Nrf1) and fission (Drp1 and Fis1) genes was lower in the placenta from the lightest females, whilst biogenesis-related gene Tfam was greater in the placenta of the lightest male fetuses. In addition, placental morphology and steroidogenic gene (Cyp17a1 and Cyp11a1) expression were aberrant for the lightest females, but glucose transporter (Slc2a1) expression was lower in only the lightest males versus their heaviest counterparts. Differences in intra-litter placental phenotype were related to changes in the expression of hormone-responsive (androgen receptor) and metabolic signaling  pathways. Thus, in normal mouse pregnancy, placental structure, function, and mitochondrial phenotype are differentially responsive to the growth of the female and male fetus. This study may inform the design of sex-specific therapies for placental insufficiency and fetal growth abnormalities with life-long benefits for the offspring.Fetal growth depends on placental function, which requires energy from mitochondria. Here we investigated whether mitochondrial function in the placenta relates to the growth of the lightest and heaviest fetuses of each sex within the litter of mice. Placentas from the lightest and heaviest fetuses were taken to evaluate placenta morphology (stereology), mitochondrial energetics (high-resolution respirometry), mitochondrial regulators, nutrient transporters, hormone handling, and signaling pathways (qPCR and Western blotting). We found that mitochondrial complex I and II oxygen consumption rate was greater for placentas supporting the lightest female fetuses, although placental complex I abundance of the lightest females and complexes III and V of the lightest males were decreased compared to their heaviest counterparts. Expression of mitochondrial biogenesis  that relate to fetal growth in the litter.  A successful pregnancy strongly depends on balancing resource allocation between the genetically determined fetal drive for growth and the mother who needs resources to support the pregnancy state. As a functional interface between mother and fetus, the placenta plays a key role in balancing fetal and maternal resource needs. Amongst its plethora of functions, the placenta executes the metabolism and secretion of hormones that have physiological effects on the mother and fetus and transfers nutrients and oxygen from the mother to the fetus . Thus, iTo enable normal placental growth and function, the placenta depends on the energy supplied by mitochondria. Mitochondria are the primary source of ATP, which is produced by oxidative phosphorylation (OXPHOS) using substrates derived from β-oxidation and the tricarboxylic acid cycle. ATP is used by the placenta to fuel growth and placental endocrine and transport functions. Mitochondria are also the place within the cell where steroidogenesis occurs; they contain several key proteins and enzymes such as steroidogenic acute regulatory protein (STAR) and cholesterol side-chain cleavage enzyme (CYP11A1), which are required for glucocorticoid and sex-steroid synthesis . MitochoIn this study, we employed an integrative approach to evaluate placental morphology, mitochondrial OXPHOS capacity and mitochondrial regulator expression  complexes and biogenesis and fission-fusion regulators), in relation to the growth of the lightest and heaviest female and male fetuses within the litter of normal wildtype mice. Importantly, since the mouse is a polytocous species, normal variation of fetal weight is expected within the litter, even in a normal, healthy gestational environment. We also examined the activity of signaling pathways governing placental growth and metabolism, as well as the expression of nutrient transporter and steroid hormone handling genes to further understand how placental phenotype is modulated by fetal weight for each sex within the litter. Analyses were conducted on the labyrinth zone (Lz) of the mouse placenta as it is responsible for controlling the transport of nutrients, oxygen, and hormones from mother to fetus.ad libitum water and chow food  during the study. At 4 months of age, females were mated overnight and the day a copulatory plug was found was designated as the gestational day (GD) 1. On GD18, pregnant dams were killed by cervical dislocation, uteri were recovered, and fetuses and placentas were cleaned from fetal membranes. All fetuses and placentas from the litter were weighed. Litter size ranged from 6 to 10 pups, with a mean ± SEM of 8.00 ± 0.34 fetuses and overall, the percentage of female and male fetuses was 48.3% and 51.7%, respectively (2O and pH 7.5) and frozen at −80 °C until respirometry analysis. The remaining half of the Lz was snap-frozen and stored at −80 °C for molecular analyses (RT-qPCR and Western Blot). Placental samples from the additional five litters were kept whole, placed into 4% paraformaldehyde, histologically processed, and used for structural analysis. The fetal brain and liver were dissected and weighed from each fetus. Fetal tails were kept for sex determination by detection of the Sry gene using the Taq Ready PCR system (Sigma), specific primers  and agarose gel electrophoresis.All experiments were performed under the U.K. Animals (Scientific Procedures) Act 1986 after ethical approval by the University of Cambridge. A total of 13 C57BL/6 J virgin female mice were housed in the University of Cambridge Animal Facility using a 12/12 dark/light system and received ectively . For sev2+, 1 mM free Mg2+, 20 mM imidazole, 20 mM taurine, 50 mM K-MES, 0.5 mM dithiothreitol, 6.56 mM MgCl2, 5.77 mM ATP, and 15 mM phosphocreatine, pH 7.1) containing saponin  for 20 min on ice. Samples were then washed by three 5-min washes in respiratory medium MiR05  in ice to remove all endogenous substrates and contaminants. Oxygen concentration (μM) and flux per tissue mass (pmol O2/s/mg) were recorded in real-time using calibrated oxygen sensors and Datlab software . Respiratory rates were corrected for instrumental background by DatLab, considering oxygen consumption of the oxygen sensor and oxygen diffusion out of or into the oxygraph chamber measured under experimental conditions in miR05 medium without any tissue present.High-resolution respirometry  was used to assess the capacity for respiratory substrate use and ETS function. Cryopreserved Lz samples (10–15 mg) were gently thawed in ice-cold sucrose solution . Samples were then permeabilized in respiratory medium BIOPS . Next, ADP (5 mM), pyruvate (20 mM), and glutamate (10 mM) were added to obtain complex I oxygen flux under OXPHOS state (CIP). Then, succinate (10 mM) was added to provoke complex I and II dependent OXPHOS (CI + ÍIP). Fatty acid oxidation (FAO) was calculated immediately after ADP addition . Trifluoromethoxy carbonyl-cyanide phenylhydrazone  was added to obtain total ETS capacity (uncoupled state). To activate complex IV (CIV) dependent respiration, the first three complexes of the ETS were inhibited by adding rotenone , malonic acid and myxothiazol  and antimycin A . Sodium ascorbate (2 mM) and N, N, N′, N′-tetramethyl-p-phenylenediamine  were then added to stimulate CIV supported respiration, which was then inhibited by adding sodium azide (200 mM). ETS excess capacity was calculated using the formula: 1- CI + IIP/total ETS (1-P/E). Cytochrome c (10 μM) was added to check mitochondrial membrane integrity and data were excluded if respiration increased by >30%. All substrates used were at their saturating concentrations to assess maximal mitochondrial respiratory capacity.A substrate-inhibitor titration protocol was performed under the presence of octanoylcarnitine and using approximately 10–15 mg of permeabilized Lz tissue placed into each oxygraph chamber. Briefly, complex I substrate malate (2 mM) was added first to determine LEAK respiration , and Ubiquitin C (Ubc)), which were stable in the placental Lz between the lightest and heaviest fetuses of each sex. Data were then displayed relative to the average mRNA expression value for the heaviest fetus of each sex.Placental Lz RNA was extracted using the RNeasy Plus Mini Kit  and the quantity of RNA obtained was determined using a NanoDrop spectrophotometer . A total of 2 μg per sample was reverse transcribed using high-capacity cDNA reverse transcription kit  according to the manufacturer’s instructions. Three dilutions of each cDNA sample  were run as a triplicate along with non-template controls in the 7500 Fast RT PCR thermocycler System  for gene expression quantification using gene-specific primer pairs  and SYBRMorphology of the Lz was assessed by double-labeling placental sections with cytokeratin and lectin antibodies to identify trophoblast and fetal capillaries, respectively. Details about the staining protocol have been described in detail elsewhere . Stainedt-tests. Retrospective comparisons between the lightest females and lightest males and heaviest females and heaviest males within the litter were also performed by paired t-tests. Relationships between data were undertaken using Pearson’s correlation coefficient (r). Values are expressed as individual data points and/or mean ± SEM and P-values < 0.05 were considered statistically significant. The number of samples per group for each analysis is shown in each figure and described in the legends of figures and footnotes of tables.Statistical analyses were performed using GraphPad Prism version 8  and SAS/STAT . To check whether each data set was normally distributed, Shapiro–Wilk tests were performed and identified that all displayed a normal Gaussian distribution. To compare conceptus biometry between males and females across different litters, linear mixed model analyses were performed (which considers each fetus as a repeat in the litter) and used the number of viable pups per litter as a covariate. Our primary objective was to assess differences between the lightest and heaviest fetuses for each sex within the litter. To achieve this, differences in placental structure, mitochondrial respiration, and gene and protein expression between the heaviest and the lightest fetuses of each sex within the litter were assessed by paired P = 0.06, relative to body weight), including the fetal brain to the liver ratio . Similarly, when data were separated into females and males, again no correlation was detected . However, when data were segregated to only assess the heaviest and lightest fetuses per sex within the litter, a positive correlation between placental and fetal weight was found for the lightest females , but not for the lightest males  or the heaviest fetuses of either sex. These data suggest that the placenta may be supporting the growth of the female and male fetuses in different ways within the litter.Placental weight was not significantly correlated with fetal weight when analyzing all conceptuses within the litter collectively , and the mean weight difference between them were similar for females and males . Fetal brain and liver weights as a proportion of body weight did not vary, which