Reichardia Roth is a small Mediterranean genus comprising ten homogeneous species with basic chromosome numbers of 7, 8, and 9. To assess the plastid genome evolution and differentiation of Reichardia species, we assembled the complete plastome sequences of seven Reichardia and two Launaea species and conducted various phylogenomic analyses comparatively with nuclear ribosomal DNA ITS sequences. Reichardia and Launaea plastomes were highly conserved in gene content and order, containing 130 genes. Plastid phylogenomic reconstruction strongly suggested that Reichardia was a sister to Launaea, and its common ancestor initially diverged into two major lineages: the first containing species with n = 8 chromosomes exclusively, and the other with n = 9, 8, and 7 chromosomes. Although the ancestral Reichardia karyotype was suggested to most likely be n = 9 from ancestral chromosome number reconstruction, the pattern of descending dysploidy indicated by the phylogenetic trees based on nuclear ribosomal DNA ITS was less evident in the trees based on the plastome. Possible reasons for these findings are discussed. Reichardia is a small genus in the subtribe Hyoseridinae Less. (formerly known as Sonchinae) from the tribe Cichorieae Lam. & DC. (Asteraceae). It comprises ten herbaceous species, of which seven are perennials with woody bases ; a biennial-perennial cycle [R. gaditana (Willk.) Samp.]; and two are annuals [R. tingitana (L.) Roth and R. intermedia (Sch. Bip.) Samp.]. Although all of these are native to the Mediterranean and/or Macaronesia (North Atlantic), those with wider distribution, namely R. tingitana and R. picroides, also colonize Middle East Asia. In addition, R. tingitana reaches Pakistan, northwestern India, and some areas of tropical and subtropical eastern Africa, showing a range roughly coinciding with the paleo-geographical limits of the Mediterranean or ‘Madrean-Tethyan’ region2. These two widely distributed species were introduced in Hawaii and Australia6. R. intermedia is also a circum-Mediterranean taxon but is quite common in the western area. R. gaditana is limited to the western Mediterranean region, whereas R. dichotoma is exclusively present in the eastern Mediterranean region. Five species show endemic distributions: the close relatives R. macrophylla and R. albanica, which are restricted to the Balkan Peninsula7; and R. crystallina, R. famarae, and R. ligulata, which are endemic to the Canary Islands. R. famarae occurs exclusively in five localities in Lanzarote and Fuerteventura9. The genus Reichardia is quite homogeneous in morphology and is easily recognizable , the base of the ligule is deep purple. Achenes are four-ribbed, outer transversely rugose, while compressed inner ones are paler and variably smooth. The pappus consists of numerous fine scabrid bristles.ble Fig. , mainly Reichardia presents three basic chromosome number groups of n = 7, 8, and 916. All the species are diploid, and no cases of polyploidy have been documented. The chromosome numbers n = 9 and 2n = 18 have been reported for the Western Asiatic R. dichotoma and the Balkan R. macrophylla, which can be considered vicarious species7 and perhaps tertiary relicts17. The recently described Albanian endemic R. albanica also showed a chromosome number of 2n = 18. Several chromosomes count in the widespread species R. tingitana, R. gaditana, and R. intermedia have shown n = 8 and 2n = 16, which are also the numbers found in the three species endemic to the Canary Islands, namely R. crystallina, R. famarae, and R. ligulata. R. picroides showed chromosome numbers of n = 7 and 2n = 14. Although some authors have reported a somatic number of 2n = 14 for R. intermedia19 in samples from the Iberian Peninsula, we chose to apply the somatic numbers from a monographic study of the genus by Gallego13 based on their subsequent reconfirmation from the Iberian materials15.Despite its low species diversity, 22, as no polyploidy numbers have ever been reported for them, despite it being a widespread phenomenon reported in Asteraceae23. Previous studies have shown a correlation between this diversity and morphological characteristics6, chemical compounds24, and phylogenetic lineages19, claiming both ascending and descending patterns of dysploidy in Reichardia. Exceptional chromosomal diversity in Reichardia has been the subject of evolutionary studies as a good model for the analysis of genome evolution and differentiation. For example, Lӧve and Kjellqvist11 suggested that the most primitive number was n = 7, and the variation from primitive to higher chromosome numbers  was derived by means of the appearance of B chromosomes. However, Gallego13 proposed an alternative hypothesis that the primitive basic chromosome number was n = 9, and the secondary basic numbers of n = 8 and n = 7 arose from the loss of chromosomes or fragmentation. The analysis of phenolic compounds in R. tingitana and R. picroides did not support any specific pattern, neither ascending nor descending, but found that chemo-taxonomic differences related to morphological and karyological characteristics were in agreement with the separation of the Reichardia species into two major groups24. Recently, the chromosomal and genomic evolution of Reichardia species has been investigated within a molecular phylogenetic framework. Siljak-Yakovlev et al.19 plotted the karyological, cytogenetic, and pollen traits of Reichardia species onto the nuclear ribosomal DNA (nrDNA) internal transcribed spacer (ITS) phylogeny and suggested that the descending dysploidy in Reichardia chromosomal numbers was accompanied by changes in heterochromatin patterns and modifications of the location and organization of ribosomal genes. They also reported smaller pollen sizes in R. picroides and R. intermedia, which correlated with their smaller genome sizes and the reduced chromosome number within the genus (n = 7 for both species); however, in our view, these reports for R. intermedia may be erroneous. Moreover, these cytological studies did not include appropriate closest relatives or progenitors of the genus. Without robust phylogenetic relationships between Reichardia and other closely related genera and precise species relationships within the genus, it may be unsuitable to explore genome evolution and differentiation.The diversity observed in chromosome numbers in the genus is presumed to be the result of dysploidy during karyotypic changes in the basic chromosome numberReichardia was previously inferred using nrDNA ITS and chloroplast DNA (cpDNA) psbA-trnH intergenic spacer and matK gene sequences28. In all phylogenies, the genera Launaea and Reichardia represented early diverging lineages within the subtribe Hyoseridinae, and Reichardia was monophyletic, which is concordant with the first comprehensive molecular analyses of the tribe Cichorieae29. The ITS tree strongly supported the monophyly of Reichardia28, which was sister to the clade containing the genus Launaea and the remaining genera of the subtribe Hyoseridinae, that is, the currently circumscribed Sonchus sensu lato (s.l.). The cpDNA trees based on either the psbA-trnH intergenic spacer27 or matK28 gene regions also supported the monophyly of Reichardia and the basal position of Reichardia and Launaea. However, cpDNA phylogenies in previous studies28 were too limited to determine the phylogenetic position of Reichardia and robust species relationships due to insufficient molecular markers and species representation. The relationships between species and genera were either highly unresolved or weakly supported, specifically in the psbA-trnH tree27. Unlike the nrDNA ITS-based phylogeny, Reichardia and Launaea shared the most recent common ancestor, and these two genera were sister to the remaining genera of the subtribe Hyoseridinae, which was also weakly supported (< 50% in the psbA-trnH tree)27 or moderately supported (86% in the matK tree)28. These conclusions are consistent with subsequent studies based on ITS datasets30.The phylogenetic position of 31. Comparative genomic analysis using whole plastome sequences is now an efficient option to improve phylogenetic resolution at lower taxonomic levels that are currently hindered by limited sequence variation due to recent divergence, rapid radiation, and conservative genome evolution of plastomes32.With the advent of high-throughput sequencing technologies for next-generation sequencing (NGS), massive amounts of data have now become available based on the considerable genome-wide variation of entire plastid genomes through plastome sequencing. The benefits of genome-wide variation could increase phylogenetic resolution tremendously and significantly enhance our understanding of plant evolution and diversity in the field of plastid genetics and genomicsReichardia species in addition to R. ligulata, sequenced in our previous study33. The plastomes of two additional species from the closely related genus Launaea were also sequenced28. Based on these complete plastome sequences, we first tested the previous phylogenetic hypotheses proposed by nrDNA ITS and cpDNA markers; plastome-based phylogeny was reconstructed using genome-wide variation and compared with the nrDNA ITS phylogeny, newly reconstructed in this study. Second, through the ancestral karyotype reconstruction, we inferred the karyotype evolution in the dysploidy process of Reichardia species in the framework of the plastome and nrDNA ITS-based phylogenies. We also performed several comparative plastome analyses to determine the structure, gene content, and rearrangements of the plastid genomes of Reichardia and two Launaea species. In addition, we identified