problem in several regions worldwide, especially in Brazil – a fire-prone region, the epidemiological evidence shown in our study should be of great concern to the public health community and policymakers.Alt-text: Unlabelled boxEnvironmental exposure has been associated with numerous adverse pregnancy outcomes, including low birth weight.There are wide disparities in the prevalence of low birth weight among different population groups. About 20.5 million infants worldwide  were born in 2015 with low birth weight, of which 95% of them were born in developing countries.3 of PM2.5 and 5.7 g per ppm O3.3 in mean PM2.5 was associated with a reduction of 4 g in birth weight.2.5, NO2, and O3 in the first and last month of the pregnancy was associated with an increase in the risk of low birth weight.3 in the third semester of pregnancy was associated with a decrease of 17% in birth weight.Among the environmental factors related to low birth weight, a large body of literature has pointed out mother's exposure to air pollution during pregnancy as a significant public health concern. To our knowledge, the first study on the relationship between air pollution and birth weight was performed by Alderman et al.2.5 on birth weight are not well understood. The current body of evidence has focused on the toxicologic and physiologic effects of ambient air pollution on fetus development, without accounting for specific particulate matter sources – e.g., wildfire. The existing evidence shows that ambient air pollution may impact the anatomy and morphology of the placenta, causing reduced oxygen transport and increased blood viscosity,Biological mechanisms involved in the effect of wildfire-related PMOverall, most of the previous epidemiological investigations indicate that the evidence on the relationship between air pollution and birth weight suggests causality, but more studies are needed to assess the importance of exposure in different periods of pregnancy and the influence of different pollutants, including the variation by air pollution sources. Given that (i) ambient air pollution represents a heterogeneous mixture of chemical elements from different sources - e.g., fossil fuel combustion, industrial emissions, and biomass burning and (ii) health effects of air pollution is driven by the chemistry of air pollution toxicity which varies through air pollution sources over space and time, we focused on the exposure to wildfire smoke as an indicator of air pollution emission with a distinct characteristic in the chemistry of air pollution toxicity, and episodic occurrence. We estimated the association between birth weight following pregnancy and wildfire smoke exposure in more than 1.5 million infants in Brazil.2 of vegetation area is burned each year globally. Brazil is a very fire-prone region where, according to the National Institute of Spatial Research - INPE (http://queimadas.dgi.inpe.br/queimadas/), between January/2020 and August/2020 there were about 120,000 km2 of burned area. Wildfires emit substantial amounts of air pollutants that can travel over large distances, affecting air quality and human health far from the originating fires.2.5) is the major pollutant emitted by wildfires. About 12–16% of global wildfire-related particulate emissions occur across Brazil.Further epidemiological studies on this topic are essential, given that wildfires have burned a large number of areas in the past years. Mouillot and FieldBirth data were provided by the Ministry of Health in Brazil. This data includes individual records of birth in Brazil between 1 January 2001 and 31 December 2018. Birth data include event date, birth weight , mother's home municipality, mother's age, mother's race , gestational age (categorized as the number of weeks of gestation), date of the last menstrual period, and sex of the infants. This data was obtained from publicly available database curated by the Ministry of Health in Brazil. The Brazilian Ministry of Health does not require ethical approval or informed consent for secondary analysis of anonymized health data at municipality level.,For this analysis, our inclusion criteria were: (1) infants born between the 37th and the 41st week of gestation; Given that preterm births  are correlated with low birth weight, this inclusion criteria will remove the effect of preterm birth on low birth weight; This inclusion criteria have been used by numerous studies.Regarding the mother's home location, the smallest spatial information included in the data is at the municipality level. There are 5572 municipalities in Brazil, which represent the smallest areas considered by the Brazilian political system. The government groups the municipalities by five regions, including the North, Northeast, Midwest, Southeast, and South. In Appendix 1 we show the spatial distribution of all municipalities and regions in Brazil. This Appendix gives a perspective about the size of the municipalities and regions in Brazil.Instituto Nacional de Pesquisas Espaciais - INPE (http://queimadas.dgi.inpe.br/queimadas/). The data include the date of wildfire records and its geographical location. These data are derived from seven satellite remote sensing observations, including NOAA-18, NOAA-19, METOP-B, MODIS (NASA TERRA and AQUA), VIIRS (NPP-Suomi and NOAA-20), GOES-16, and MSG-3. All images from these satellites are