suggests that the lightest fetuses are symmetrically smaller when compared to the heaviest fetuses . While CIP state was not different between the lightest and heaviest fetuses of either sex (P = 0.01); a difference that was not observed for the males . In addition, calculation of 1-P/E indicated that ETS excess capacity was lower in the lightest females compared to the heaviest (P = 0.03), again a difference not identified for the males , and mitochondrial fission genes, dynamin-related protein (Drp1) and Mitochondrial fission factor (Fis1), was lower in the Lz of the lightest females, when compared with the heaviest females (Tfam), a mitochondria biogenesis transcription factor gene, was greater in the Lz of the lightest males versus the heaviest males , biogenesis, fusion and fission genes, and additional mitochondrial regulatory proteins in the placental Lz of the lightest and heaviest fetuses for both sexes . These a females . Whereasst males . Mitochost males . In addist males . Howeverst males .Slc2a1 and Slc2a3), amino acid , solute carrier family 38, member 4 (Slc38a4), Solute carrier family 7, member 5 (Slc7a5) and Solute Carrier Family 3 Member 2 (Slc3a2)) and lipids fatty acid transporter member 1–6 ) were quantified in the placental Lz zone by RT-qPCR , and cytochrome P450 family 17 subfamily A member 1 (Cyp17a1), glucocorticoid metabolism (Hsd11b1 and Hsd11b2), and steroid hormone signaling (estrogen receptor beta (Esr2) and androgen receptor (Ar)) in the placental Lz using qPCR. These analyses showed that the expression of Slc2a1 mRNA was ~20% lower for the lightest compared to the heaviest males (P = 0.021), however, this difference was not observed for the lightest versus the heaviest females. In addition, no differences were found between the lightest and the heaviest fetuses within the litter for any of the other nutrient transporter genes quantified in either fetal sex (Cyp11a1 was ~63% greater (P = 0.038), while Cyp17a1 ~ 20% lower (P = 0.035) in the lightest compared to the heaviest female fetus, with no differences in these steroidogenic genes detected in the males (Ar) a steroid-hormone activated transcription factor was ~91% greater (P = 0.046) in the lightest compared to the heaviest males only , and Star in the Lz were not different between the lightest compared to the heaviest fetuses in either sex.Since the energy provided by mitochondria helps to fuel placenta transport and endocrine function, we evaluated whether variations found in mitochondria functional capacity  are associated with the expression of nutrient transporter and steroidogenic genes between the lightest and heaviest for each sex within the litter. In particular, the mRNA expression of key transporters for glucose solute carrier family 2 facilitated glucose transporter member 1 and 3 , however, this was not related to a significant change in activation of AMPKα , but no difference was found for the females . In addition, we evaluated the expression of imprinted genes Placental specific insulin growth factor 2 (Igf2P0) and Dlk1, which are involved in feto-placental growth and allocation of maternal resources [To provide information related to the differences in placental morphology and mitochondrial function between lightest and heaviest fetuses, the abundance of key growth and metabolic signaling proteins, namely protein kinase B (AKT), AMPKα , p44/42 mitogen-activated protein kinase (MAP) (ERK1/ERK2), p38 MAPK and peroxisome proliferator-activated receptor-gamma (PPARγ) were evaluated by western blotting . The abu females . The abu females . Interesesources , 31, andP < 0.05). The placenta of the heaviest males in the litter was also greater by ~13% when compared to the heaviest females (P = 0.04), with a tendency for this to also vary with sex for the lightest female littermates (P = 0.054). The placental expression of glucose  and lipid  transporter genes were also differentially expressed between the lightest (but not heaviest) male and female fetuses of the litter. Placental respirometry rates associated with CIOxphos  and with CI + CIIOxphos/Total ETS  together with biogenesis  and dynamic  genes were all greater in the lightest males compared to the lightest females. Meanwhile, biogenesis genes Pparγ  and Tfam  were decreased in the heaviest males compared to females. Finally, the expression of the steroidogenic gene Cyp11a1 was lower  in the lightest males compared to females and in heaviest fetuses, gene expression of Cyp17a1  was decreased in males compared to females. There was also no effect of fetal sex in the placental Lz morphology of the lightest and heaviest fetuses.To gain further insight into the intra-litter differences in placental phenotype, retrospective comparisons between the lightest females and lightest males and heaviest females and heaviest males within the litter were performed . These dIn line with other studies, this study in mice showed that feto-placental weight is on average, greater for males compared to females. Furthermore, the relationship between fetal weight and placental weight varied for each fetal sex within the litter. The principal aim of this study was therefore to understand how fetal weight differences in normal physiological mouse pregnancies relate to placental function in the two sexes separately. We showed that placental mitochondrial functional capacity does indeed alter with respect to natural differences in the weight of females and male fetuses within the litter. The placental Lz of the lightest female and male fetuses showed the altered abundance of ETS complex proteins and mitochondrial biogenesis genes when compared with their respective heaviest counterparts, however, the specific nature of these changes differed in females and males. Moreover, the morphology, respiratory capacity, mitochondrial fission, and abundance of misfolded protein regulators of the placental Lz differed between the lightest and heaviest females, but not males. Furthermore, the level of nutrient (glucose) transporter genes varied between the lightest and heaviest males, but not females, whereas the ability to produce steroid hormones (informed by expression of steroidogenic enzyme genes) differed only between the lightest and heaviest females within the litter. There were also changes in the expression of hormone-responsive genes, and growth and metabolic signaling proteins in the placental Lz between the lightest and heaviest fetuses of each sex. Despite dissimilarities in the changes seen for each sex, the average weight difference between the lightest and heaviest fetuses was similar for both sexes. Together, these data suggest that in normal mouse pregnancy, placental structure, function, and mitochondrial phenotype appear to respond differentially to the genetically determined growth demands of the female and the male fetus .Slc38a2) by the lightest versus the heaviest placentas [Slc38a2 gene causes FGR [In our study, placental efficiency, and other fetal biometry parameters  were not different between the lightest and heaviest fetuses of each sex. This is relevant since placental efficiency indicates the capacity of the placenta to support fetal growth and alterations in this measure, as well as the symmetry of fetal body growth, enhances the risk for chronic diseases in later life through developmental programming . Similaruses FGR . Similaruses FGR . Variatiuses FGR . Howeveruses FGR .While there was no difference in placental fetal capillaries, trophoblast volume or barrier thickness between the lightest and heaviest of either fetal sex, maternal blood space volume, and placenta surface area were lower in the lightest compared to the heaviest female fetuses. These differences in placental structure suggest mal-perfusion of the placenta of the lightest females and would be expected to decrease the delivery of nutrients and oxygen to the fetus, and could explain the weight discrepancy with the heaviest females in the litter. Indeed, previous studies in pigs have shown that compared to placentas supporting fetuses weighing closest to the litter mean, placentas supplying the lightest fetuses within the litter have impaired angiogenesis . MoreoveTfam, as well as elevated complex I and IV dependent respiration in the placenta [Nrf1, fission regulator genes Drp1 and Fis1, and mitochondrial complex I protein were lower, yet complex I LEAK and complex I + II OXPHOS rates were greater in the placenta supporting the lightest compared to the heaviest females. Whereas in males, biogenesis gene Tfam was greater, yet mitochondrial complexes III and V proteins were lower and LEAK and OXPHOS rates were not different in the placenta supporting the lightest compared to the heaviest males. Therefore, for both sexes, the placentas of the lightest fetuses appear to increase mitochondrial respiratory efficiency , although the underlying mechanisms and extent to which this may occur are different for females and males. Indeed, our results suggest that mitochondria in the placenta sustaining the lightest female fetuses in the litter are more responsive to adaptative mechanisms, as they exhibited increased mitochondrial respiration rates. This enhanced adaptive response may have been beneficial in providing the energy to sustain the expression of glucose transporters for the lightest fetuses. Previous work has demonstrated there is a reduced abundance of all mitochondrial complexes and lower OXPHOS respiration rates in placental trophoblast from obese women who deliver high birth weight babies [Previous studies have shown that in human pregnancies associated with FGR, although mRNA expression of ETS complexes  is lower, there is higher mitochondrial DNA (mtDNA) content and higher oxygen consumption related to mitochondrial bioenergetics in the placenta . Similarplacenta . These dt babies . Moreovet babies . Thus, tIn the lightest female, but not lightest males, there was lower CLPP protein abundance when compared to the heaviest female fetuses of the litter. CLPP is also decreased along with mitochondrial complex abundance in the placenta of preeclamptic women delivering FGR babies . The bioCyp11a1 while lower Cyp17a1 gene expression in the lightest compared to the heaviest female fetus, would be expected to enhance the synthesis of the steroid hormone precursor pregnenolone, but also limit the synthesis of sex steroids. Indeed, other work on the placenta has shown that mRNA expression of steroidogenic enzymes is associated with the synthesis of steroid hormones [The placental expression of key steroid synthetic enzyme genes was differentially altered between the lightest and heaviest females only. The greater hormones . CYP11A1hormones , 42. In hormones . Thus, fhormones . Intereshormones , 46. Addhormones . Thus, dThe mechanisms underlying the differences in intra-litter placental mitochondrial function for each fetal sex are unknown. However, intra-litter differences in placental morphology and mitochondrial functional capacity likely stem from variations