highly variable plastid regions that could be utilized as useful markers for further population genetic or phylogeographic studies of Reichardia and closely related genera.In this study, we sequenced and assembled the whole plastid genomes of seven Reichardia species  and two species of Launaea (L. arborescens and L. nudicaulis) were highly conserved in terms of gene content and arrangement, with 98.6% pairwise sequence similarity (99.2% among eight Reichardia species only)  to 152,652 (R. famarae) base pairs (bp) with an overall guanine-cytosine (GC) content of 37.6%, whereas two plastomes of Launaea were slightly longer than Reichardia plastomes, that is, 153,119 bp for L. nudicaulis and 154,086 bp for L. arborescens with an overall GC content of 37.4%. The plastomes of Reichardia and Launaea species consisted of four typical regions: LSC, SSC, and a pair of inverted repeat regions (IRs), sharing exactly the same genes and similar gene contents at all boundaries among the four regions, with slight changes in the length of intergenic regions. They all contained the functional protein-coding gene ycf1 at SSC/IR with its pseudogene copy, ycf1Ψ at IR/SSC, and functional rps19 at LSC/IR with pseudogene copy rps19Ψ at IR/LSC endpoints  Table . The totReichardia and two Launaea cp genomes contained 130 genes, including 87 protein-coding genes (excluding pseudogenes), six rRNA genes, and 37 tRNA genes  than species with chromosome numbers of n = 9  or n = 7 , and R. intermedia . Codon usage values are described by relative synonymous codon usage (RSCU), which reflects how often a particular codon is used relative to the expected number of times that codon would be used in the absence of codon usage bias. All the RSCU values for each amino acid considered in this study were similar among the ten plastomes. The highest RSCU value was indicated in the usage of the UUA codon for leucine (1.9–1.92), followed by AGA for arginine (1.84–1.86), while the lowest were indicated in the usages of the AGC codon for serine (0.33–0.36) and CUG for leucine (0.35–0.38).The frequency of codon usage in the eight ces Fig. . The codReichardia and Launaea plastomes ranged from 43 to 48 in the 19 genes of the 35 protein-coding genes  than species with chromosome numbers of n = 7 (45 for R. picroides) and n = 9 , with 47 sites showing the highest number in the genus. Genes with predicted sites included photosynthesis-related genes , self-replication genes , and others . We detected no RNA editing sites in atpB, atpF, atpI, clpP, petD, petG, petL, psaB, psaI, psbB, psbE, rpl2, rpl23, rps8, rps16, and ycf3. The highest numbers of potential editing sites were found in the NADH dehydrogenase genes, which was consistent with previous findings in tobacco, maize, rice, and other plants37; the ndhB gene was the highest at 9–10 sites, followed by the ndhD gene at 4–5 sites. The highest conversions in the editing frequencies of codons associated with the corresponding amino acid changes were represented by the changes from serine (S) to leucine (L) (average confidence score of 14.52), followed by proline (P) to leucine (L) (average confidence score of 8.76).The possible RNA editing sites predicted among the nes Fig. . We founReichardia and Launaea plastomes was compared using DnaSP38 and visualized by plotting with mVISTA39. The results showed a high degree of synteny and gene order conservation in the mVISTA graph  belonging to the same subtribe Hyoseridinae33. Genetic polymorphisms in different regions of the chloroplast genome varied substantially. The SSC region, where the most variable gene, ycf1, was located, was the most divergent , whereas the two IR regions were highly conserved . Six divergence hotspots among the Reichardia and Launaea plastomes are suggested as potential chloroplast markers: five intergenic regions  and one protein-coding gene (ycf1)  Fig. .Figure 540. We identified that seven genes potentially evolved under positive selection in ten Reichardia and Launaea plastomes by calculating the dN/dS ratio using various site-specific substitution models implemented in EasyCodeML (Table 42. These genes included NADH-dehydrogenase subunit genes (ndhB and ndhG), subunit genes of photosystem I (psaI) and II (psbH), and large subunit genes of ribosomal protein (rpl20), ycf2, and ycf15. Positively selected sites were suggested based on the posterior probability calculated using the Bayes empirical Bayes (BEB) method43 with a cutoff > 0.95 and > 0.99 indicated with asterisks  in Table rps2, rbcL, and ndhG) have been identified in Paulownia44, five  in Panax45, three  in the tribe Sileneae46, and six  in Rosaceae37.Selective pressure in genes or genomic regions is inferred by the proportion of amino acid substitutions, and the ratio (ω = dN/dS) of nonsynonymous substitution (dN) and synonymous substitution rates (dS) has been widely used as a genomic signature of selective pressure acting on a protein-coding gene, that is, ω = 1 indicates neutral mutations, ω < 1 indicates purifying selection, and ω > 1 indicates diversifying positive selectionML Table 41,42. ThReichardia and the closely related species in the subtribe Hyoseridinae, with Lactuca sativa as an outgroup. The topological structures of ML phylogenetic trees constructed by IQ-TREE47 and BI trees by MrBayes 3.2.7a48 were consistent, and BI trees showed higher support values than ML trees  shared the most recent common ancestor with R. picroides and R. intermedia with chromosomal numbers of n = 7 and 8 robustly . The remaining five species  with chromosome numbers of n = 8 formed a clade with full support (100% BS for ML and PP 1 for BI phylogenies), which was sister to the clade of n = 7, 8, and 9. On the other hand, the nrDNA ITS phylogeny showed incongruence in inter- and intra-generic relationships compared with the plastome phylogeny . Reichardia was monophyletic in both nrDNA ITS ML and BI phylogenies , but differed in intra-generic relationships from the plastome trees; R. intermedia (n = 8), and R. picroides (n = 7) displayed a closer affinity with the other species with a chromosome number of n = 8  than species with a chromosome number of n = 9  (98% BS in ML and 1 PP in BI trees).Phylogenetic trees were constructed using maximum likelihood (ML) and Bayesian inference (BI) methods based on the plastome and nrDNA ITS sequences to infer the phylogenetic relationships among ees Fig. . The besces Fig.  of 18 reReichardia, Launaea, and Sonchus, was inferred to be equivocal with 2n = 14, 16, 18, and 32, and 2n = 36 for both plastome (node P21) and nrDNA ITS (node 29) datasets , suggesting a decrease in dysploidy , and the other with 2n = 14 (R. picroides), 16 (R. intermedia), and 18  species  shared by three genera, idy Fig. . Unlike ies Fig. .Figure 8Reichardia and closely related genera. All three genera, Reichardia, Launaea, and Sonchus, were confirmed as monophyletic with strong support values (BS 100% each) in both phylogenies of full plastome sequences and concatenated sequences of plastid protein-coding genes , life form variability , and chromosome variation 50, the phylogenetic position of the genus Launaea remains to be clarified in future studies.This study provides, for the first time, greater resolution with high support for the plastome-based inter- and intra-generic relationships among nes Fig. . Howeverhus Fig. a, while eny Fig. b. It is Reichardia also demonstrated incongruences between the cp genome and nrDNA ITS phylogenies. One novel finding of the current plastid phylogenomic study was that species with different chromosome numbers of n = 7 (R. picroides), n = 8 (R. intermedia), and n = 9  were resolved in the same clade. This clade was sister to another clade with n = 8 species exclusively. In the nrDNA ITS phylogeny reconstructed previously19 and in this study  was resolved as sister to a clade of the species with chromosome numbers of n = 8 and n = 7, clearly supporting the descending dysploidy process for chromosomal evolution in Reichardia. One of the derived groups included two species with n = 7 and 8 , whereas the other was exclusively comprised of five n = 8 species . However, the current study strongly suggested a close relationship among n = 7, n = 9, and a representative with n = 8 (R. intermedia) species on both plastome-based phylogenies  initially diverged into two lineages: one with n = 8 species and the other including n = 9, n = 8, and n = 7 species. The hypothesis that n = 9 is the ancestral chromosome number in Reichardia coincides with the closely related genera Launaea and Sonchus, where this number is the most common and widely distributed51. Furthermore, it has been assumed as the ancestral basic chromosome number of the tribe Cichorieae54.Species relationships within ies Fig. . AccordiR. intermedia (n = 8) and R. picroides (n = 7) based on plastome sequences is an unexpected finding. The last two species show a wide distribution in large circum-Mediterranean regions in Africa, Asia, and Europe which hardly overlapped with those of n = 9 species and only partly with R. dichotoma in certain regions of West Asia (Turkey and Lebanon-Syria)5. In contrast to these two species, n = 9 species are currently found in very limited and isolated areas with a disjunctive geographical distribution pattern; Albanian endemic R. albanica is found only in Mount Çika in southern Albania at altitudes between 1000 and 1700 m above sea level (a.s.l.), Balkan endemic R. macrophylla in the central part of the Balkan Peninsula (East Dinarides) at altitudes between 500 and 1750 m a.s.l., and Western Asiatic R. dichotoma in Turkey, Lebanon-Syria, Iran, and North and Trans-Caucasus7. Considering