processed by INPE to estimate wildfire records. This estimate is based on a specific satellite as reference – AQUA satellite. Each wildfire record estimated here indicates the existence of fire within a pixel of 1 × 1 km. The minimum size of the wildfire to be detected by the satellite sensor is about 30 × 30 m. The quality of the data over time is high, given that the data is based on seven satellite, and then the estimates are derived from a reference satellite. We accounted for all wildfire records in Brazil based on the reference satellite in the period between 2000 and 2018. Then, we summed the daily wildfire records within each municipality in Brazil to merge the wildfire data with the health data. Note that the wildfire data is a continuous variable representing the number of wildfire records per day and per municipality.Wildfire data were provided by the National Institute of Spatial Research of Brazil – We adjusted the analysis for ambient air pollution, meteorological variables, and socioeconomic variables included in the birth data (as mentioned above).2.5, given the substantial amount of PM2.5 emitted by wildfires. At the global scale, approximately 32% of the total tropospheric organic aerosol in biomass burning aerosol originates from direct particulate emissions, whereas the rest is formed in the atmosphere.2.5 in Brazil.,For ambient air pollution, we accounted for PM2.5 (μg/m³) were obtained from a global model based on satellite observations. The data was accessed from the Copernicus Atmosphere Monitoring Service (CAMS), which include CAMS-Reanalysis predictions for the period between 2000 and 2018. The CAMS service runs ensemble models using several satellite observations and emission inventories amongst other predictors. The predictions were retrieved at a spatial resolution of 0.125° (approximately 12.5 km) and a temporal resolution of 6 h, including daily measurements for 00, 06, 12, and 18 UTC . We calculated the daily mean temporal resolution from 2000 to 2018 and the daily mean concentration within each Brazilian municipality. Description of the validation of the CAMS global model is presented in Appendix 2.Concentrations of PMFor the meteorological data, it was accessed from the European Centre for Medium-Range Weather Forecasts (ECMWF). Weather data include surface temperature (°C), humidity (%), wind speed (m/s), wind direction (°), and precipitation (mm/day). Temperature, humidity, wind speed, and wind direction were derived from Era-Interim reanalyses, with a spatial resolution of 0.125° and temporal resolution of 6 h. This reanalysis was performed by the ECMWF. Precipitation data was accessed from the Climate Prediction Center (CPC) and the National Ocean and Atmospheric Administration (NOAA). This data has an original spatial resolution of 0.50° (approximately 50 km), with interpolation to 12.5 km, and a temporal resolution of 6 h. We accounted for daily mean values of weather variables within each Brazilian municipality.2.5 and meteorological covariates for each trimester within the boundaries of the mother´s municipalities.The wildfire exposure window was based on the gestational intervals, which included the first (week 1 to week 12), the second (week 13 to week 28), and the third trimesters (week 29 to week 37). For each trimester, we estimated the sum of wildfire records – the exposure variable. This estimate was based on the number of wildfire occurrences within the boundaries of the mother's home municipality. We also calculated the average PMNote that in our study design we considered birth weight because of a gradual crossover, meaning that it is plausible that several weeks of exposure are needed to manifest the effect. We highlight that the use of trimester exposure can be used to compare the weekly averages with the trimester averages. If they are similar, we can assume that there are no bias and trimester exposures are more clinically relevant.We applied a case-control study design using logistic regression model to estimate the odds ratio (OR) for low birth weight associated with wildfire occurrence during a specific trimester of pregnancy (1–3 trimester). The cases were defined as newborns with weights between 1000 and 2500 g and controls were newborns with weights more than 2500 g to 6000 g.2.5, meteorological variables , mother's age, mother´s home state , year of birth , and latitude/longitude (centroids) of the mother's home municipality. We adjusted de model for PM2.5 because of the biological pathway. There may be at least two types of etiologic pathways possibly linking prenatal wildfire exposure with low birth weight – biological pathway (exposure to air pollution from wildfires) and psychosocial pathway (stress caused by direct or indirect consequences of the wildfires). It is also possible a mixture of these two pathways. The Centers for Disease Control and Prevention (CDC) has recognized these aspects and released guidelines (https://www.cdc.gov/air/wildfire-smoke/pregnancy.htm). Therefore, we used the wildfire records as the main exposure because we cannot measure the ecological indicator of maternal stress . Controlling for PM2.5 we can capture the effects from exposure to air pollution from the fires . Consequently, our results may reflect the potential conjoint impact of these two pathways. The mode was given by:LBW is the probability of low