in the abundance of AMPK, AKT, and PPARγ proteins between the lightest and heaviest female and male fetuses . IncreasSlc2a1, encodes GLUT1) in their placenta, when compared to the heaviest males. Insulin is a major fetal growth factor that signals via AKT to mediate its metabolic effects. Reduced activation of AKT in the placenta of the lightest males may therefore reflect reduced fetal insulin signaling to the placenta [Slc2a1 gene in the lightest relative to the heavier male fetus may explain their fetal growth discrepancy.Activation of AKT (phosphorylated AKT protein) was lower in the Lz of the lightest compared to heaviest male fetuses only. The AKT–mTOR signaling pathway plays a crucial role in the regulation of placental transport function and it was shown to be upregulated in pregnancies from obese women delivering LGA babies  and downplacenta . These dplacenta . Moreoveplacenta . Conversplacenta . Since gSlc7a5 and Slc3a2 genes, respectively) and plays a key role in the control of fetal growth [Differences in placental phenotype for each fetal sex may also relate to changes in PPARγ protein abundance; PPARγ was greater in the lightest females, but lower in the lightest males compared to their respective heaviest counterparts. In humans, PPARγ expression was found to be reduced in the placenta of SGA fetuses and to associate positively with fetal and placental weights within this subpopulation . In addil growth . PPARγ il growth . While nl growth . Hence, Igf2 [paternally expressed 3 (Peg3), zinc finger, imprinted 1 (Zim1), pleiomorphic adenoma gene-like 1 (Igf2), imprinted maternally expressed transcript (H19), and Zac1 [Igf2 and H19) affects placental endocrine capacity in females and males differently [Igf2P0 and Dlk1, imprinted genes reported to be linked with feto-placental growth and PPARγ, respectively [Slc38a4, an imprinted gene that was measured to inform on alterations in placental amino acid transport capacity, also did not vary between the lightest and heaviest fetuses of each sex. Thus, further work is required to ascertain the specific contribution of imprinted genes to differences in placental phenotype between the lightest and heaviest fetuses for each sex in the litter.Differences in placental phenotypes and signaling pathways, like PPARγ with fetal weight in the two sexes, may be related to imprinted genes. Other work which did not assess the fetal sexes separately and also instead assessed placental phenotype between the lightest and heaviest placentas within the litter found altered expression of imprinted genes, including Igf2 . Moreoveand Zac1 . Finallyferently . Howeverectively , 65, 66 Previous work has shown there are ontogenic changes in placental Lz morphology, function, mitochondrial respiration, and mitochondrial-related regulators that support the growing demands of the fetus during normal late mouse pregnancy . In thisin vivo transport assessments [Our study has clear strengths. It provides a comprehensive analysis of the structural, functional, mitochondrial, and molecular differences in the isolated transport Lz for the lightest and heaviest fetuses of each sex in litters of normal, healthy pregnant mice. However, the Lz is composed of numerous cell types, and the contribution of each cell population to the specific placental alterations seen is unknown. We also do not know if there are alterations in the endocrine junctional zone, which is also important for the support of fetal growth . Moreoveessments  or hormoessments . FinallyIn summary, our data show that the placental transport zone (Lz) adopts different strategies, at the level of morphology, nutrient transport, steroid handling, and mitochondrial function to support the growth of the lightest and the heaviest fetuses within the litter in normal physiological mouse pregnancy. These adaptations are likely mediated via metabolic  and endocrine cues  within the fetus that trigger signaling pathways  in the placenta, initiating pleiotropic effects. Further work is required to test the mechanisms underlying phenotypic differences in the placenta and to ascertain the relevance of our findings for pregnancies with adverse conditions, such as maternal malnutrition, obesity, or reduced oxygen availability where the maternal ability to provide resources to the fetus for growth are constrained. From a clinical perspective, our data may be important for understanding the pathways leading to placental insufficiency and fetuses not reaching (FGR/SGA) or exceeding their genetically determined growth potential (LGA). They may also have significance in understanding the discordance in weight and perinatal outcomes between babies of multiple gestations in women. Moreover, since the spectrum of pregnancy outcomes and the factors causally involved are likely to be many, determining how placental phenotype interacts with the weight of female or male fetuses within normal mouse litter may be useful to the design of sex-specific therapeutic agents to improve pregnancy outcomes in humans. This is highly relevant given the profound impacts of fetal growth and pregnancy complications on the immediate and life-long health of the child.ESP, JLT, and ANSP designed the study. ESP, JLT, and DPC performed the experiments and analyzed and graphed the data. ESP and ANSP wrote the paper. All authors contributed to data interpretation and performed final editing checks and approved the final manuscript.All data are available upon reasonable request.The authors have declare that no conflict of interest exists.Supplementary_Table_1_ioac056Click here for additional data file.


This study aimed to evaluate the safety and efficacy of innovative retinoic acid (RA) eluting stents with bioabsorbable polymer. Sixty stents divided in ten groups were implanted in the iliac arteries of 30 rabbits. Two polymers  and “B”, polylactic acid), and three doses  of RA  were used on cobalt chromium stents (Rontis Corporation), one group of bare stent (C), one group (D) of Everolimus eluting stent , and two groups of Rontis Everolimus eluting stents coated with polymer A (EA) and B (EB). Treated arteries were explanted after 4 weeks, processed by methyl methacrylate resin and evaluated by histopathology. None of the implanted stents was related with thrombus formation or extensive inflammation. Image analysis showed limited differences between groups regarding area stenosis . Group BH had lower intimal mean thickness than AH . Stents eluting RA, a non-cytotoxic drug, were not related with thrombus formation and had an acceptable degree of stenosis 4 weeks post implantation. RA dose and type of polymer may play role in the biocompatibility of the stents. The devices release an antiproliferative drug locally, thereby acting against the proliferation of smooth muscle cells, directly in the endothelium preventing restenosis. Currently used drugs on DES include paclitaxel, a cytotoxic drug and rapamycin (sirolimus) and its derivatives such as everolimus, a group of cytostatic drugs2. Although the wide use of DES significantly reduced restenosis compared with bare metal stents (BMS), restenosis still is a major complication3. Moreover, the late and very late stent thrombosis, the need for dual antiplatelet therapy and the chronic inflammatory response to polymer and/or the cytotoxic and cytostatic drugs that are currently in use, justify further investigation and development of technologies which will improve the safety and efficacy of DES4.Implantation of new-generation drug eluting stent (DES) is the standard treatment strategy in percutaneous coronary intervention5. RA is a product of oxidation of retinol or retinal. The main circulating form of RA in humans is all-trans-retinoic acid, while 13-cis- and 9-cis-isomers are also present in small concentrations. RA constitutes the drastic form of vitamin A that acts through specific nuclear receptors [retinoic acid receptors (RAR) and retinoic X receptors (RXR)], affecting the expression of specific genes5. Retinoids contribute to the homeostasis of cell differentiation and proliferation and have been used successfully in the treatment of diseases related to cell proliferation as well as in the treatment of various malignancies, such as acute promyelocytic leukemia6. Studies have shown that RA has a regulatory effect on the maturation and differentiation of vascular cells, inhibition of cell proliferation, migration, inflammation and extracellular remodeling synthesis of vascular smooth muscle cells (VSMCs)7.Retinoid acid (RA) belongs to the group of retinoids, which are derivatives of vitamin A9. Moreover, local application of RA in blood vessels reduced neointimal hyperplasia in experimental models of restenosis11. The doses however that are needed to obtain these effects may cause serious side effects12. We hypothesized that local delivery of relatively low doses of retinoic acid using stents as the delivery platform/device, may result in reduction of restenosis after stent implantation, potentially without causing the inflammatory effects that are common with stents coated with cytostatic or cytotoxic drugs such as rapamycin and its derivatives and paclitaxel. In order to test this hypothesis, we developed innovative stents coated with bioabsorbable polymers and all-trans-retinoic acid. The aim of this study was the evaluation of the safety and efficacy of stents that release retinoid acid, in a rabbit iliac artery model.Oral administration of RA resulted in reduction of vascular stenosis in experimental in vivo stenosis modelsAll the stents were successfully expanded into the target arteries and the apposition was confirmed in all cases by angiography. The apposition was also evaluated by OCT after implantation in four stents. A typical OCT image of a RA coated stent of the BM group is presented in Fig. One of the animals that had received two bare metal stents did not survive until the 4-week study period and therefore was not evaluated in this study. There were no gross lesions noted at the necropsy.None of the implanted stents was related with thrombus formation or necrosis of the vascular wall. Further, the arterial wall did not appear dissected, and only multifocal ruptures of the elastic laminae  were observed. The host reaction was considered low with all the treatments. There were unstained particles detected within the tunica intima in association with macrophages and giant cells in all the groups, except for group C (BMSs).Markers of local inflammation were included in the histological analysis of target/stented arterial segments. All implant sites showed a low-level arterial reaction consisting of one or more of the following parameters, summarized in Table The summarized average scores for all evaluated parameters and the total arterial reaction are summarized in Table The total arterial reaction average score was lower with treatments BL, BM and BH, compared to groups AL, AM, AH. The decrease in average score was mostly due the less severe vascular wall findings and