the putative ancestral Reichardia karyotype (n = 9) and relic species restricted to the eastern Mediterranean17, it is highly plausible that Reichardia originated in the eastern Mediterranean. The mean divergence time based on the nrDNA ITS sequences for the two closely related genera Launaea and Sonchus was estimated to be 6.4 million years (myr)55. The estimated divergence time of Reichardia from its close relatives could be earlier than 6.4 myr based on the ITS tree topology  or younger than 6.4 myr on the plastome tree . Thus, according to the proposed divergence estimation, we can hypothesize that Reichardia originated around the Messinian salinity crisis in the Mediterranean basin56. It is plausible that after its origin in the eastern region, Reichardia rapidly expanded its range and diversified during dramatic climatic changes in the Mediterranean, forming n = 8 (R. intermedia) and n = 7 (R. picroides) lineages. In addition, the rapid speciation of n = 8 species in the western Mediterranean region, including species endemic to Macaronesian Islands , most likely occurred after initial splitting from the n = 9 lineages. The Macaronesian Islands are well known for adaptive radiation and the diversification of explosive insular species of numerous plant lineages, including Aeonium57, Argyranthemum59, Echium61, Tolpis62 and woody Sonchus alliance28. The uncertain phylogenetic position of Reichardia relative to Launaea and Sonchus and n = 9 likely ancestral may also suggest that the common ancestor of Reichardia existed widely in the circum-Mediterranean region in the late Miocene and subsequently diversified during climatic turmoil, forming a relictual n = 9 lineage in the eastern Mediterranean and speciating in the western part of the Mediterranean. Furthermore, we cannot ignore the role of Quaternary climatic oscillations in shaping distribution areas, particularly in current relicts. The wide distribution range of R. tingitana, which roughly coincides with the paleo-geographical limits of the Mediterranean, seems to indicate, at least partially, that the expansion and diversification of this lineage may not have been recent and must have taken place prior to the present geologic configuration of the Mediterranean region. Given the phylogenetic position of R. tingitana , we cannot completely rule out the possibility of its recent rapid range expansion during climatic oscillation in the region.Given their current geographical distribution ranges, the affinity of n = 9 species with Reichardia species based on plastome sequences were also supported by morphological and chemotaxonomic data. Achene heteromorphy was less marked in the n = 9 species than in the other groups. R. intermedia and R. picroides showed plainly smooth inner achenes with differentiated shapes . The separation of Reichardia species into two major groups seems to be corroborated further by differences in chemical compositions between R. picroides  and R. tingitana 24.The phylogenetic relationships among pes Fig. c. In addReichardia species. Hybridization and introgression often appear to be responsible for the phylogenetic incongruence between organellar and nuclear markers, as observed in many groups of plants; however, other factors, such as phylogenetic sorting or the retention of ancestral polymorphisms, could also play a role in generating incongruences64. The R. intermedia and R. picroides group shared the most recent common ancestor with n = 9 species in plastome-based phylogenies with strong support values (BS 99% or 97% in ML and PP 1 in BI trees) , R. x canariensis Gallego & Talavera (R. tingitana × R. ligulata), and R. × sventenia Gallego & Talavera (R. tingitana × R. famarae)6. While the R. × sventenia and R. × canariensis cases are examples of hybridization between widely distributed R. tingitana and the locally restricted species endemic to the Canary Islands, the hybrid origin of R. × baetica occurred between two divergent n = 8 lineages with a much wider geographical distribution.Lastly, the phylogenetic incongruence between the plastome and nrDNA ITS phylogenies could be the result of past gene flow occurring in es) Fig. , whereasReichardia and Launaea. We achieved the first full resolution regarding inter and intra-generic phylogenetic relationships based on the complete plastome sequences of most Reichardia species with variable chromosome numbers . The plastid phylogenomics strongly suggested the early divergence of two major lineages of Reichardia, one with a group of species with exclusively n = 8 chromosome numbers and the other with n = 9, 8, and 7 species together. Although the plastome-based relationships were not in full agreement with the stepwise descending dysploid hypothesis in Reichardia, the ancestral Reichardia karyotype was highly likely to be n = 9 species. It is necessary to utilize whole nuclear genome data and a thorough synteny analysis to elucidate convincingly the species relationships and chromosomal evolution within the genus Reichardia. Based on thorough characterization and comparative analyses of the plastomes, we discovered several informative mutation hotspots, which will increase the efficiency and feasibility of phylogenetic reconstruction among Reichardia and closely related genera.In conclusion, we found highly conserved plastomes, including gene order and content, in Reichardia and Launaea species were collected in the field (see plant sources in28), except for R. albanica, which was obtained from herbarium specimens . Total genomic DNA was isolated using CTAB65 and a DNeasy Plant Mini Kit  following the manufacturer’s protocol. An Illumina paired-end (PE) genomic library was constructed and sequenced using the Illumina HiSeq platform  at Macrogen Corporation . Sequence reads of the chloroplast genomes were assembled using the de novo genomic assembler Velvet 1.2.1066. Annotation was performed using Geneious R10 67 and ARAGORN v1.2.3668. The annotated plastome sequences were deposited in GenBank under the accession numbers listed in Table R. albanica, MN882723 for R. crystallina, MN893254 for R. famarae, MZ665535 for R. gaditana, MZ665534 for R. intermedia, MN893256 for R. picroides, MZ665533 for R. tingitana, MN877944 for L. arborescens, and MN877945 for L. nudicaulis. R. ligulata (GenBank accession number NC051919) was obtained from our previous study33. The annotated GenBank  format sequence file was used to draw a circular plastid genome map . The codon usage frequency was calculated using MEGA770 with the relative synonymous codon usage (RSCU) value, which is the relative frequency of occurrence of the synonymous codon for a specific amino acid. The online program predictive RNA editor for plants (PREP) suite71 was used to predict the potential RNA editing sites for annotated protein-coding genes with 35 reference genes available with known edit sites, based on a cutoff value of 0.8 . The overall sequence divergence was estimated using the Lagan alignment mode72 in mVISTA39. The nucleotide diversity (Pi) was calculated using sliding window analysis  to detect the most divergent regions  in DnaSP38. To evaluate the natural selection pressure in the protein-coding genes of the ten plastomes, site-specific models implemented in EasyCodeML41 were used in the preset running mode based on CodeML algorithms42. Seven codon substitution models with heterogeneous ω values across sites were investigated and compared to detect positively selected sites based on likelihood ratio tests .We performed several comparative plastome analyses of the eight Reichardia and Launaea assembled in this study were investigated in the context of their relationships with closely related species in the subtribe Hyoseridinae, including the outgroup species Lactuca sativa from the same tribe (Cichorieae). We further compared the plastome phylogeny with the nrDNA ITS phylogeny to explore phylogenetic incongruence and gain insights into chromosome evolution. To reconstruct the plastome phylogenies, we analyzed 19 plastomes of major genera of the subtribe Hyoseridinae and one outgroup based on whole plastome sequences, as well as concatenated sequences of 80 common protein-coding genes, including only nine newly sequenced species and downloaded plastomes from GenBank  scores and weights by testing 88 DNA models of ModelFinder49 implemented in IQ-TREE v. 1.4.2. The evolutionary model of GTR + I + Г  was selected for Bayesian inference. The Markov chain Monte Carlo simulation (MCMC) had a length of 4,000,000 generations, sampling every 100 generations, for both plastome and nrDNA ITS phylogeny. The average standard deviation of the split frequencies reached below 0.01, which indicates that two runs reached stationarity. The first 25% of samples were discarded as burn-in, and the remaining samples were retained for the construction of a 50% majority-rule consensus tree with clade frequencies.The phylogenetic positions of the newly sequenced plastomes of nk Table . The nrD74 and MrBayes ancestral states with R (MBASR)75. The coded trait scores of chromosome numbers of each taxon were mapped onto both phylogenies, and the ancestral states and shared-derived karyotypes were inferred using MrBayes 3.2.7a48. The parameters in the analyses were set to “character.type = unordered” and “n.samples = 500”.The reconstructed ML trees for the plastome and nrDNA ITS sequences were used as phylogenetic hypotheses to perform ancestral state reconstruction of chromosome numbers using the R toolkitSupplementary Legends.Supplementary Figure S1.Supplementary Table S1.Supplementary Table S2.Supplementary Table S3.Supplementary Table S4.Supplementary Table S5.