birth weight in the region j, during the trimester of exposure t; β values are regression coefficients for the exposure variable wildfire (during the trimester t); s are the smoothing spline function to characterize nonlinear relationships between LBW and the confounders PM2.5, weather parameters, age (mother's age), year (year of birth), and latitude/longitude  during the trimester t; e is the error term spatially and temporally dependent based on the regions j and trimester of exposure t. We used R - version 2.13.1  to perform the statistical analysis. We performed the logistic regression models using the GLM function. To allow the inclusion of spline function in the model, we used the package “splines”. We report the results as percentage change in risk (and 95% CI) of low birth weight associated with an increase of 100 wildfire records. The percentage change in risk was calculated as (OR−1) × 100%. We used this approach to describe the health burden associated with exposure, which may be a better information for policy makers and general readers (Steenland and Armstrong 2006). We conducted a sensitivity analysis by stratifying the analyses by sex and by race (white and black). We conducted the analyses for each one of the five Brazilian regions . We performed this subgroup analysis by region to capture the regional heterogeneity of landscape in Brazil , which is strongly correlated with wildfire occurrences.We adjusted the model for the following confounding variables: PMRole of the funding source: The funding bodies did not play any role in the study design, data collection, data analyses, results interpretation, writing of this manuscript or decision to publish.2.5 is also in the North region, with an average value between 37 and 39 μg/m³ over the trimesters. Overall, the summary statistics for wildfire and covariates throughout the trimesters are similar. In Summary statistics for wildfire and covariates stratified by regions and trimesters are shown in The subgroup analysis by sex and race (only white and black) are shown in https://www.healtheffects.org/about/strategic-plan). Our study is in line with this essential concern by contributing to the scientific evidence on health effects on fetus development (birth weight) due to exposure to a specific particulate source (wildfire smoke). This contribution is essential because, on an equal-mass basis, wildfire-related PM2.5 may be more toxic than ambient PM2.5 in the same region during non-fire periods,2.5 elemental constituents in Massachusetts and Connecticut in the US. They found that an interquartile range increase in exposure was associated with low birth weight for various PM2.5 chemical constituents, including elemental carbon (13% increase in risk). Particulate carbon  has been the most indicated as trace elements of wildfires.Estimating the health effects of different particulate matter sources and constituents is suggested as a priority by the environmental health community. For example, this is one of the priorities listed by the Health Effects Institute (HEI) in the strategic plan for understanding the health effects of air pollution  of the true association between wildfire exposure and birth weight. Third, our results only suggest an association between exposure to wildfire and low birth weight – this approach does not capture the cause-effect between wildfire exposure and birth weight.Our study, however, has several strengths. First, our findings add strength to the evidence that mother's exposure to air pollution during pregnancy may cause low birth weight. In our study, we found this evidence accounting for wildfire-related air pollution, which, to our knowledge, was not explored to date in South America. Second, our sample was relatively large, including more than 1.5 million records of birth nationwide for over 18 years. This is the study with the largest sample size and the largest study period in Brazil. Due to this large sample, our statistical analysis had enough power to be able to detect differences in outcome measures. Third, the birth dataset included many individual-specific variables for the assessment of potential confounding.In conclusion, this study demonstrates that wildfire exposure may increase the risk of infants with low birth weight. Given that wildfire is a growing problem in several regions worldwide, especially in Brazil – a fire-prone region, the epidemiological evidence shown in our study should be of great concern to the public health community and policymakers.WJR contributed to the conceptualization, methodology, data curation, funding acquisition, and prepared the original draft. HA contributed towards the exposure assessment, health analysis, and review and editing of the manuscript. MDA participated in reviewing and editing of the manuscript and contributed towards the exposure analysis. JDS contributed towards the methodology and reviewing and editing of the manuscript.Researchers who are interested in using the data should contact Dr. Requia (weeberb.requia@fgv.br). There will be an assessment for the data request by a committee including stakeholders from the Brazilian Ministry of Health. If approved by the committee, the researcher can gain access to the data.The Lancet Group takes a neutral position with respect to territorial claims in published maps and institutional affiliations.None.