the lack or lower severity grade of peri-strut hemorrhages in BL, BM and BH groups. EF and EG groups showed less total arterial reaction score than C group. Group EF showed slightly higher average score than group EG.The results of image analyses are summarized in Table 2 respectively, p = 0.0235). Moreover, Group AH showed the highest Stenosis (26.2%) and Intimal Mean Thickness values (105.1 μm2). Also, Group AH showed statistically significant difference with Group D for stenosis .Group AH showed statistically significant difference with Group BH for Intimal Mean Thickness . No statistically significant difference was observed between Groups BL and BH for all parameters measured.Group AL showed statistically significant difference with Group AH for intimal mean thickness  and Stenosis  and between Groups EG and C  and Groups EF and C for Intimal Mean Thickness .No statistically significant difference between Groups EF and EG was present for Medial Area, Intimal Area, Stenosis and Intimal Mean Thickness. No statically significant difference was observed between Groups EF and EG and Group D, for none of the tested parameters. However, a statistically significant difference was observed between Groups EG and C for Intimal Area  none of the implanted stents was related with thrombus formation or necrosis of the vascular wall; therefore, 4 weeks post implantation, none of the treatments seem to increase the risk of a thrombotic event; (2) all the groups presented multifocal ruptures of the elastic laminae , but without further medial/transmural rupture, which suggested a low risk of arterial transmural dissection induced by the stent implantation at this time-point; (3) the host reaction associated with the implant struts was considered low with all the treatments with only minimal to slight differences among them; and (4) the dose of RA and the type of the polymer may play a role in the biocompatibility of the device.13. All-trans-retinoic acid induces a dose-dependent inhibitory effect on the proliferation of human arterial smooth muscle cells in vitro15. A similar dose-dependent inhibition of the growth activity was also observed in human vein smooth muscle cells9.Retinoids exhibit complex effects on vascular smooth muscle cells including regulation of proliferation, growth, differentiation and migration, as shown by in vitro and in vivo experimentsLactobacillus casei cell wall extract8. Similarly, oral administration of RA (10 mg/kg/day) significantly mitigated the stenosis extent of vein grafts in a rabbit autogenous vein grafts model after 2, 4 and 8 weeks of treatment9. Furthermore, Leville et al. reported that oral administration of RA (10 mg/kg/day) decreases cell proliferation, increases apoptosis, and decreases the expression and activity of matrix metalloproteinases in an animal model vein bypass grafting17. Some years later, Gregory et al. used peripheral bypass graft able to immobilize RA through porous poly membranes in a rat aortic bypass model. Results indicated reduced intimal hyperplasia, percent stenosis, and greater lumen area10.Experiments with various in vivo stenosis models have shown that oral administration of retinoic acid resulted in reduction of stenosis. Oral administration of RA (30 mg/kg) for 14 weeks, in a mouse model of coronary stenosis, resulted in significant suppression of inflammation and reduced the incidence of coronary artery stenosis of mice in which inflammation and stenosis were induced with 18. Similarly, DeRose et al. showed inhibition of vessel remodeling as measured by increases in luminal diameter, using 10 mg/kg all-trans retinoic acid19. In another study, Wiegman et al. investigated the effect of orally delivered RA  in plaque size and vessel geometry after balloon angioplasty performed on rabbits with focal femoral atherosclerosis. Histomorphometry measurements showed that per os administration of RA resulted in larger lumen area. However, no differences in absolute plaque area were observed20.Previous studies have examined the role of RA in reducing neointimal formation after vascular injury. Miano et al. showed that orally administered all-trans-retinoic acid increased the luminal area (by 35% to 37%) after balloon injury in rat carotid arteries12. Herdeg et al., after performing plaque formation in rabbits right carotid artery and undergoing balloon angioplasty, administered RA locally  by using a double-balloon catheter. Results showed a non-significant limitation of restenosis formation 4 weeks after intervention (p = 0.0937)11. In another experimental model of restenosis after balloon injury, citrate-based polyester membranes containing retinoic acid, were wrapped around the carotids of rats that had undergone balloon injury. The membranes inhibited neointimal formation after balloon injury and decreased restenosis21.Other studies involved local administration of RA aiming to prevent the adverse effects caused by the higher doses of systematic delivery of RA  and one of the most popular drug eluting stent in the last decade (Xience Pro).The presence and severity of inflammation was investigated as part of the histological analysis of target arterial segments. Inflammatory responses were expected to be observed in light of the endothelial damage & subsequent healing mechanisms caused by stent implantation that induced mechanical stress on the vascular wall when expanded or apposed, respectively. However, a biocompatible stress material & coating would be expected to inhibit further/chronic inflammation, while the presence of RA might inhibit inflammatory responses further due to its therapeutic effects, if confirmed. For that reason, markers of inflammation discussed in Results and shown in Table In the comparative evaluation, differences among the groups in total arterial reaction average score were limited, which translates into limited differences in biocompatibility and performance among the treatments. The histopathological evaluation showed that the total arterial reaction average score was numerically lower in groups with polymer B compared to polymer A in RA drug eluting stents. The difference was mostly associated with less severe vascular wall changes. Histomorphometrically, RA drug eluting stents with polymer B had numerically lower stenosis and intimal mean thickness values when compared to groups with RA drug eluting stents with polymer A. Moreover, stents with polymer A and high dose resulted in larger stenosis and intimal mean thickness values . These results indicate that stents with polymer A were deemed to induce more vascular changes and stenosis than stents with polymer B. Regarding dose–effect, the result from the combination with the lower dose and polymer A was statistically significantly different from the AH group for intimal mean thickness, suggesting a drug delivery type A dose-related increase of the intimal layer thickness. No such dose effect was observed for drug delivery type B and no statistically significant differences were present between BL and BH groups for all measurements.Groups EF and EG showed less total arterial reaction score than group C (BMS). Histomorphometrically, Group EG was significantly different compared to reference item Group C for Intimal Area, Stenosis and Intimal Mean Thickness. Group EF showed significant difference compared to reference item Group C for Intimal Mean Thickness. Despite the fact that no statistically significant difference between Groups EF and EG was present by histomorphometric analysis, Group EF showed slightly higher total arterial reaction average score than group EG and Group EF showed greater percentage of stenosis when compared to Group EG. This trend aligned with the superiority observed with BL, BM and BH compared to groups AL, AM, AH.The unstained particles detected within the tunica intima in all groups, except for group C, were associated with scattered macrophages, giant cells and considered to elicit a minimal “foreign-body” reaction. The origin is unknown, however, it is likely debris from the stent polymer or from the implantation procedure. Generally, this was considered a minimal change, but these particles might contribute to the triggering of the local immune response and neointimal proliferation.23. Further investigation is needed in order to examine the possible long term effects of RA eluting stents. Finally, we did not measure the RA concentrations in the iliac wall.Our study has some limitations. First, all stents were implanted in healthy iliac arteries (without atherosclerotic lesions). Second, iliac arteries (elastic-natured) are different from muscular coronary arteries. Third, observation period in our study was 4 weeks. This period was chosen according to current guidelines for a first evaluation of the feasibility and safetyRA, a non-cytotoxic drug, is known to inhibit neointimal formation after balloon injury in various experimental models.The present research is the first to investigate the safety and efficacy of a RA eluting stent in an experimental model of restenosis. We showed that RA eluting stent implantation is not related with thrombus formation or medial/transmural rupture and has an acceptable degree of stenosis 4 weeks post implantation in a rabbit iliac artery model. The dose of RA and the type of polymer may play a role in biocompatibility of the device. Further investigation is needed in order to clarify the role of RA in vascular injury after DES implantation.24 and the European Union legislation25 following the principle of 3Rs  to ensure: a reduction in the number of animals used, the improvement of experimental techniques and their living conditions so that they are kept to a minimum pain or suffering. The experiments were performed at the Biomedical Research Foundation of the Academy of Athens, Greece / Athens. The protocol was approved by the Protocol Evaluation Committee of the Biomedical Research Foundations of the Academy of Athens, Greece and the Directorate of Agricultural and Veterinary Policy of the Prefecture of Attica, Greece, a State authority. All methods were performed in accordance with the relevant National and international guidelines and regulations.The study was carried out in compliance with the ARRIVE guidelines26. The animals were individually housed in stainless steel cages. Two stents of the same group were implanted in each rabbit, one stent in each iliac artery. Sixty stents were used in total, which were divided in ten groups, six stents per group, three animals per group. The observation period after implantation was 4 weeks.Endovascular stents were implanted in the iliac arteries of 30 male New Zealand (2.5 to 3.5 kg) rabbits. The rabbit iliac artery model was chosen as a first test for the preclinical evaluation of the RA eluting stents because the size and injury response of rabbit iliac arteries are relatively comparable to human coronary arteries27. In brief, the animals were placed in supine position, and immobilized. Both auricular dorsa were shaved and