Background: Direct-acting antiviral (DAA) agents for the treatment of hepatitis C virus (HCV) infection have been proven safe and effective in cirrhotic patients awaiting liver transplantation (LT). However, in the long term, data remain minimal regarding the clinical impact of viral eradication on patients listed for decompensated cirrhosis or hepatocellular carcinoma (HCC). We aimed to elucidate the clinical outcomes of patients regarding delisting and the evolution of HCC during the long-term follow-up. Methods: An observational, multicenter, retrospective analysis was carried out on prospectively collected data from HCV-positive patients treated with an interferon-free regimen while awaiting LT in 18 French hospitals. Results: A total of 179 patients were included in the study. The indication for LT was HCC in 104 (58.1%) patients and cirrhosis in 75 (41.9%) patients. The sustained virological response was 84.4% and the treatment was well tolerated. At five years, among 75 patients with cirrhosis treated for HCV, 19 (25.3%) were delisted following improvement after treatment. Predictive factors for delisting highlighted an absence of ascites, MELD score ≤ 15, and Child–Pugh score ≤ 7. No patients with refractory ascites were delisted. Among patients with HCC, 82 (78.9%) were transplanted. The drop-out rate was low (6.7%) and few recurrences of HCC after LT were observed. Conclusions: DAAs are safe and effective in patients awaiting LT for cirrhosis or HCC. A quarter of patients with cirrhosis can be delisted because of clinical improvement. Predictive factors for delisting, as a result of improvement, may assist prescribers, before initiating HCV infection therapy in the long-term perspective. Hepatitis C virus (HCV) infection has long been one of the main causes of end-stage liver disease and indications for liver transplantation (LT) worldwide . NonetheThe first report evaluating treatment for HCV infection (sofosbuvir and ribavirin) in 61 patients with decompensated cirrhosis or hepatocellular carcinoma listed for LT encouraged hepatologists to treat HCV infection before LT. This strategy helped to prevent a recurrence of HCV positivity after LT and led to improvements in liver function that sometimes resulted in delisting . AfterwaFirstly, an improvement in liver function allowing for delisting is not always observed after HCV infection treatment; additional data are required for the identification of patients who could benefit from this treatment before LT ,11. SecoThe aims of our study were therefore primarily to assess the long-term efficacy and safety of DAA antiviral therapy in patients awaiting LT with decompensated cirrhosis or HCC. Subsequently, we aimed to determine the probability of patient delisting, as well as elucidate the clinical outcomes of delisted patients and the evolution of HCC during the long-term follow-up.We performed an observational, multicenter, retrospective analysis of data prospectively collected from 18 hospitals throughout France. All HCV-positive patients having received antiviral therapy with an IFN-free regimen while awaiting LT between November 2013 and June 2015 were consecutively enrolled in this study. Data on comorbidities  were collected.The antiviral regimen and treatment duration were decided at the physician’s discretion based on current guidelines and treatment availability. Data on any prior treatments for HCV infection were also collected. For patients who failed an initial course of antiviral therapy with DAAs and then received a second DAA regimen, only the second antiviral regimen was analyzed. Liver and renal function tests, as well as blood sample data were analyzed at: each visit during DAA therapy, end of treatment (week 24), post-treatment week 12, post-treatment year 1, post-treatment year 2 and post-treatment year 5. MELD and Child–Pugh scores were calculated at each visit using standard formulas based on measured parameters. HCV RNA levels were monitored at baseline, at end of treatment, and at post-treatment week 12.An end of treatment response was defined as undetectable HCV RNA levels at end of treatment. A sustained virological response (SVR) was defined as undetectable HCV RNA at week 12 after treatment discontinuation (before or after LT). All patients were monitored for adverse events at each visit. Clinical and biological responses of patients to antiviral treatment were defined according to changes in Child–Pugh score from between treatment start and 12 weeks after the end of treatment as follows: complete response: Child–Pugh B/C to A; partial response: Child–Pugh C to B; stable response: Child–Pugh A to A; no response: Child–Pugh B or C, or a deteriorating condition. In patients with decompensated cirrhosis, any decision to delist a patient was at the physician’s discretion. For patients with HCC, the number of nodules, the diameter of the largest nodule, and alpha-fetoprotein (AFP) levels were recorded before treatment, at the end of treatment, and at the end of follow-up (AFP score). Treatments for HCC were also recorded. The onset of HCC during follow-up was also reported for patients on the list with decompensated cirrhosis.The French liver transplantation allocation system is based on the MELD score. There are some exceptions for cases of hepatic complications despite a low MELD score. In a context of HCC, the criteria for LT are based on the AFP score, which must be ≤2, with the HCC at tumor-node-metastasis (TNM) classification stage 2 . The avep-value < 0.20). In addition, three procedures for selecting variables  were used to obtain the most appropriate logistic-regression model. All procedures led to the same final model. The Hosmer–Lemeshow test was used to assess the goodness of fit of the logistic model. In addition, Kaplan–Meier method was used to generate overall survival and LT-free survival estimates, and the log-rank test was applied to compare groups. A p-value <0.05 was considered to be statistically significant and all statistical tests were two-sided. All statistical analyses were performed using SAS software V.9.4 .Descriptive statistics are presented as means (±SD) and medians (interquartile ranges) for quantitative variables and counts (percentages) for qualitative variables. The Wilcoxon rank sum test was applied to compare the distribution of continuous variables and Chi-squared test (or Fisher’s exact test when appropriate) was used to test the association of categorical variables. We performed a ROC curve to determine the optimal cut-off values of MELD and CHILD scores for delisting (maximum of Youden’s index). A multivariate Logistic regression was established to determine predictive factors for delisting  based on selected clinical and biological characteristics , diabetes , and dyslipidemia . In the overall study population, 59 (33.0%) patients presented with moderate or refractory ascites and 31 (17.3%) patients presented with encephalopathy. At baseline, the median MELD and Child–Pugh scores were 11  and 7 , respectively.The flow chart presented in n = 111; 62.0%)  . The ove; 62.0%) .p = 0.63) (p = 0.0001)  patients had undergone LT: 82 (78.9%) from the HCC group and 39 (52.0%) from the decompensated cirrhosis group. Among patients who did not undergo LT, 19 were delisted for an improvement and 29 for a deterioration in their condition. Ten patients were still inactive on the transplant list at the end of follow-up. The median follow-up period was 84.5 months . Forty-four (24.6%) patients died during follow-up, but there was no significant difference in overall survival between patients enrolled for HCC or decompensated cirrhosis  A. Transp 0.0001) B.n = 4), bladder cancer (n = 1), lost to follow-up or refusal by the patient (n = 2), sepsis (n = 1), extensive portal thrombosis (n = 1), road accident (n = 1), or deterioration in condition (n = 2). Nine of these 12 delisted patients subsequently died . Under multivariate analysis, only the MELD score (OR: 0.820 [0.710–0.949]) was found to be an independent predictive factor of delisting for improvement. Furthermore, no patients with refractory ascites were delisted a.Predictive factors for LT were also analyzed. Following univariate and multivariate analysis, we identified the following liver transplantation predictive factors: HCC (OR: 7.32 [3.077–17.431]); bilirubin (OR: 1.025 [1.008–1.042]), and refractory ascites (OR: 0.275 [0.076–0.998]) b.n = 1), LT refusal (n = 2), extra-hepatic cancer (n = 1), alcohol relapse (n = 3), and others (n = 3). Twelve of the 22 not transplanted patients subsequently died. Among the 82 transplanted patients, 67 were still alive at the last follow-up. The treatments most frequently administered were trans-arterial chemoembolization (TACE) and radiofrequency ablation.One hundred and four patients were included for study in a context of HCC . Their cTreatment for chronic HCV infection improves liver function in patients with decompensated cirrhosis. This improvement is not consistent and is sometimes insufficient for avoiding LT in these patients. Predictive factors for delisting or LT are therefore necessary to decide whether or not to initiate HCV infection treatment. The problem is slightly different for patients awaiting LT for HCC, but likewise, the indication and timing for HCV infection treatment still needs to be clarified for these patients ,18. Our The results concerning overall survival at five years were outstanding 75.4%). The tolerance of DAA therapy was satisfactory SAEs: 19%) and comparable to the recently reported results in the real-world experience of the HCV-TARGET cohort % and com,7,9,19. 5.4%. TheIn our study, the delisting rate as a result of improvement in patients with decompensated cirrhosis was 25.3%. This is comparable to previously reported findings ranging from 19.2%–30.9% ,7,20. AfAmong the different study cohorts found in the literature, most patients have been enrolled on LT lists for decompensated cirrhosis, but data are scarce with respect to patients on LT lists for HCC, and particularly with a long follow-up ,7,9. In There are limitations to our study inherent to its retrospective nature and study design, and notably to the lack of data available for determining changes to Child–Pugh and MELD scores after HCV infection treatment. Another issue arises with respect to the classification of patients as “inactive on the list” or “delisted” given this is an arbitrary decision that may vary from one LT center to another.In summary, this large French patient cohort under HCV infection treatment while awaiting LT for decompensated cirrhosis or HCC with a long-term follow-up confirms the high efficacy and safety of antiviral therapy with DAAs. Overall, HCV infection treatment allows for LT avoidance in one out of four (25.3%) patients with decompensated cirrhosis, particularly for cases with absence of ascites, MELD score ≤ 15, and Child–Pugh score ≤ 7. The risk is insufficient improvement in liver function that can result in patients falling in a situation of “MELD purgatory” . The res