In sub-Saharan Africa, infant anemia, stunting and low birth weight remains major public health problems. It is unclear whether prenatal iron supplementation and/or deworming can reduce the risk of infant stunting, anemia and low birth weight. The aim of this study was to investigate the relationship between iron supplementation and/or deworming and stunting, anemia, and low birth weight in infants under two years of age in sub-Saharan Africa.Our studies examined pooled data from Demographic and Health Surveys (DHS) in twenty-three African countries collected between 2014 and 2020. childhood anemia and stunting in infants under the age of two were the primary outcomes. Iron supplementation and deworming during prenatal visits were the main exposure variables. A multivariate logistic regression model was used to investigate these relationships.The prevalence of stunting was 29.9%, severe stunting was 10.6%, childhood anemia was 74.3%, childhood severe anemia was 3.2%, and low birth weight was 16.4%, respectively. The use of prenatal iron supplementation alone was associated with a significant reduction of childhood anemia [aOR (95% CI) = 0.9 (0.8–1.0)]. Prenatal deworming alone was associated with a significantly reduced risk of stunting [aOR (95% CI) = 0.7 (0.8–1.0)], childhood anemia [aOR (95% CI) = 0.7 (0.8–0.9)], and low birth weight [aOR (95% CI) = 0.7 (0.8–1.0)]. Prenatal iron supplementation plus deworming or iron supplementation (with or without deworming) were not associated with childhood anemia, infant stunting and low birth weight.In Sub-Saharan Africa, prenatal deworming alone has the potential to improve infants’ outcomes. Childhood anemia was improved with prenatal iron supplementation alone. Our recent findings indicate the necessity for prospective studies on the association between prenatal iron supplementation plus deworming and childhood anemia, stunting and low birth weight.The online version contains supplementary material available at 10.1186/s12884-023-05399-7. The nutritional health of the woman, especially before and during pregnancy, is essential for the proper development of the infant . The mothttp://dhsprogram.com/). Each survey gathered information on sociodemographic, health, and nutrition indicators from a nationally representative sample of households chosen through multistage cluster probability sampling. The survey procedures were thoroughly described [Our studies used nationally representative survey data from DHS in twenty-three Sub-Saharan African countries collected between 2014 and 2020. We used information from women's most recent live birth in the two years preceding the interview in each of these surveys. The DHS data were obtained from the DHS program website (escribed . In brieOur main outcomes are listed and defined follows: stunting in infants: proportion of infants who have a z-score for height-for-age that is less than two standard deviations below the median of the 2006 WHO reference population . Severe The ‘Iron supplementation (with or without deworming)’ variable was classified as yes or no depending on whether women took iron tablets or syrup during their most recent pregnancy. To investigate the impact of the number of days of iron intake on the outcomes, a variable was constructed. ‘Number of days of iron intake’ was defined as all women who had given birth in the previous two years and had taken iron tablets or syrup for at least two days during their past pregnancy, regardless of whether they received deworming medication. There were four categories for the variable: none, less than 60, 60 to 90, and more than 90 days of intake. The variable ‘Deworming (with or without iron supplementation)’ was classified as yes or no depending on whether women received deworming medication during their most recent pregnancy. Deworming treatment (Albendazole or Mebendazole) targets soil-transmitted helminthiases, of which the most common species infecting humans are Ascaris lumbricoides (roundworm), Trichuris trichiura (whipworm) and Ancylostoma duodenale or Necator americanus (hookworms) [The covariates and potential confounders considered were the mother’s age , Currently in union , Type of place of residence , Highest educational level , Wealth index , Number of ANC visits , Improved Water Source , Sex of infants  and infant's age in months.SPSS version 24 was used for statistical analysis. All P-values were two-tailed, and statistical significance was defined as P ≤ 0.05. Using clusters, strata, and weight statements, all analyses were corrected for complicated survey design effects. The sampling weights defined/derived from the original national surveys were used. Descriptive statistics for participants were presented as means (Standard Error (SE)) for continuous and frequencies (percent) for categorical outcomes. We utilized multivariable logistic regression, which was adjusted for country, residence area, maternal marital status, maternal education levels, drinking water sources, household wealth index, mother age, infant’s sex, and infant’s age. Potential confounders were selected based on knowledge from previous studies .In Sub-Saharan Africa Table , half ofMore than two thirds of women (79.7%) had taken iron supplements (with or without deworming) during their last pregnancy, with 36.4% reporting use for 90 days or more Table . 47.7% wFigure This study, based on nationally representative data from 36,879 mother-infant pairs from 23 sub-Saharan African countries, found that 16.4% of children were born with low birth weight, 29.9% were stunted, 10.6% were severely stunted, 74.3% were anemic, and 3.2% were severely anemic, highlighting the persistence of these outcomes in this setting. Daily iron supplementation alone during pregnancy was associated with a reduced risk of childhood anemia. Similarly, deworming alone during pregnancy was associated with a reduced risk of infant stunting, anemia and low birth weight. In contrast, the use of iron supplementation plus deworming or iron supplementation (with or without deworming) for any duration was not associated with the above-listed five infant outcomes. To our knowledge, the current study is the first sufficiently powered study to report a protective effect of prenatal deworming on infant stunting in sub-Saharan Africa.Our findings are consistent with those of China  and NepaOur findings are consistent with previous studies in which deworming (with or without iron supplementation) during pregnancy reduces the risk of infant stunting , 36 and Our findings are in contradiction with an Indian study in which iron supplementation (with or without deworming) during pregnancy reduces the risk of childhood anemia . In IndiIn contrast, our findings that prenatal deworming (with or without iron supplementation) does not have a beneficial effect on childhood anemia are consistent with a meta-analysis . BecauseThe study's strength lies in the use of pooled data from nationally representative surveys that offered a large sample of mother-infant pairs. The same standard methods were followed for the infant's anthropometric measures and hemoglobin testing. To reduce recall bias, live births from two years prior to the interview were chosen. There are several limitations to this study as well: we tried to reduce recall bias in our analyses by providing women samples of iron tablets/syrup to assist them to remember their prenatal iron intake. Other sorts of supplements, however, may be remembered by women. Because women were not randomly assigned to iron supplements, it's probable that confounding factors exist after our adjustments for several of the potential confounders.In Sub-Saharan Africa, prenatal deworming alone have the potential to improve infant’s outcomes. Childhood anemia was improved with prenatal iron supplementation alone. Strengthening health systems in countries with high prevalence of iron deficiency and parasitic infections; population health education to reduce behavioral barriers to accessing health services; and improving adherence and enabling initiation of iron supplementation as early as possible in pregnancy can all help to maximize the positive effects of these interventions. Our recent findings indicate the necessity for prospective studies in low- and middle-income countries to investigate effects of combined iron supplementation and deworming during pregnancy on childhood anemia, infant stunting and low birth weight.Additional file 1: Supplementary Table 1. Countries in Sub-Saharan Africa and the number of participants (mother and infant pairs) included in the study.