sterilized with povidone-iodine. A novice trainee and an experienced interventional cardiologist performed the trans-auricular vascular access, elective vessel catheterizations, and stent implantation in the iliac arteries.The rabbits were anesthetized by an intramuscular injection of ketamine (35 mg/kg) and xylazine (7 mg/kg) and were monitored by peripheral pulse oximetry during the procedure. We used a published method of percutaneous transauricular endovascular access28 optical coherence tomography (OCT) was performed post-procedural in a small number of stents (Ν = 4). The preparation and materials used for the process were slightly modified for those cases that OCT was performed.For the initial implantations and in order to establish an accurate stent placement, as well as for collecting data required by a parallel study aiming to develop an in silico simulation of the expansion processThe central auricular artery was punctured with a 22-gauge intravenous catheter approximately at the distal half of its subcutaneous course. Then the central needle of the catheter was removed. A 0.018-inch  hydrophilic guide wire was carefully advanced into the external carotid artery and aortic arch / descending aorta and then into the thoracic aorta. The catheter was removed, and a 3-cm-long incision of the dermis was performed at the point of the initial puncture along the course of the guide wire to the base of the dorsa. In cases where OCT was scheduled to be performed, a 5-F compatible 0.035-inch, slender introducer sheath  was advanced into the external carotid artery after serial step-by-step dilations with the sheath’s own dilator. Repeated over-the-wire dilations were performed in order to remove the tight and narrow peripheral segment of the artery prior to sheath insertion. Then, heparin (100 IU/kg) was administered intra-arterially. Upon access gaining a 5F guiding catheter  was advanced to the distal aorta to get direct negotiation with targeting vessels and an angiography was performed to visually evaluate the vessel diameter and as roadmap for accessing femoral vessels. Following angiography, the wire was exchanged to an 0.014-inch wire , and it was advanced to the femoral artery. Finally, the stent was forwarded and placed, a few millimeters after the origin of the iliac artery . Following the stent implantation, OCT was performed without contrast media but via washing out with saline solution only.In cases where the OCT was not scheduled, the process was exactly the same as above, up to the point of the introducer sheath use entry. A 4-F, 0.018-inch compatible Radifocus Introducer II sheath  was used to gain the access point and the following activities were the same as described above. Another differentiation was on the guiding catheter where the above referred catheter was replaced with a 4-F angiographic catheter . A final angiography was performed for evaluating the stent apposition, the vascular patency and the presence of possible complications . The animals did not receive any antiplatelet medication.For the preparation of the drug eluting stents, Cobalt-Chrome bare metal stents  with dimensions 12 mm length × 2.50 mm diameter were coated with coatings consisting of a mixture of polymer and drug Table . Two difRontis Cobalt-Chrome bare metal (uncoated) stents and commercially available Everolimus eluting stents  were also used of the same size . The types of stents, compositions of the coatings and the drug doses of the tested items are included in Table The animals were euthanized with sodium pentobarbitone injection. Necropsies were performed by an experienced veterinary doctor. The treated artery segments were collected and preserved in 10% neutral buffered formalin and transferred for histological processing, image analysis and histopathological evaluation. Histology and morphometry were performed at an independent Good Laboratory Practice certified laboratory  by a board-certified veterinarian pathologist. Samples from implants sites were processed by methyl methacrylate (MMA) resin embedding, sawed by a diamond band saw in transversal sample direction (EXAKT System), ground and polished to a final thickness of approximately 40–60 µm (EXAKT System). One section from each sample was stained with adapted Paragon stain following Standard Operating Procedures. Evaluation of the slides for quality check was performed, and then slides were transferred to the study pathologist for pathology evaluation.The parameters evaluated by histopathology examination adapted to the scoring system described by ISO 10993-6:2016 were: arterial reaction , artery inflammation , medial smooth muscle cell (SMC) loss , artery cell replacement tissue , host reaction associated with the implant , elastic lamina (EL) rupture , medial hypertrophy . An adapted scoring system, described by ISO 10993-6:2016, was applied. Each parameter scored from 0 to 4 according to the histological findings. Defined by the ISO 10993-6:2016, a score difference between 0.0 to 2.9 is considered no or minimal host reaction, 3.0 to 8.9 slight host reaction, 9.0 to 15.0 moderate host reaction and ≥ 15.1 severe host reaction compared to a reference material.An Olympus Slideview VS200 slides scanner using an Olympus U-TV1XC camera and 20× objective was used to scan the arterial samples. Quantitative evaluation was performed using Olympus imaging and image analysis software cellSens v1.18.2), area within internal elastic lamina , lumen (µm2), intima (µm2) , media (µm2) , stenosis (%) [calculation:100 − (100 × Lumen/IEL)], intimal thickness (µm) . These arithmetic mean values were used for further descriptive statistics. Illustrative images with the measurements, one for each group, are displayed in Fig. Quantitative evaluation by image analysis on each artery was performed, including the following parameters: Area within external elastic lamina . Descriptive statistics were used for medial area, intimal area, stenosis and intimal thickness. The Shapiro–Wilk test for normality was performed. When the data followed normal distribution, the comparisons were performed with the unpaired t-test. When data did not follow normal distribution, the Mann–Whitney test was used. A p value less than 0.05 was considered significant.Supplementary Information.


We show that highly dynamic regions such as large estuaries have thicker SMLs than most oceanic sediments. Organic carbon preservation and SMLs are inversely related as mixing stimulates oxidation in sediments which enhances organic matter decomposition. Sites with SML thickness >60 cm usually have lower organic carbon accumulation rates (<50 g C m−2 yr−1) and total organic carbon/specific surface area ratios (<0.4 mg m−2). Our global scale observations reveal that reworking can accelerate organic matter degradation and reduce carbon storage in coastal sediments.The sediment-water interface in the coastal ocean is a highly dynamic zone controlling biogeochemical fluxes of greenhouse gases, nutrients, and metals. Processes in the sediment mixed layer (SML) control the transfer and reactivity of both particulate and dissolved matter in coastal interfaces. Here we map the global distribution of the coastal SML based on excess  The authors map the global distribution of the mixed layer in coastal ocean sediments, based on a neural network model. These observations reveal that mixing can accelerate organic matter degradation and reduce carbon storage in the coastal ocean. These sediment records can be altered by physical and/or biological mixing which can modify sedimentary structures and obscure record interpretations2. Constraining the thickness and location of the sediment mixed layer (SML) is essential for resolving key pathways in marine biogeochemical cycles4. For instance, the SML is an important driver of the exchange of nutrients, organic carbon, redox-sensitive elements and greenhouse gases between the seafloor and the overlying seawater6.Coastal sediments record detailed historical changes of land-use and climate, which can impact source-to-sink particle dynamics across the land-ocean boundary210Pb  is a natural “tracer” for sediments accumulating over time. Pioneering work using 210Pb for dating marine sediments7, established chronologies in estuarine and coastal sediments10. In rapidly changing coastal environments, sediments are commonly remobilized as stationary fluid muds, and/or resuspended and laterally transported as dense suspensions12. Particle reworking and remobilization can result in loss of chronological information15 and is highly heterogeneous spatially and temporally. Thus, the distribution and thickness of the SML are important to improve global flux estimates of dissolved and particulate constituents of key biogeochemical cycles in Earth System Models16.Quantifying the thickness of the SML is challenging, particularly in highly dynamic coastal sediments. Atmospherically derived 18. Some examples of neural networks applications in the earth sciences include: hydrology, including flooding forecasts, and water quality modeling20; geophysics/geomorphology, including earthquake predictions, and simulating land-use change22; and atmospheric sciences, by modeling cloud formation and temperatures24. Recently, machine learning  has been applied to resolve important questions in oceanography, including the global distribution of seafloor total organic carbon, benthic properties, sediment porosity/density and sediment accumulation rates30.Neural networks have emerged as a powerful tool to resolve complex spatial and temporal patterns that are common in large datasets in the geosciences15, global-scale datasets remain sparse. A tracer-identified surface mixed-depth global mean value of 9.8 ± 4.5 cm was obtained in the 1990’s32 and later updated to 5.75 ± 5.67 cm33. Nevertheless, there remains a significant gap in our knowledge of the distribution patterns of SMLs in the global coastal ocean. Hence, we posit that a more accurate estimate of SMLs is needed for better incorporation into global biogeochemical ocean models.While the SML has been assessed on local and regional scales210Pbex profiles that has been reworked over time scales of months35. We estimated SML thicknesses in the global coastal ocean by compiling data from 742 globally distributed sediment cores. First, we evaluated the spatial patterns and drivers of sediment mixing. We then used a neural network model to estimate the thickness of SMLs in the entire global ocean shallower than 200 m, linked it to organic carbon burial in marine sediments. This global assessment of coastal SMLs improves our ability to estimate and interpret the burial capacity and remineralization of organic matter and nutrients in ocean sediments.Here, we define the SML as the sediment thickness captured by 210Pb (210Pbex) in coastal shelf and estuarine sediments, devoid of sediment mixing, typically reflect exponential decay with depth1. Vertical 210Pbex profiles have previously been divided into eight types to cover