Thyroid dysfunction is common in patients with nephrotic syndrome, especially patients with primary membranous nephropathy (pMN). In view of both MN and thyroid dysfunction are associated with autoimmunity, the current study aimed to elucidate the significance of thyroid dysfunction in patients with pMN.Four hundred and twenty patients with biopsy-proven pMN from 2018–2021 were retrospectively enrolled. Clinical and pathological parameters, and treatment response of patients with and without thyroid dysfunction were analyzed.P = 0.003). Similar to the urinary protein and the positivity of anti-PLA2R antibody, multivariate COX analysis showed thyroid dysfunction was also identified as an independent risk factor for the failure to remission .Ninety-one (21.7%) patients with pMN suffered from thyroid dysfunction, among which subclinical hypothyroidism (52.7%) was the main disorder. Compared to patients with normal thyroid function, patients with thyroid dysfunction presented with a higher level of proteinuria, a lower level of serum albumin, a higher level of serum creatinine and more severe tubulointerstitial injury at the time of biopsy. But the positive rate and level of circulating anti-phospholipase A2 receptor (PLA2R) antibody were comparable between these two groups. Though following the similar treatment, the percentage of no response to treatment were significantly higher in the patients with thyroid dysfunction (38.6 vs. 20.0%, In conclusion, thyroid dysfunction is common in the patients with pMN and might predict a severe clinical manifestation and a poor clinical outcome, which indicated that the thyroid dysfunction might be involved in the disease progression of pMN. It is commonly known that hypothyroidism usually occurs in patients with proteinuria, in particular nephrotic syndrome. In addition to a series of cases , 2, seveThe main cause of thyroid dysfunction in patient with proteinuria was usually thought as the urinary loss of thyroid hormone , for itsFour hundred and ninety-one consecutive patients with biopsy-proven pMN, diagnosed in Tianjin Medical University General Hospital from 2018 to 2021 were reviewed retrospectively. Patients with membranous nephropathy secondary to known factors, such as malignant tumors, hepatitis virus B/C, systemic lupus erythematosus or heavy metal poisoning, were carefully excluded. Patients with known autoimmune thyroid disease, hypothalamic-pituitary axis dysfunction, pituitary tumors, absent thyroid gland, thyroid gland tumors before the onset of membranous nephropathy were also excluded from the study. Among them, thyroid function of 71 patients were not evaluated at the time of biopsy. Thus, 420 patients remained in this study -conjugated antibodies  on frozen tissues. PLA2R expression in glomeruli was detected by indirect immunofluorescence with rabbit polyclonal anti-PLA2R antibodies as the primary antibody  on frozen tissues.For light microscopy, paraffin sections were stained with hematoxylin and eosin, periodic acid-Schiff, periodic acid-silver methenamine, and Masson’s trichrome. Pathologic findings in the glomeruli, tubules, interstitium, and vessels were described in detail. Stages of MN were defined according to the Churg and Ehrenreich’s classification criteria. The chronic tubulointerstitial injury was defined as tubular atrophy and interstitial fibrosis. The scoring of chronic tubulointerstitial injury were graded semi-quantitatively from 0 to 2: 0, normal; 1, < 25.0% of interstitia affected; 2, > 25.0% of interstitia affected.Circulating anti-PLA2R antibody was detected in the automatic analyzer, using a commercial quantum-dots based fluorescence-linked immunosorbent assay kit  with the recombinant human PLA2R as the antigen, following the standard instructions recommended by the manufacturers. The level of anti-PLA2R antibody > 14RU/ml was designated as positivity.2 or dependence on dialysis) and eGFR decreasing more than 25% from baseline were used to evaluate the renal function.Patients with pMN were treated according to the Kidney Disease: Improving Global Outcomes (KDIGO) guidelines for membranous nephropathy. During follow-up, for evaluation of therapeutic responses, complete remission was defined as urinary protein excretion < 0.3 g/24 h with normal renal function. Partial remission was defined as urinary protein excretion < 3.5 g/24 h but > 0.3 g/24 h, or at least a 50% reduction from peak values with stable serum creatinine (no more than 25% increase from baseline). Treatment failure was defined as not reaching the criteria of partial remission. For evaluation of treatment outcomes, treatment failure was used as the primary endpoint. End-stage kidney disease(ESKD) . Differences of quantitative parameters were assessed using the t-test for data that were normally distributed or nonparametric test for data that were not normally distributed. Differences of qualitative data were compared using the chi-square test/Fisher’s exact test, otherwise, the Kruskal–Wallis or Mann–Whitney U tests were performed. Correlations of quantitative parameters were analyzed using the Pearson correlation coefficient analysis for data that were normally distributed and Spearman correlation coefficient analysis for data that were not normally distributed. Kaplan–Meier curves were used to analyze the clinical treatment outcomes of patients (Log-rank test). If the P < 0.001) and FT4 , but a higher level of TSH   patients suffered from thyroid dysfunction, including 48 (52.7%) patients with subclinical hypothyroidism, 16 (17.6%) patients with overt hypothyroidism, and 27 (29.7%) patients with low T3 and/or low T4 syndrome. The other 329 (78.3%) patients presented with normal thyroid function. Compared to the patients with normal thyroid function, patients with thyroid dysfunction had significantly lower levels of FT3 . Compared to the patients with normal thyroid function, we found patients combined with thyroid dysfunction had a significantly higher percentage of nephrotic syndrome , presenting with more proteinuria , a higher level of serum cholesterol , while a lower level of serum albumin . In addition, the kidney function was poorer in pMN patients combined with thyroid dysfunction, with a significantly higher level of serum creatinine  and lower level of eGFR . The level of hemoglobin of patients combined with thyroid dysfunction was 128.6 ± 21.2 g/L, significantly lower than that of patients with normal thyroid function . Among all the patients, circulating anti-PLA2R antibody was detected in 347 patients, including 75 patients with thyroid dysfunction and 272 patients with normal thyroid function. The positive rate of circulating anti-PLA2R antibody at the time of diagnosis was similar in pMN patients with and without thyroid dysfunction . There was no significant difference in the level of anti-PLA2R antibody between these two groups, either   and correlated positively with serum albumin    and serum albumin , but did not related to serum creatinine  , while it had no significant correlation with urinary protein  and the parameters of renal function   and anti-thyroid peroxidase antibody (TPOAb) detection record. Five of seventeen (29.4%) patients were TGAb positive and 6/17 (35.3%) patients were TPOAb positive. In the 17 patients, anti-PLA2R antibody was detected in 13 patients, and 3/9 (33.3%) patients with anti-PLA2R antibody positivity were TGAb/TPOAb positive, while in the 4 anti-PLA2R antibody negative patients, only 1 (25.0%) patient was TGAb/TPOAb positive. Nine of ninety-one patients had anti-thyrotropin receptor antibody (TRAb) detection record, but all of them were TRAb negative.P = 0.674). By light microscopy, the Churg stages of the 91 patients with thyroid dysfunction consisted of 29 (31.9%) patients of stage I, 33 (36.2%) patients of stage II and 29 (31.9%) patients of stage III, with no significant difference with that of patients with normal thyroid function (P = 0.629) (Table P = 0.860) (Table P = 0.004). . During follow-up, of the 57 patients with thyroid dysfunction, 48 (84.2%) patients received steroids and/or immunosuppressive drugs, which was similar to that of those with normal thyroid function   patients with thyroid dysfunction and 109 (35.1%) patients with normal thyroid function were lost to follow-up. The mean follow-up time was similar between patients with and without thyroid dysfunction  (Table P = 0.003). Kaplan–Meier curve also revealed that the cumulative survival of failure to treatment in the patients with thyroid dysfunction was significantly lower than that of those with normal thyroid function (P = 0.010)  patients with dysfunction obtained remission, including 10 (17.5%) patients with complete remission and 25 (43.9%) patients with