Foot-and-mouth disease virus (FMDV) can persistently infect pharyngeal epithelia in ruminants but not in pigs. Our previous studies demonstrated that persistent FMDV infection in cattle was associated with under-expression of several chemokines that recruit immune cells. This report focuses on the analysis of differentially expressed genes (DEG) identified during the transitional phase of infection, defined as the period when animals diverge between becoming carriers or terminators. During this phase, Th17-stimulating cytokines (IL6 and IL23A) and Th17-recruiting chemokines (CCL14 and CCL20) were upregulated in animals that were still infected  compared to those that had recently cleared infection (terminators), whereas chemokines recruiting neutrophils and CD8+ T effector cells (CCL3 and ELR+CXCLs) were downregulated. Upregulated Th17-specific receptor, CCR6, and Th17-associated genes, CD146, MIR155, and ThPOK, suggested increased Th17 cell activity in transitional carriers. However, a complex interplay of the Th17 regulatory axis was indicated by non-significant upregulation of IL17A and downregulation of IL17F, two hallmarks of TH17 activity. Other DEG suggested that transitional carriers had upregulated aryl hydrocarbon receptor (AHR), non-canonical NFκB signaling, and downregulated canonical NFκB signaling. The results described herein provide novel insights into the mechanisms of establishment of FMDV persistence. Additionally, the fact that ruminants, unlike pigs, produce a large amount of AHR ligands suggests a plausible explanation of why FMDV persists in ruminants, but not in pigs. Picornaviridae (genus Aphthovirus). Susceptible hosts include domestic and wild cloven-hoofed animals such as ruminants and pigs. Infection in cattle begins in the respiratory tract. During this primary infection, the virus replicates locally in the nasopharynx or lungs, depending on the route of exposure [Foot-and-mouth disease (FMD) is one of the most contagious and economically devastating viral diseases of livestock; the disease is caused by FMD virus (FMDV), a positive-sense single-stranded RNA virus of the family exposure ,2. The iexposure ,4,5,6,7.exposure .The site of persistent FMDV infection in cattle has been localized to the epithelial cells of the nasopharyngeal mucosa, including the dorsal soft palate and roof of the nasopharynx ,9,10,11.To understand the mechanisms involved in FMDV persistence, a previous study applied an experimental, hypothesis-free functional genomics and bioinformatics approach to identify candidate mechanisms based on genes differentially expressed in tissues targeted and not targeted for persistent FMDV infection  and betwThe current study provides further analysis of DEG from previously published data  collecteThe probes with differential expression at FDR ≤ 0.1 showed that 1274 and 598 known genes were upregulated and downregulated, respectively, in nasopharyngeal epithelia of transitional carriers compared to terminators. The functional analysis of the upregulated DEG using DAVID tools detected significant enrichment in an immune-related gene ontology (GO) term (GO:0006955) and seven KEGG immune processes related to infection in T cells and epithelia, immune cell migration, phagocytosis, and four KEGG signaling pathways involved in immune regulation including (1) PI3K-Akt, (2) NFκB, (3) HIF-1, and (4) Wnt signaling pathways . The dowAnalysis of the DEG gene list using the IPA pathways analysis identified the top five inferred upstream regulators for differential expression as (1) estrogen receptor 1 (ESR1), (2) beta-estradiol (an estrogen hormone), (3) KRAS, (4) dexamethasone, and (5) TNF A. Similap = 0.05) in transitional carriers than terminators. The expression level of eight genes known to be induced by AHR signaling including B7H4 [p ≤ 0.05).AHR and HIF1α compete to form heterodimers with aryl hydrocarbon receptor nuclear translocators (ARNTs). AHR was expressed at a significantly higher level (8.1-fold), and HIF1α expression was significantly downregulated by 1.8-fold in transitional carriers compared to terminators . Among ting B7H4 , CCL20 [ing B7H4 , CD8A [2ing B7H4 ,24, CD39ing B7H4 ,26, CYP1ing B7H4 , IL6 [22ing B7H4 , IL23A [ing B7H4 , and STAing B7H4  were siging B7H4 , were alp = 0.04, FDR= 0.05 and p = 0.03, respectively) in carriers than in terminators. In contrast to enhancing glucose uptake and glycolysis of HIF1A, an enzyme (ACSS3) that catalyzes the first reaction of fatty acid metabolism was upregulated 9.6-fold. HIF1A is activated by the AKT-mTOR signaling pathway via extracellular ATP and TCR signaling [HIF1 α expression is inducible by STAT3 and NFκB . Three iignaling . Althougignaling ,36 were Several genes playing a critical role in NFκB signaling were differentially expressed in nasopharyngeal tissues between transitional carriers and terminators . IKBKB ip ≤ 0.05. Complement membrane attack complexes can activate non-canonical NFκB by forming an Akt+ NIK+ signalosome on Rab5+ endosomes [On the other