multiple mixing possibilities12. Here, we simplified this classification into five common types . A Type III profile reflects constant 210Pbex activities along the upper layers of the core overlaying an exponentially decaying trend, usually attributed to mixing from sediment resuspension or reworking very recently (months to years)36. A Type IV profile also reveals an intense reworked surficial layer during an episodic disturbance event such as a storm, leading to deep homogenous 210Pbex activities in the upper section, overlying sediments with no excess 210Pb. The Type V profile can be used as evidence of repetitive reworking and deposition in a highly reworked mud stratigraphy37. The criteria used here in defining the thickness of the SML is based on the upper homogenized layer of 210Pbex in Type III and Type IV profiles, indicative of mud layers reworked on a timescale of months. Since any exclusion of other 210Pbex profile types can result in an underestimation of mixing depths. Thus, our use of Type III and Type IV profiles represents a conservative estimate of SML thicknesses on a global scale. Furthermore, it is possible that an exponential decrease in the upper section of a Type I profile could originate from slow/deep bioturbation. This kind of reworking is indistinguishable from profiles originating from sediment accumulation and/or a combination of sediment accumulation and bioturbation.Profiles of excess pes Fig. . A Type 210Pbex profiles from the global continental shelf . So, gravity-core data were included when analyzing SMLs in Amazon Shelf sediments. Overall, 742 cores had complete 210Pbex profiles and recognizable SMLs to be used in our model.We compiled 1093 published 39. However, thick SMLs demonstrate the heterogeneity of depositional environments in LDEs, and the need for careful site selection of sampling locations for paleo-reconstruction work, which has proven to be successful in some LDEs 41. These LDEs include the Yangtze  were found in North America away from large-river sources. In some enclosed marginal seas such as the Gulf of Mexico  usually have higher SML thicknesses due to stronger hydrodynamic forcing and larger watersheds with more extensive and varied human disturbances.The SML thickness is controlled by (1) environmental factors such as precipitation, temperature, and water depth; 2) physical factors including winds, tides, waves, and bottom stress; (3) biological activities including feeding and burrowing; and (4) sediment sources. We identified 12 influencing factors (Methods) and analyzed their relationship to SMLs . Since it is well established that SMLs largely occur in fine-grained deposits Our neural network simulation of SMLs in the global coastal ocean with water depths shallower than 200 m predicts that >50% of the SMLs have a thickness of 0-5 cm, with only 3% thicker than 30 cm Fig. . The max33 and 9.8 cm32. The timescale of SML formation varies depending upon the environmental conditions. Individual particles within the SML, both organic and inorganic, will range in age depending upon the sediment accumulation rate and intensity of physical and biological mixing45. For example, mudflats adjacent to the Amazon River estuary have deposition rates reaching 1 cm per day46. Similarly, continental shelf sediments near the mouth of the Yangtze River, have short-term deposition rates of 4.4 cm per month47. In the northern Gulf of Mexico, near the Mississippi/Atchafalaya LDE complex, SMLs with thicknesses of 10–20 cm, were formed within 1 year48. Many reported SMLs deposit over seasonal to inter-annual timescales49.Based on a probability distribution of modeled average SML thicknesses , biological  and human  driving forces. The presence of hypoxia/anoxia will limit the presence of infauna which, in turn, will limit the thickness of SMLs driven by biological factors. For example, in cases where bottom waters are anoxic, such as the basins off California, SMLs are absent8. Hypoxia/anoxia is increasing in many areas around the world such as in the northern Gulf of Mexico53, where SML thicknesses are likely to decline with smaller, more opportunistic benthic species dominating in sediments54. Our neural network model allows for a more comprehensive and quantitative understanding of reworked muds, induced by physical, biological, and anthropogenic factors, in the coastal ocean.Marine sediments host significant levels of biogenic materials that drive biogeochemical exchange, carbon storage and regulation of greenhouse gases2 sequestration and carbon storage in sediments has received considerable attention55. Coastal margins including mangroves and saltmarshes cover ~16% of the global seabed area but account for > 90% of total ocean OC burial56, thereby playing a central role in the global carbon cycle. Reworked muds, including mobile muds, resuspended sediments, and bio-mixed muds, are subject to long-term hydrodynamic sorting, which has significant impacts on OC transport, degradation, and deposition on ocean margins, as well as interpretations of related climate records57.As anthropogenic climate change modifies the global ocean, the importance of CO58. Enhanced remineralization of sedimentary OC can reduce OC accumulation rates (OCAR)59; in contrast, to more quiescent sedimentary environments, that typically favor OC preservation6. Total organic carbon/specific surface area (TOC/SSA) ratios, commonly used as an indicator of OC preservation that normalizes grain size effects, are usually lower in mobile-mud deposits and higher in high productivity/upwelling regions61.Continuous resuspension and redeposition drive periodic oxidation-reduction cycles that accelerate the degradation of organic matter, making the mixed zones effective “incinerators” of organic matter62. The average thicknesses of SMLs, in these four morphotypes, are 5.8, 3.6, 6.5 and 9.9 cm, respectively. Intense sediment reworking is mostly found in IM coastal-shelf settings, with >22% of SML deeper than 10 cm and about 2% >60 cm  preservation in the coastal ocean. The distribution patterns of OCAR Fig.  and TOC/ cm Fig. . Most of cm Fig. . An inve cm Fig.  implies −2 yr−1, almost all SMLs are thinner than 10 cm. For example, the mangrove-dominated Indonesian coast has OCAR = 1722 g C m−2 yr−1 with an SML of <10 cm , and lower TOC/SSA ratios in areas with thick SMLs   Fig. .63, resulting in local changes in the density of deposits and sources of OC64. Weak sediment reworking is associated with OC preservation in polar continental margins. For example, coastal sediments in north-east Greenland, characterized by sediments with shallow SMLs had OCAR at 1540 g C m−2 yr−1 .In the other three coastal morphotypes, <10% of SMLs have thicknesses >10 cm Fig. . In the r−1 Fig. 65. There66. Interestingly, reported TOC/SSA ratios are all >0.4 mg m−2  have thicker SMLs. Sedimentary OC in these mobile-mud regions experience frequent sediment reworking with rapid Fe redox cycling and long-distance transport, resulting in low OC burial  coastal oceans minimize mineralization of carbon along the transmission pathm−2 Fig. , consistm−2 Fig.  and had 210Pb profiles, provide a strong connection for assessing carbon storage. These correlations provide global-scale evidence that refreshed exposure of sedimentary OC to the overlying water column reduces OC accumulation. Specific mechanisms will vary among sites and will have different impacts among shelf morphotypes. The simulation presented here provides a first step establishing a more global characterization of SMLs in the current global coastal ocean. This characterization can lead to more insights about hotspots of organic matter cycling in marine sediments56, which can more broadly support Earth System Models. Finally, as many marine macrofaunal benthos in the coastal ocean are undergoing poleward range expansion due to global warming70, SMLs will likely undergo additional change that will also need to be included in ongoing modeling efforts.The estimated thicknesses of SMLs here are based on a decadal time scale from 210Pb with coastal and/or estuary; and reworked muds and/or mixed layer. The data repository created by Solan et al.71 provided an excellent base to show relationships between benthic faunal community distribution and sediment characteristics, and we also obtained additional 210Pbex profiles from references therein. We compiled 238 studies with 1093 sites having 210Pbex profiles distributed globally. Data source references are presented in the Supplementary Data The Web of Science , Google Scholar, and Bai Du Scholar were utilized to search the literature using the following key words or phrases: Environmental factors, physical dynamics and biological activities were collected for each site and presented in Supplementary Data 73 and water depths from the ocean bathymetry database . Tropical cyclone effects and hazard risks were based on the Global Cyclone Hazard Frequency and Distribution, v1 dataset (1980–2000), and assessed on a 2.5 min’ global grid. More than 1600 storm tracks were assembled and modeled, through the period January 1st, 1980, to December 31st, 2000, for the Atlantic, Pacific, and Indian Oceans, at UNEP/GRID-Geneva PreView (https://www.ldeo.columbia.edu/chrr/). Sediment types were collected from seafloor lithology in the GplatesPortal74. We added about 7800 points in areas with poor data coverage and reproduced a new digital map of seafloor lithology . Annual average chlorophyll-a concentration and primary production were originally calculated using these climatology data, which were then linearly interpolated for each location. Part (165 sites) of the organic carbon accumulation rates (OCAR) data for global tidal marshes and mangroves was extracted from the study of Wang et al.75. The remaining estimates of OCAR and TOC/SSA ratios were collected from the literature and both the actual data and relevant references are listed in the Supplementary Data Tidal ranges were extracted from a global tidal range dataset81, was employed to assess the distribution of SML in the global coastal ocean. Briefly, a neural network is a set of neurons that can be connected and combined in one or multiple layers. The first layer, called the input layer, consists of the source data. There are then intermediate layers, called hidden layers. The resulting output, in this case the thickness of the SML, is obtained in the last layer. Two key issues in MLP design are the specification of the number of hidden layers and the number of neurons in these layers. Once the number of layers and number of neurons in each layer have been selected, the network’s weights and thresholds must be set to minimize the prediction error made by the network; this task is the role of the training algorithms.A supervised multilayer perceptron (MLP), the most commonly applied type of neural networks82. After data collection, correlation analysis and data normalization were used to train the neural network more efficiently. For correlation analysis, several