partial remission. The rate of remission was significantly lower than that of patients with normal thyroid function . Furtherly, multivariate analysis identified thyroid dysfunction as one of the independent risk factors for worse treatment response . The other independent risk factors included the positivity of anti-PLA2R antibody , the higher level of urinary protein . Compared with other pathological parameters, the severity of chronic tubulointerstitial injury on renal biopsy was also verified as a risk factor for clinical outcome of patients with pMN . However, it was not the independent risk factor after multivariate analysis. . Among the 57 patients with thyroid dysfunction, the eGFR of 4 (7.0%) patients showed a reduction of more than 25%, with no significant difference with that of patients with normal thyroid function . . Table .P = 1.000) and proteinuria . Nineteen patients got normal thyroid function during follow-up, including 14/23 patients with proteinuria remission and 5/11 patients with consistent proteinuria .During follow-up, 34/57 patients with thyroid dysfunction had the thyroid function re-evaluated. Among them, we found 10 patients received the thyroid hormone replacement, 7 of whom were patients with overt hypothyroidism. Compared to the patients with replacement, patients without replacement had a similar remission of both thyroid dysfunction  patients with pMN suffered from thyroid dysfunction, and subclinical hypothyroidism 48/420 (11.4%) was the dominant disorder. These results were consistent with other studies in patients with proteinuria , 6, 7. H% patientUrine loss of thyroid hormones linked with plasma protein was thought as the original reason for the hypothyroid dysfunction in patients with proteinuria –16. In tPrevious studies found membranous nephropathy was also the most common pathological type in the autoimmune thyroiditis associated glomerulonephritis . Both thMegalin is a protein expressed on the apical surface of thyroid epithelial cells , which iBased on previous studies and the findings in our study, thyroid dysfunction is common in MN. But whether thyroid dysfunction had a worse impact in the clinical outcomes of patients with pMN was also meaningful. In the current study, our results showed that patients with thyroid dysfunction had worse renal function, which might be the result of change in renal blood caused by the hypothyroidism . BecauseThough our study also showed patients without thyroid hormone replacement had a similar remission of proteinuria to that of patient with replacement. A meta-analysis found thyroid hormone replacement therapy significantly increased the remission of nephrotic syndrome in patients with combined euthyroid sick syndrome . And ShiAs a retrospective study, there were some limitations in the current study. First, we did not perform IF/IHC of PLA2R or THSD7A, or other antigens on all the patients’ kidney tissue. Second, the autoantibodies associated with autoimmune thyroiditis were detected in only a few patients. Third, almost half of the patients with thyroid dysfunction were lost to follow-up and the change of thyroid hormones during the follow-up were not detected regularly. And this is a monocentric and single ethnicity study, multiple center studies were needed to validate our results.In conclusion, thyroid dysfunction is not uncommon in patients with pMN. Patients with combined thyroid dysfunction present with more severe proteinuria, lower level of serum albumin and worse kidney function at the baseline. And thyroid dysfunction might also predict a poor treatment response during follow-up. These findings indicated that thyroid dysfunction may be caused by the massive proteinuria or other immune factors, and could also exacerbate the glomerular damage in return.


The influence of thyroid-stimulating hormone (TSH) on gestational outcomes have been studied and checked whether differing TSH levels are relevant on human reproduction outcomes. International guidelines recommend TSH values <2.5 mIU/L in women trying to conceive, since values above this level are related to a higher frequency of adverse reproductive outcomes. This study aimed to evaluate whether TSH values correlate with different gestational outcomes in euthyroid infertile women without autoimmune thyroid disease.in vitro fertilization (IVF) treatment. The participants were divided into two groups: TSH 0.5-2.49 mIU/L (n=211) and TSH 2.5-4.5 mIU/L (n=45). The clinical data, hormonal profiles and reproductive outcomes were compared between groups. Additionally, a systematic review with meta-analysis following the PRISMA protocol was carried out in PubMed/MEDLINE, EMBASE, and SciELO, with no time or language restrictions, for articles comparing TSH groups named “low TSH”  and “high TSH” (≥2.5 mIU/L). A meta-analysis of proportions was performed with pooled estimates expressed as relative risk (RR) of events and a random effects model.A retrospective cohort study was conducted involving 256 women who underwent I²: 87%; τ2: 0.0544; p<0.01). The number of deliveries was not significantly different between groups, despite a trend towards higher frequency in the high-TSH group .Age, BMI, free thyroxine levels (FT4) hormonal profile and IVF outcomes were not different between groups, neither gestational outcomes (p=0.982). Also, no difference was observed when the TSH and FT4 levels were compared between patients with positive or negative gestational outcomes (p=0.27 and p=0.376). Regarding the systematic review with meta-analysis, 17 studies from 2006 to 2022 were included, and added by this original retrospective research comprising 13.247 women undergoing IVF. When comparing the proportions of clinical pregnancy between the TSH groups, no significant difference was found , with high between studies heterogeneity (Variation in TSH levels within the normal range was not associated with pregnancy and delivery rates in women, without autoimmune thyroid disease, who underwent IVF treatment.https://www.crd.york.ac.uk/prospero/, identifier CRD 42022306967. The mother is responsible for supplying the needs of the embryo and the foetus for thyroid hormones until the gland matures  has been associated with adverse reproductive and pregnancy outcomes, including infertility, miscarriages, prematurity, and congenital defects, even when FT4 is within values , 4, 5. I.5 mIU/L TSH and FT4 are used as the main parameters to evaluate thyroid function, taking into account that both undergo changes during pregnancy, and therefore their concentrations must have been well established before the gestational period , 8. AccuThere are treatment recommendations for TSH values >4 mIU/L; however, for values between 2.5 and 4.0 mIU/L, replacement therapy with LT4 is still considered, despite inconsistent or limited evidence reporting interference with gestational outcomes in this TSH range , 10.in vitro fertilisation/intracytoplasmic sperm injection (IVF/ICSI). The present original study was also followed by a systematic review of the literature with meta-analysis to investigate a possible association of TSH values in the 0.5–2.49 mIU/L and 2.5–4.5 mIU/L ranges with gestational outcomes in women undergoing IVF.In this context, a retrospective cohort study was conducted to evaluate whether the serum TSH values and other variables of interest are related to the gestational outcomes in euthyroid women without autoimmune diseases who underwent This retrospective study was conducted at the Geare Centre for Reproductive Medicine in Recife, Brazil, with data from patients seen between January 2010 and December 2017. The Research Ethics Committee of the Centre for Medical Sciences of the Federal University of Paraíba  was approved before to start the enrolment. Considering the retrospective nature of the study, and the consistency with the REporting of studies Conducted using Observational Routinely-collected health Data (RECORD) Statement without any experimental intervention by the researchers, and the anonymized data collection, a formal informed consent was waived from by the Research Ethics Committee. The study was not advertised, and no remuneration was offered to the patients to enter or continue the study. An independent data safety and monitoring committee evaluated the ad interim and final results of this study. Over the study period, there were no significant differences in the facilities available for patient care and in the referral patterns of our service.All the women evaluated in this study underwent IVF/ICSI. The clinical data, hormonal profiles, and reproductive outcomes relevant to the research were extracted from the participants’ medical records. The inclusion criteria were women aged ≤40 years, follicle-stimulating hormone (FSH) ≤12.0 IU/L, TSH ≥0.5 mIU/L and ≤4.5 mIU/L, both ovaries present without morphological abnormalities, and no history of endocrine diseases, such as hyperprolactinemia and polycystic ovary syndrome. The exclusion criteria were patients with autoimmune disease, with previous history of ovarian surgery, who had undergone radio/chemotherapy, or who had received previous hormonal treatment, including Levothyroxine Sodium (LT4) tablets.post-hoc power analysis, considering a two-tailed test, with an effect size of 0.5 and alpha 0.05 that resulted in a Power (1 – beta) of 0.843.The sample size used was based on convenience, according to the digital medical records database available to the authors. A The cause of infertility was investigated according to established diagnostic procedures for infertile couples, including semen analysis, serological tests, hormonal profile, thyroid peroxidase antibody test, tests for sexually transmitted infections, and imaging examinations  , 12. HysThe serum FSH and luteinising hormone (LH) concentrations were obtained during the follicular phase of the menstrual cycle, whereas the prolactin levels