hand, the transcription factors, receptors, or receptor ligands in non-canonical NFκB signaling such as RELB, NFKB2/p100, CD27, LTB, LTBR, and TNFRSF8/CD30  were expndosomes . These rndosomes ,41,51.p = 0.02). These results, together with the Wnt signaling pathway significantly overrepresented by DEG in the KEGG pathway analysis  was significantly higher in transitional carriers than in terminators , and WNTanalysis , indicatp < 0.05). These results suggest that the transitional carriers expressed higher levels of both Th17 stimulatory and Th17 suppressive cytokines, which may explain why three Th17 cytokines were not upregulated in carriers.Expression levels of IL6, IL16, IL23A, IL34, and TNFSF15 were significantly higher  in transitional carriers compared to terminators . Of thesThere were also eight cytokine receptors  that had significantly upregulated expression and two (ACVR2B and SIGIRR) that were downregulated in transitional carriers . Among tThere was significant differential expression of chemokines in epithelia of transitional carriers compared to terminators. Six chemokines, CCL11, CCL14, CCL20, CXCL12, CXCL14, and CXCL13, were significantly upregulated in nasopharyngeal epithelia of transitional carriers, and one (CCL3) was downregulated . CCL11, p = 0.01 and 0.06, respectively), supports the increased recruitment of monocytes. Upregulated CCR6, the receptor of CCL20, strongly suggests increased Th17 recruitment in transitional carriers. Similarly, CCR3 (high-affinity receptor of CCL11) [p ≤ 0.05) in transitional carriers. CCR7 is expressed on naive T and B cells, central memory T cells (Tcm), and mature DC [p = 0.03). Therefore, the results of chemokines and their receptors suggest that the epithelia of transitional carriers recruited more monocytes, eosinophils, and Th17 cells, reduced recruitment of neutrophils and CD8+ T cells, decreased antigen-cross presentation to CD8+T cells, and promoted Th17 to Treg transition and macrophage M2 activation compared to terminators.Three chemokine receptors, CCR1, CCR6, and CCR7, were upregulated in transitional carriers compared to terminators . CCR1, Cf CCL11)  and CXCRf CCL11) ,95,100 aature DC . The expature DC , while Xature DC . The recp = 0.02), CD8A [p = 0.01) [p = 0.08) (p = 0.03). However, there are three Th17-suppressing DEG  [p = 0.01) in transitional carriers , CD8A , CD40L ( = 0.01) , and ThP = 0.08) . A markeNFRSF6B) ,111,112 carriers .p ≤ 0.05) in transitional carriers (Treg marker genes such as FOXP3  and IL10carriers . Four upcarriers . ADCY4 cThe expression of eight genes with immune inhibitory effects on macrophages or antigen-presenting cells (APC)  was significantly upregulated in transitional carriers compared to terminators . SignaliTransitional carriers had a generally downregulated expression of defensin genes with two genes (DEFB1 and DEFB103A) significantly downregulated by 3.4- and 10.7-fold and one defensin gene (DEFB4B) significantly upregulated by 10.8-fold in transitional carriers . DEFB103Historically, it has been reported that approximately 50% of FMDV-infected ruminants remain persistently infected 28 days after infection ,4,5; howPersistent infection does not occur in pigs , indicatThe differential expression of ELR+ CXCLs and CCL3 is consistent with the microscopic analyses of the nasopharynx of the animals included in this study, wherein there were reduced quantities of CD8+ T cells in the epithelia of transitional carriers compared to terminators . These rIL-17RA signaling in the epithelium, activated by two Th17-specific cytokines, IL17A and IL17F, is required for neutrophil recruitment. During the transitional phase, the involvement of Th17 cells in the FMDV infection of pharyngeal epithelia was strongly supported by upregulated expression of Th17-promoting cytokines and chemokines  in transitional carriers ). The list of upregulated and downregulated genes associated with each human Entrez Gene ID was analyzed with Ingenuity Pathway Analysis (IPA)  and the NCBI Functional Annotation Bioinformatics Microarray Analysis program (DAVID Bioinformatics Resources version 6.8) to identify the biological pathways significantly over-represented among DEG. The biological functions of DEG were determined based on scientific publications (included as cited references) or on the NCBI Gene database (https://www.ncbi.nlm.nih.gov/gene/ (accessed on 1 June 2022)).The methods of pathway analysis of DEG have been described . All bovp-values along with the FDR. Expression levels of genes downregulated or upregulated in transitional carriers compared to terminators are shown as negative and positive values (fold changes), respectively. Immune regulatory mechanisms especially involved in mucosal immunity and its association with ruminant physiology were also taken into consideration in the formulation of the hypothesis.The biological inferences have been described , which w