highly correlated factors were selected, and transformed into values between 0 and 1 using data normalization. The selected data were divided into two randomly selected groups, the training group which corresponded to 70% of the patterns, and the test group, which corresponded to the remaining 30% of the patterns.Four essential steps were used in designing the neural network: (a) collecting data, (b) preprocessing data, (c) building the network, and (d) training and test performance of the model , which handles all the data that enters a neuron2 score to evaluate the neural network model. There are other algorithms with very different principles that may also be suitable for predicting SMLs, such as K-nearest neighbor (KNN), Random Forest (RF) and support vector machine (SVM). We compared each algorithm based on their performance and found that the MLP was the most applicable algorithm for predicting global SML , the root mean-square error (RMSE), the mean absolute error (MAE), and percent correct (PC) between the measured values and the predicted values. Here, we used MAE and RML Table .The optimal architecture of the developed artificial neural network revealed three hidden layers of 20, 40 and 10 neurons  obtained were constant at a level of 3000. When epochs reached 3000, the MAE decreased to 0.32  and remained stable, and the R2. The operational steps in ArcGis included attributes extraction, fishnet creation, location selection, and data export. Finally, we entered the grid into the trained neural network model to predict the thickness of SML with the same grid resolution. The model prediction results are available as a downloadable file , resulting in about 50,000 sites along the coastal zone and continental shelf to water depths of 200 m. Each grid is 20 × 10 arcmin with an area of ca. 510 km, resultiSupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2


Brassica napus L.), phacelia (Phacelia tanacetifolia Benth.) and sunflower (Helianthus annuus L.) at 2%, 5% and 10% levels. The macronutrient composition of the biscuits was determined: crude protein content (Kjeldahl method), crude fat content (Soxhlet extraction), ash content (carbonization), moisture content (drying), carbohydrate content (formula). Their total phenolic content (TPC) and in vitro antioxidant capacity  were determined spectrophotometrically. The colour of the biscuits was measured using a tristimulus-based instrument, and their texture was characterized by using a texture analyser. Sensory profile of biscuits was determined by qualitative descriptive analysis (QDA). The consumer acceptance and purchase intention of the biscuits were also evaluated, based on the responses of 100 consumers. Additionally, an external preference map was created to illustrate the relationship between consumer preference and the sensory profile of the biscuits, and penalty analysis was conducted to identify directions for product development. Phacelia pollen appeared to be the most effective for improving the nutritional quality of biscuits. The addition of phacelia pollen at the 10% substitution level increased the protein content and TPC of the control biscuit by 21% and 145%, respectively. Significant changes (p < 0.05) were also observed regarding the colour and texture of biscuits. The results of the QDA revealed that biscuits containing pollens of different botanical sources have heterogeneous sensory attributes. The biscuit containing sunflower pollen at the 2% substitution level was preferred the most , and purchase intentions were also the highest for this product. Based on the results of the present study, it is recommended to use sunflower pollen for developing pollen-enriched foods in the future.Bee pollens are potential functional food ingredients as they contain essential nutrients and a wide range of bioactive compounds. The aim of this study was to investigate the effects of enrichment with monofloral bee pollens on the nutritional properties, techno-functional parameters, sensory profile, and consumer preference of biscuits. Biscuits were prepared according to the AACC-approved method by substituting wheat flour with pollens of rapeseed ( Apis mellifera L.), which moisten pollen grains with nectar and salivary secretions, then transfer the formed pellets to the hive. Pollen pellets can be harvested by using pollen traps placed at the entrance of the hives [There is a growing trend towards consuming foods enriched with health-beneficial substances of natural origin. Apicultural products, including bee pollens, are well applicable for this purpose, as they contain various macro- and micronutrients that are essential for or have beneficial effects on the human body . Bee polhe hives . Approxihe hives , and thihe hives . Bee polhe hives .The nutritional and sensory properties of pollens depend on their botanical composition ,7,8. Pol(Brassica napus L.), phacelia (Phacelia tanacetifolia Benth.) and sunflower (Helianthus annuus L.), based on their nutritional properties, colour, texture, sensory profile, consumer preference and purchase intention. Substitution levels of 2, 5, and 10% were chosen in order to obtain results that are comparable with literature data. Pollens of the selected plant species can potentially be produced monoflorally because they are grown as monoculture, are very attractive pollen sources for honeybees, and have economic significance in several countries.Biscuits are popular bakery products, primarily because they can be consumed quickly, and they have a long shelf life and a varied taste and texture. The essential ingredients of these products are flour, fat/oil, sugar, water, and chemical leavening agents, such as baking soda. These ingredients are considered to be unhealthy by consumers; therefore, a large number of studies have been conducted in recent decades in order to improve the nutritional quality of biscuits . Bee polCommercial wheat flour , ground sucrose , margarine with a fat content of 70% , glucose , salt , and baking soda  were purchased from a retail store in Hungary. Dried honeybee-collected bee pollens originating from rapeseed, phacelia and sunflower were provided by a local beekeeper. These products were harvested between April and July 2021 in Pest county, Hungary. Pollen loads were sorted by colour, shape, and size to remove pellets of unknown botanical origins. Subsequently, the botanical composition of these products was identified by microscopic pollen analysis. Margarine and glucose syrup were stored in 4 ± 0.5 °C, while other ingredients were stored in a dark place at room temperature until use.Microscopic pollen analysis was used to identify the botanical origin of pollen pellets. The determination was performed by an expert of the Melissopalynological Group of the International Honey Commission. After homogenization, ten pollen loads were selected randomly and suspended and dispersed in 10 mL distilled water. Subsequently, 30 µL of the suspension was transferred onto two slides using a micropipette. After drying on a hot plate, they were covered with glycerin gelatin mixture or glycerin gelatin mixture stained with fuchsine. Pollen grain identification was performed for both slides by examining the entire area of a 20 mm × 20 mm cover slip . Pollen Biscuits were prepared according to the AACC-approved method 10–50D  using thFor crude protein determination, the classical Kjeldahl method was applied. A nitrogen-to-protein conversion factor of 6.25 was used during the calculations. Crude fat content was determined by Soxhlet extraction using petroleum ether as a solvent. Ash content was determined gravimetrically by carbonization at 525 ± 25 °C in a laboratory furnace until constant weight is achieved. Moisture content was also determined gravimetrically by drying biscuits at 105 ± 2 °C in a laboratory air-oven until constant weight. The total carbohydrate content of the pollens was calculated using the following formula (Equation (1)):Extracts of biscuit samples were prepared as follows: 1.00 g of ground and homogenized biscuits were weighed in centrifuge tubes and were dissolved in 10 mL of solvent. Distilled water was used as a solvent to extract compounds that are water-soluble. The mixtures were homogenized by vigorous shaking for 30 s, then treated in an ultrasonic bath  for one hour. Subsequently, samples were centrifuged  at 11,000 rpm for 10 min. Then, 1.5 mL of supernatants were transferred into Eppendorf tubes and stored at −20 ± 2 °C until analysis.2CO3 solution (0.7 M) was also added. Test tubes were vortexed, and warmed in a 50 °C water bath for 5 min. Absorbances of the solutions were measured at 760 nm against a blank solution. The results are expressed in mg GAE /100 g dry weight.For the determination of the total phenolic content (TPC), the Folin–Ciocalteu method developed by Singleton and Rossi (1965) was used . FirstlyThe FRAP assay was conducted as proposed by Benzie and Strain (1996) . The FRAThe TEAC assay was performed by applying the method of Miller and co-workers (1993) . As a fiThe measurement of DPPH radical-scavenging activity was carried out according to the method of Blois (1958)  and modiThe colour parameters of the baked biscuits were measured using a Konica Minolta chroma meter CR-410 device . Results are expressed as CIELAB colour coordinates, where L* indicates the lightness from black (0) to white (100), while a* describes the red–green colour , and b* describes the yellow–blue colour  . Hue is After cooling to room temperature, the dimensions of biscuits were measured. Their volumes were calculated by the following equation (Equation (4)), where r is the radius and h is the height of biscuits:1 is the weight of the biscuit prior baking (g) and w2 is the weight of the biscuit after baking (g):The weight of the biscuits was measured with an accuracy of four decimal places before and after baking. Baking loss (%) was determined using the following equation (Equation (5)), where wThe texture of the biscuits was characterized by applying a Brookfield CT3 Texture Analyzer . Nine biscuits were selected randomly from each type. Measurements were conducted on the centre of the selected biscuits. The texture profile analysis was performed with a TA44 probe . The following test parameters were adjusted: total cycles: 2; test speed: 1 mm/s; target value: 4 mm; trigger load: 4 g. Data recording and analysis of the texture profile were performed using TexturePro CT v1.9 build 35 software . The units for certain texture parameters are given in the default form provided by the software. Based on the texture profile, the following parameters were determined: hardness (g), adhesive force (g), fracturability (g), quantity of fractures (-), cohesiveness (-) and springiness (mm). Gumminess (g) and chewiness (mJ) were calculated using the following equations (Equations (6) and (7)):Sensory tests were carried out in a sensory laboratory  that met the standard requirements . The worSensory profiling was conducted by 12 trained