were measured during the luteal phase. The serum levels of TSH, FT4 were detected before controlled ovarian stimulation (COS) for IVF/ICSI.Controlled ovarian stimulation (COS) was initiated between the first and third day of menstrual cycle using follitropin alpha 300 IU/day + lutropin alpha 150 IU/day  for an average period of 10 days. From the sixth day of COS, when at least one follicle with a minimum diameter of 14 mm was identified, a gonadotropin release hormone (GnRH) antagonist was initiated, namely cetrorelix acetate 0.25 mg/day , concomitantly with the use of gonadotropin. When at least three follicles of size 17–22 mm were identified, oocyte maturation was triggered with GnRH analogues leuprorelin acetate 1 mg subcutaneously . After 36 h, an ovarian puncture was performed for oocyte retrieval.Four hours after oocyte retrieved, IVF/ICSI was performed, and the embryos were kept in a culture medium until the blastocyst stage, lasting between 5 and 7 days. The embryos were subsequently frozen by the vitrification method.The patients were prepared for the embryo transfer with oestradiol valerate 6 mg/day  starting between the first and third day of the menstrual cycle, after ultrasonographic evaluation. After 10–12 days, a new transvaginal ultrasonographic evaluation was performed. Additionally, if the endometrial thickness was above 7 mm, oestradiol levels were between 200 and 300 pg/mL, and the progesterone levels were <1 ng/mL, then vaginal progesterone 800 mg/day  was started 5 days prior to the transfer of the thawed embryo. A maximum of two embryos were transferred in each procedure.The primary gestational outcome was clinical pregnancy, confirmed by the presence of a gestational sac and foetal heartbeat by a transvaginal ultrasound performed four weeks after the embryo transfer. The secondary gestational outcome was the number of live births.Statistical analyses were performed using RStudio software, version 1.1.383 . Data normality was assessed using the Shapiro–Wilk test. The descriptive analysis was presented as absolute and relative frequencies, and numerical variables were presented as medians and interquartile ranges (IQR), as none of the data presented normal distribution. Either the Mann–Whitney U test or the chi-square test was used to analyse the differences in the hormone levels and reproductive outcomes between the groups. Logistic regression was used considering the primary and secondary outcomes as response variables. The level of statistical significance was set at p<0.05.A systematic review was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA)  guidelinWe selected observational studies with women undergoing IVF/ICSI for those were available data about treatment outcomes and hormonal profiling that presented dosages of at least TSH and FSH for subgroups with different TSH levels. The exclusion criteria were studies comprising patients with autoimmune disease or were under hormonal treatment for thyroid diseases. The primary outcome was clinical pregnancy, and the secondary outcome was the number of live births.The databases searched were MEDLINE, EMBASE, and SciELO using their respective search engines.In Vitro Fertilization”(Title/Abstract) OR “ICSI”(Title/Abstract) OR “Intracytoplasmic sperm injection”(Title/Abstract) OR “Assisted reproduction techniques”(Title/Abstract) OR “ART”)(Title/Abstract)) NOT (“autoimmunity” OR “cancer” OR “neoplasia” OR “neoplasm” OR “carcinoma”). Search strategy for SciELO is closely related to the MEDLINE. Search strategy for Embase is presented as Supplement.The search term and the combination were ((“Thyroid”(Title/Abstract) OR “TSH”)(Title/Abstract) AND (“infertility”(Title/Abstract) OR “IVF”(Title/Abstract) OR “Titles and/or abstracts of studies retrieved were screened independently by two review authors (BB and EM) to identify studies that potentially meet the aims of the systematic review. The full text was retrieved and independently assessed by two authors (EM and RAMN). Any disagreement over the eligibility articles was resolved through discussion with a third collaborator (CPB). No translation was needed during the process. All the process was performed without any kind of automated or machine learning process.Data extraction was performed using an electronic form by two independent investigators (RN and LE), with disagreements resolved by a third investigator (VR). No automation tools were used.The data retrieved were author and date, study design, total and stratified sample in the study groups, primary and secondary outcomes of the groups of interest. No data imputing was performed.th, 2022.Studies considered eligible for synthesis were those containing data according to the eligibility criteria of patients and that were possible to extract data, disregarding the primary study aim. No restriction to language or publication date was imposed, retrieveing studies up to May 18The absolute number of observed events was used to calculate the proportions and the random effects model. The measures of concentration were standardised to the same unit, and the values were transformed into means and standard deviations when presented in a different unit .The data was presented in a summary of evidence and synthesis as forest plots, with studies ordered by publication year.Two analyses were performed. The first one compared groups according to the TSH level, <2.50 mIU/L (labeled ‘low TSH’) and ≥2.5 mIU/L (labeled ‘high TSH’) as thresholds, considering clinical pregnancy as the main outcome, using the Risk Ratio for binary outcomes. The second analysis was a calculation of an overall mean of TSH levels presented in each study as continuous variables, using the log transformed means and backtransformed to the original measurement scale.2) with 95% confidence intervals (95% CI). The meta-analysis of single means was performed using the inverse variance method, the Hunter-Schmidt method to estimate the between-study variance (τ2) and its square root (τ), presented with 95% confidence intervals (95% CI). In both cases, the Knapp-Hartung adjustments were used to calculate the confidence interval for the summary effect due to the expected variance for the limited number of studies. In both cases, heterogeneity was estimated by the I2  clinical pregnancy, (b) mean TSH, (c) mean FSH, and (d) live birth rate. The results were presented as regression coefficients with 95% confidence intervals (CI), association meta , using the meta  and the packages .The risk of bias assessment was performed by two independent investigators, with disagreements resolved by a third part (CPB). We used the ROBINS-E tool for risk of bias assessment, and the results are presented as charts according to the defined by the authors . CertainA total of 1,016 women undergoing IVF/ICSI were evaluated for inclusion in this study. Of these, 256 women had the required data and met the inclusion and exclusion criteria. The thyroid function of euthyroid patients was evaluated by categorising the patients into two groups according to serum TSH levels: low-TSH group (<2.50 mIU/L) and high-TSH group (≥2.5 mIU/L). The clinical and hormonal parameters and gestational outcomes according to the TSH levels are presented in To analyse the profile of women with favourable and adverse gestational outcomes, the patients were dichotomised according to the primary reproductive outcome into the groups: Clinical Pregnancy Yes and No. The data are shown in So, we stratified the women according to the age into two groups: women aged ≤35 years (n=154) and women aged >35 years (n=102). As expected, women aged ≤35 years presented significantly lower FT4 (p=0.002), and greater AFC (p=<0.001), oocytes retrieved (p=0.029), MII (p=0.015), embryos (p=0.002); clinical pregnancy rate (p=0.019), and live births (0.041) (data not shown).The Spearman’s correlation test for serum TSH and FT4 levels of euthyroid women and variables of interest associated to clinical pregnancy showed no statistically significant correlations Table 3,A total of 17 articles were included in the meta-analysis, comprising 13.247 women undergoing IVF/ICSI . According to the ROBINS-E tools, only one study presented critical risk of bias . The dom2: 78%; τ2: 0.0544; p<0.01).An analysis of proportions between the 0.5–2.49 mIU/L and 2.5–4.5 mIU/L TSH groups in relation to the primary outcome of clinical pregnancy revealed a very small difference between the two groups’ gestational outcomes , although there was a greater tendency for pregnancy in patients with TSH between 2.5 and 4.5 mIU/L , as observed in The comparison between the TSH groups regarding deliveries showed a higher occurrence of this outcome in the high-TSH group , with moderate heterogeneity   and the Meta-analysis of Observational Studies in Epidemiology (MOOSE) guidelines. This study is registered in International Prospective Register of Systematic Reviews (PROSPERO) under the CRD 42022306967 and it was approved by the Research Ethics Committee of the Centre for Medical Sciences of the Federal University of Paraíba . Signing the Informed Consent Form was waived by the Research Ethics Committee.Conceptualization: VD’A and BB. Methodology: VD’a, EM, RO and BB. Software: EM. Validation: EM, RO and BB. Formal analysis: VD’A, EM and BB. Investigation: VD’A, EM, LD’A, MC and RN. Resources: MC, EM and BB. Data curation: VD’A, EM and RN. Writing – original draft preparation: VD’A, LD’A and RN. Writing – review and editing: BB, RO, DC, AL and CB. Supervision: BB and RO. Visualization: VD’A, EM and BB. Project administration: BB. All authors critically reviewed the manuscript and approved the final version of the manuscript. All authors contributed to the article and approved the submitted version.We would like to thank all the individuals who participated in this study and supported this research.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.