Diffusion tensor imaging (DTI) is a specialized imaging technique that measures the strength and vector of water molecule movement, which is analyzed using a variety of parameters. This method has the potential to depict pathological conditions that cannot be studied using conventional magnetic resonance imaging methods. However, most reports on DTI parameters measured only fractional anisotropy and apparent diffusion coefficient values in veterinary medicine. Therefore, we performed DTI on five adult beagles under anesthetic maintenance and analyzed the various DTI parameters obtained. After post-processing, DTI parameters were calculated along the entire spinal cord. Among DTI parameters, fractional anisotropy, relative anisotropy, and axonal diffusivity significantly decreased in the caudal direction. However, the apparent diffusion coefficient, radial diffusivity, and mean diffusivity values were not significantly correlated with vertebral levels. We provide evidence on the existence of segment-dependent DTI parameters in the canine cervical spinal cord. Therefore, comparisons of DTI parameters between lesions at different vertebral levels should be avoided unless normative data are available. Furthermore, the DTI data obtained in this study may contribute to the development of a clinical reference for spinal cord evaluation in dogs using DTI parameters.This study aimed to determine the characteristics and reference values of each vertebra in the cervicothoracic region by performing diffusion tensor imaging (DTI) scans and analyzing DTI parameters in normal Beagle dogs. In five adult Beagles under anesthetic maintenance, DTI was performed using a 1.5-T magnetic resonance imaging (MRI) scanner. Axial DTI was performed using three overlapping slabs to cover the cervical and thoracic spinal cords. After post-processing, DTI parameters were calculated along the entire spinal cord. Among DTI parameters, fractional anisotropy, relative anisotropy, and axonal diffusivity significantly decreased in the caudal direction. However, the apparent diffusion coefficient, radial diffusivity, and mean diffusivity values were not significantly correlated with vertebral levels. We provide evidence for the existence of segment-dependent DTI parameters in the canine cervical spinal cord. Therefore, comparisons of DTI parameters between lesions at different vertebral levels should be avoided unless normative data are available. Furthermore, the DTI data obtained in this study may contribute to the development of a clinical reference for spinal cord evaluation in dogs using DTI parameters. Diffusion tensor imaging (DTI) is a special imaging technique that measures the strength and vector of movement of water molecules. It has the potential to observe pathological conditions that cannot be investigated with conventional magnetic resonance imaging (MRI) methods. Normally, water molecules undergo random Brownian motion, known as isotropic diffusion. However, in vivo, especially in axonal fibers, water molecules demonstrate enhanced diffusion in a certain direction along the fibers. This is anisotropic diffusion. Because axon fibers generally exhibit stronger anisotropic diffusion when in the direction of the fiber than when perpendicular to the fiber, it is possible to measure not only the direction of the axon fiber but also the degree of myelination and demyelination due to increased or decreased anisotropy. In DTI, which measures the anisotropic diffusion coefficient, the commonly used measures are fractional anisotropy (FA), which describes the degree of directional dependence; mean diffusivity (MD) and apparent diffusion coefficient (ADC), which express the magnitude of diffusion; and relative anisotropy (RA), which describes the ratio of the anisotropic part to its isotropic part. DTI can also be used to measure parameters such as axonal diffusivity (AD), which represents the diffusivity of water parallel to the axon fiber, and radial diffusivity (RD), which represents the diffusivity of water perpendicular to the axon fiber. These parameters have been extensively studied in humans, including standardization and variation at the vertebral level ,3,4,5,6.DTI has also been investigated in veterinary medicine for its usefulness in various neurological diseases ,8,9,10. This study aimed to determine the characteristics and reference values of the spinal cord at each vertebra in the cervicothoracic region by performing DTI scans and analyzing DTI parameters in normal Beagle dogs.This prospective study strictly followed the animal care rules of the Laboratory Animal Center, Imabari Campus, Okayama University of Science, and was compliant with the Guide for the Care and Use of Laboratory Animals (8th ed.). The experimental protocols were approved by the Animal Care and Use Committee of the Okayama University of Science .2O; and end-expiratory carbon dioxide concentration, 40 mmHg). Needle catheters (22 G) were placed in the right or left cephalic veins to administer a propofol injection.Five adult Beagle dogs  free of neurological symptoms were enrolled in this study. These animals had not