panellists  with the necessary knowledge and experience in sensory descriptive analysis including techniques and practices in attribute identification and terminology development. The members of the trained sensory panel were practiced and highly skilled in sensory profiling of bakery products. Each panellist went through training that met the standard requirements ,32. The The tests were designed and implemented according to the international standard for consumer preference tests ,35. A toParticipants recorded their answers on a questionnaire consisting of four parts. Part A recorded information regarding the socio-demographics , total net household income and frequency of biscuit consumption. Part B included a nine-category monotonic ascending hedonic response scale with descriptive terms  and emoticons for each category. Ten attributes were evaluated: darkness, overall odour intensity, sweet odour intensity, margarine odour intensity, overall taste intensity, sweet taste intensity, margarine taste intensity, hardness, crumbliness and overall acceptance. In Part C, the above-listed properties  were evaluated in a 5-category just-about-right (JAR) scale . In part D, a question was included regarding the purchase intention on a 5-point structured scale  .The colour, texture and nutritional parameters were determined in twelve, nine or four parallel measurements, respectively. Data are expressed as mean ± standard deviation (SD). In the case of colour parameters, ANOVA was applied with Tukey-HSD post hoc test to determine statistical differences between biscuits (α = 0.05). In the cases of textural and nutritional parameters, statistical differences were determined by Kruskal–Wallis nonparametric test and Dunn’s pairwise procedure with Bonferroni correction (α = 0.05).Sensory profile data and consumer preference scores are expressed as mean ± standard deviation (SD). Statistical differences between biscuits were determined for each attribute by applying ANOVA with Tukey-HSD post hoc test (α = 0.05). The performance of the trained sensory panel was analysed using the mixed assessor model–control of assessor performance (MAM-CAP) table method. The MAM-CAP table was created using R-project software version 4.0.2  with the MAM-CAP-package ,38. SignThe descriptive sensory attributes were submitted to Principal Component Analysis (PCA) and the obtained sensory map was used as a plot for positioning consumer overall liking data grouped into three clusters (PREFMAP) . To idenPenalty analysis was run on just-about-right (JAR) data. Firstly, the JAR values were amalgamated into 3 groups. Categories 1–2 were labelled as “not enough”, category 3 as “JAR” and categories 4–5 as “too much”. Then, the mean overall acceptance level of samples was calculated for each group. The penalties (or mean drops) were calculated as the differences between the mean of one of the non-JAR categories and the mean of the JAR category. These values were plotted against the respondent percentage of the non-JAR category, placing each response in a so-called mean drop plot. Each statistical test was carried out at a significance level of 5% by using XLSTAT Sensory Version .Brassica napus L.), phacelia (Phacelia tanacetifolia Benth.) and sunflower (Helinathus annuus L.). The proportion of the predominant pollen was above 96% in each sample; thus, they can be referred to as monofloral based on the criteria proposed by Campos and co-workers (2008) [The results of the pollen analysis confirmed that the pollens used for enrichment originated from rapeseed  and calculated parameters  are indicated in ab*) were calculated for each pair of samples, and are presented in ab* < 1 “observer does not perceive the difference”, 1 < ΔEab* < 2 “only an experienced observer can perceive the difference”, 2 < ΔEab* < 3.5 “unexperienced observer also perceive the difference”, 3.5 < ΔEab* < 5 “clear difference in colour is perceived”, 5 < ΔEab* “observer perceives two different colours” [ab* = 1.7). Additionally, the colours of biscuits containing 2% rapeseed or sunflower pollen are also hard to distinguish by the human eye (ΔEab* = 2.1). The 10% phacelia pollen-containing sample differed most from the other samples. There is an overall tendency that the more we increase the amount of pollen, the more the samples deviate from the control. The total colour difference resulting from pollen addition is greatest for phacelia, followed by sunflower and rapeseed pollens.From the CIELAB colour coordinates, total colour differences , except for some odour attributes associated with very small quantities of ingredients . The F-Scal values were adequate , with the exception of cut hay, cabbage flavour and odour, brightness, and shape regularity. The sensory panel agreed on all sensory attributes (F-Disag p > 0.05). The panel’s repeatability was very good (RMSE ≤ 3.15).The MAM-CAP table  presentsResults of the quantitative descriptive analysis (QDA) are summarized in The overall odour intensity of biscuits increased, while the flour odour intensity decreased significantly with increasing pollen concentration. Biscuits containing high concentrations of pollen had lower margarine odour intensity. The baking soda odour intensity of the products was low and was not affected by pollen addition. The sweet odour intensity decreased significantly with increasing pollen ratio, probably because the odour of pollen overpowers the sweet odour of biscuits. The mean scores of the sour, bitter and salt odour intensities did not exceed 2.0 in any samples and were not affected by pollen addition. Based on our results, the phacelia pollen-containing biscuits were characterized by a cut hay odour, while rapeseed pollen-containing products possessed a cabbage odour, the intensity of which increased significantly with increasing pollen concentration.Brassica napus L.) belongs to the Brassicaceae family, the members of which usually contain glucosinolates and their hydrolysis products, particularly isothiocyanates [Brassicaceae family [The results showed that the overall flavour intensity of biscuits in which the wheat flour was substituted with 5 or 10% pollen was significantly higher compared to the control. Accordingly, the intensity of the flour flavour decreased significantly with increasing pollen concentration. The mean scores for the sweet taste intensity of the products varied between 43.2 and 55.0, while low mean scores were observed regarding the sour, bitter and salt taste intensities. The sweet taste intensity decreased by rising pollen ratio. Significantly higher bitter and salt taste intensities were observed for biscuits containing pollens at 10% substitution level in comparison to the control. The increase in bitter taste can be attributed to the aroma compounds of the added pollens or to the enhanced Maillard reaction, which is proposed as a pathway of bitter compound formation in thermally processed foods . The rescyanates . These ce family , and preThe hardness and adhesiveness of biscuits decreased, while chewiness increased as the added pollen ratio rose, regardless of the pollen type. Mouthcoating effect was more characteristic of pollen-containing biscuits in comparison to the control. Biscuits containing pollens at a 10% substitution level had particularly strong mouthcoating effect. The crumbliness and fracturability of biscuits fell significantly with increasing pollen concentration. Most sensory results are not in accordance with the instrumental texture analysis, which can be explained by the fact that the uncertainty is greater for the instrumental measurement due to the heterogeneity of biscuits.Consumer liking data were calculated for all consumers and per clusters of consumers. 16%, 39% and 45% of consumers belonged to cluster 1, cluster 2 and cluster 3, respectively. Means ± standard deviations of liking scores are presented in Comparing results for liking scores between the three clusters, it can be stated that their preferences showed significant differences. Consumers of cluster 1 gave similar scores to each biscuit, except for the 10% phacelia pollen-containing biscuit, which was less preferred by them based on the colour and overall liking scores. Consumers of cluster 2 gave significantly lower liking scores for biscuits containing phacelia pollen at 10% concentration level compared to other products. Additionally, they gave lower mean scores for the control sample in comparison to other clusters. An important characteristic of cluster 3 is that they typically gave lower scores for biscuits containing any type of pollen at 10% concentration level. Based on the overall liking scores, consumers of cluster 1 preferred the 2% phacelia pollen-containing biscuit and the 10% rapeseed pollen-containing biscuit. On the other hand, the product enriched with 2% sunflower pollen was preferred by consumers of the other two clusters the most. The reason for consumers’ preferences may be that biscuits enriched with rapeseed or phacelia pollens were characterized by specific “cabbage” or “cut hay” odour and flavour, while the addition of sunflower pollen did not cause any off-flavours in the products.Penalty analysis was used to gain understanding of the attributes that most affected the overall liking ratings. An external preference map was created to illustrate the relationship between consumer preference and sensory profile of biscuits, which is presented in Purchase intention was evaluated for all consumers. The results are presented in In this work, biscuits enriched with rapeseed, phacelia and sunflower pollen at substitution levels of 2, 5 or 10% were compared, based on their nutritional, physical and sensory properties. The results confirmed that pollens are applicable for improving the nutritional value of biscuits; however, further research is needed to understand the interactions between phenolic compounds and macronutrients in pollen enriched biscuits, and to improve the bioaccessibility of nutrients in these products. Consumer acceptance of biscuits was strongly influenced by the botanical origin and concentration level of pollen used for enrichment. Based on the consumer preference test, biscuits enriched with sunflower pollen had more acceptable sensory properties compared to other samples. The reason behind consumers’ preferences may be that biscuits enriched with rapeseed or phacelia pollens were characterized by specific “cabbage” or “cut hay” odours and flavours, while biscuits enriched with sunflower pollen did not have any off-flavours according to the results of sensory profile analysis. This study is the first to demonstrate that pollens of different plant species have heterogeneous effects on the nutritional properties, colour parameters, texture, sensory profile and consumer preference of biscuits. Based on the results of the present paper, it is recommended for food product developers and researchers studying the potential of pollen as a functional food ingredient to use sunflower pollen for the enrichment of foods.