been included in any previous research. The dogs were housed in the Laboratory Animal Center at the Imabari Campus, Okayama University of Science, under a 12:12-h light-dark cycle . Room temperature was maintained between 24 °C and 26 °C, and humidity was between 40–60%. For inclusion, all dogs were clinically healthy based on physical examination and complete blood count, and results from biochemical testing, abdominal radiography, and echocardiography were normal. The animals were fasted for approximately 12 h prior to anesthesia. For the anesthetic protocol, propofol at a dose of 6–7 mg/kg was administered intravenously (IV), and the dogs were intubated using a tracheal tube. General anesthesia was maintained using sevoflurane  and oxygen (2 L/min) under mechanical ventilation  with a 4-channel knee matrix and 4-channel spine matrix coil. The protocol comprised an initial T2-weighted image of the sagittal plane of the cervical and thoracic cords. Transverse DTI scans were obtained.3; slice thickness, 3 mm; repetition time, 5000 ms; and b-value, 1000. Diffusion-weighting gradient schemes with 12 non-collinear directions were used.Transverse diffusion tensor images were acquired using three overlapping slabs in the same anatomic location prescribed for T2WI to cover the C1-C4, C4-C7, and C6-T2 segments . The imaTotal MRI scanning time was approximately 1 h.2. For ADC, FA, RA, λ1, λ2, and λ3 values, these data were analyzed using a workstation built into the MRI system. The λ1, λ2, and λ3 values were used to calculate DTI parameters using the following formula:Sagittal T2 images were used to identify the relevant anatomy. ADC, FA, RA, and three types of diffusion eigenvalue  maps were created using the package built on the MRI system. To measure the ADC, FA, RA, λ1, λ2, and λ3 values, the regions of interest (ROIs) were placed manually on the spinal cord in all transverse images with special care to include the least amount of cerebrospinal fluid . The sizEach DTI value was averaged for each vertebral level.s) was calculated. A p-value < 0.05 was considered significant. A correlation coefficient >0.5 was defined as a positive correlation, whereas that <−0.05 was defined as a negative correlation.All DTI values are represented as mean ± standard deviation. Statistical tests were performed using commercially available statistical analysis software . The Spearman’s rank test was used to analyze the relationships between DTI values and body weight. Additionally, the correlation between the DTI values for the spinal cord at each vertebra was evaluated using the Spearman’s rank correlation coefficient, and the correlation coefficient . ADC, RD, and MD values were not significantly correlated with vertebral levels.All DTI data are summarized in The maximum and minimum FA values in the DTI parameters for each vertebra are indicated by the C3 and T2 segments, respectively A. The maThis study demonstrated that DTI parameters are inconsistent throughout the cervicothoracic cord. Among DTI parameters, FA, RA, and AD values significantly decreased in the caudal direction. Generally, in cervical segments, the ratio of white-to-gray matter is the highest cranially and then gradually decreases as it progresses caudally along the spinal cord . In a prTo our knowledge, this study is the first to evaluate canine DTI parameters at the cervical and upper thoracic vertebral levels as previous studies have been conducted on humans and rodents. Consistent with the findings of this study, a decreasing trend in FA, RA, and AD values toward the posterior part of the cervical spinal cord has been reported in some human studies ,3,4,5,6.In addition to segment-dependent variation, age has been reported to influence DTI parameters in humans. Fiber number, axonal diameter, and fiber density decrease with age , resultiIn this study, cardiac triggering/gating was not employed because of the significant increase in scan time. This decision was based on previous reports that cardiac triggering/gating does not improve DTI quality in the human spinal cord ,6. HowevThis study has a limitation: the whole spinal cord was evaluated without distinction between white and gray matter. It was difficult to identify the spinal cord structures from the blurred images obtained using a 1.5-T MRI. In a human study using a 3-T MRI, it has been reported that, in the normal cervical spinal cord, FA values in the white matter, but not gray matter, were reduced caudally . Fiber dIn summary, an initial understanding of dog DTI parameters was described in normal cervical and upper thoracic spinal cords, which showed a clear trend of decreased FA, RA, and AD in the caudal direction. Because DTI parameters in the canine spinal cord exhibit segment-dependent variability, comparisons of DTI parameters between lesions at different vertebral levels should be measured with this considered. We believe our results will contribute to the development of a clinical reference for canine spinal cord evaluation using DTI parameters.