HGS was independently and inversely associated with AIx in patients with type 2 diabetes. Patients with diminished HGS should be subjected to intensive exercise therapy for reducing the risk of arterial stiffness and cardiovascular disease.Handgrip strength (HGS) is a measure of overall skeletal muscle strength and is used to identify risks for cardiovascular disease and mortality. Furthermore, HGS is an indicator of arterial stiffness that leads to atherosclerotic cardiovascular disease. This study aimed to examine the relationship between HGS and augmentation index (AIx) in patients with type 2 diabetes. A cross-sectional study was conducted to examine patients with type 2 diabetes whose HGS and AIx were measured in our hospital. AIx was measured noninvasively using an applanation tonometer, and multiple regression analyses were conducted to assess the independent relationship between HGS and AIx. This study included 404 patients. After adjusting for age, gender, body mass index, duration of diabetes, smoking and exercise habit, biochemical parameters, and physiological parameters related to arterial stiffness, HGS was found to be independently and inversely associated with AIx (β = − 0.270, Trial registration: UMIN000023010. A 10-year follow-up of the Look AHEAD  study has shown that low cardiorespiratory fitness is associated with an increased risk of atherosclerotic cardiovascular disease (hazard ratio (HR) = 1.94; 95% confidence intervals (CI), 1.12–3.35) in patients with T2DM4. On the other hand, cardiorespiratory fitness is positively associated with muscle strength measured by handgrip test in women5. Albin et al.6 reported that individuals with high cardiorespiratory fitness and strong handgrip strength (HGS) had a lower prevalence ratio of arterial stiffness  than those with low cardiorespiratory fitness and weak HGS. Therefore, HGS may be an indicator for arterial stiffness that leads to atherosclerotic cardiovascular disease. HGS is a simple method for evaluating overall skeletal muscle strength and is also a useful tool for identifying risks for T2DM7, cardiovascular disease (CV), hospitalization, and mortality9, which provides important information on a variety of health outcomes in clinical practice.Type 2 diabetes (T2DM) has become a pandemic, which is a serious problem worldwide. Patients with T2DM show lower cardiorespiratory fitness10. AIx is considered a vascular parameter showing the deleterious effect on cardiac workload of increased wave reflection related to arterial stiffness11. Previous studies have reported that AIx is higher in subjects with diabetes13 and hypercholesterolemia14, and predicts myocardial ischemic threshold in patients with coronary artery disease15. Moreover, a 10% increase in AIx is associated with all-cause mortality  and CV mortality  in patients with end-stage renal failure16. Not brachial-ankle pulse wave velocity (PWV) but heart rate-adjusted AIx is reduced by hemodyalysis, suggesting that AIx reflects large artery stiffness more precisely than PWV17.The augmentation index (AIx) is a measure of systemic arterial stiffness and risk factor for CV19 and few studies have investigated it in a T2DM population. If HGS has a significant relationship with AIx in patients with T2DM, HGS could also be a useful indicator for large artery stiffness. Thus, the present study aims to examine the associations of HGS with AIx in patients with T2DM.To date, there have been several studies that examine the association between HGS and AIx; however, the results are controversialWe conducted a cross-sectional study in patients with T2DM who were treated at the National Center for Global Health and Medicine Kohnodai Hospital. A total of 406 individuals whose HGS and AIx were measured at the medical examination (the same consultation day) were included. Trained technicians at the Clinical Research Center of the National Center for Global Health and Medicine at Kohnodai Hospital asked participants at the outpatient clinic about the subjects’ history of CV events, smoking habit, and exercise habit such as resistance training and walking. Patients with type 1 diabetes (n = 2) were excluded. The opt-out method of obtaining informed consent was used as this study was a retrospective analysis of the existing data. The study protocol was approved by the Medical Ethics Committee of the National Center for Global Health and Medicine (Reference No. NCGM-G-002052), and the study was performed in accordance with the Declaration of Helsinki.75) were measured noninvasively using an applanation tonometer . Patients put their left forearm on the elbow placement board of the device in a sitting position. Left radial pulse waves and left brachial blood pressures were automatically recorded. After recording the radial and aortic pulse waveforms, cSBP was automatically estimated using a linear equation by the device20.Height was measured using a rigid stadiometer . Weight was measured using calibrated scales . Body mass index (BMI) was calculated as body weight in kilograms divided by the square of body height in meters. Waist circumference was measured at the umbilical level at the end of exhalation in a standing position. HGS was measured using a Smedley analog hand dynamometer  twice, using both hands in a standing position. We utilized the average HGS in kilograms. Peripheral arterial stiffness was examined by measuring the PWV using a pulse pressure analyzer , and central systolic blood pressure (cSBP) and the AIx corrected for a heart rate of 75 bpm  using the revised equation adjusted for the Japanese population24.We measured plasma glucose (PG), hemoglobin A1c (HbA1c), B-type natriuretic peptide (BNP), serum high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) levels. Recent studies identified LDL-C-to-HDL-C ratio (LDL-C/HDL-C) as a better biomarker to predict both atherosclerosis progression and CV than HDL-C or LDL-C alonehttps://www.psychologie.hhu.de/arbeitsgruppen/allgemeine-psychologie-und-arbeitspsychologie/gpower). Effect size was set at 0.02 according to Cohen’s definition25. The number of predictors was assumed to be 10 , the one-tailed alpha level is 0.025, and the sample size calculation indicated that at least 395 subjects were needed for a power of 0.8. This suggests that our sample size had sufficient power to detect the association between HGS and AIx.Sample size calculation was performed using G*Power (75 (dependent variable). The regression modeling was performed in three steps with age, gender, BMI, smoking habit, and duration of diabetes being included as possible confounders in the model 1, and further adjustments for exercise habit, HbA1c, and LDL-C/HDL-C (model 2), and finally, further adjustments for eGFR, BNP and cSBP (model 3). In addition, p values of < 0.05 determined by performing a two-sided test were considered as indicating statistical significance.Statistical analyses were performed using SPSS version 25 . All numerical variables are expressed as the mean ± standard deviation. Pearson’s correlation coefficient was calculated to analyze the associations of HGS with the clinical data. Mann–Whitney U test or Student’s t test, depending on the normality test results (Shapiro–Wilk test), was performed to detect significant differences between men and women, as appropriate. χ2 test was also performed to identify significant differences in categorical variables between men and women. Multiple regression analysis was performed to test independent associations between HGS (independent variable) and AIx2. Sixty patients (14.9%) had a history of CV. Eighty-six patients (21.3%) were current smokers. Two-hundred and three patients (50.2%) engaged in habitual exercise. Two-hundred and sixty-seven patients (66.1%) received glucose-lowering drugs, one hundred and twenty-three patients (30.4%) received statins, one hundred and twenty-four patients (30.7%) received renin-angiotensin system inhibitors, and fifty-eight patients (14.4%) received antiplatelet drugs. Demographic, anthropometric, and clinical data are listed in Table This study enrolled 404 patients (231 men and 173 women) with T2DM. The mean age and duration of diabetes of study participants were 63.1 ± 14.3 and 11.2 ± 11.8 years, respectively. The mean BMI was 25.7 ± 5.7 kg/m75; on the other hand, women had lower HGS than men. The number of smokers and cigarettes per day were larger in men than in women.Table p < 0.001), duration of diabetes , HDL-C , PWV , AIx75  . In contrast, HGS was positively correlated with BMI , waist circumference , eGFR , and LDL-C/HDL-C .HGS was inversely correlated with age .The adjusted associations between HGS and AIxp = 0.009).Furthermore, HGS was significantly lower in subjects with a history of CV than in subjects without  6, IL-8, IL-15, and myostatin) are altered in human skeletal muscle cells cultured from patients with T2DM39. A recent study showed that aerobic exercise training increased a myokine, irisin secretion, which was associated with improvement of arterial stiffness in rats with obesity40. To elucidate the common mechanism for decline in muscle fitness and progression of arterial stiffness, further investigations are required. However, given that muscle strength is a modifiable risk factor for CV, exercise therapy aiming to increase muscle strength can be effective for reducing the risk of atherosclerotic CV in patients with T2DM41.AIx measures the augmentation of central aortic pressure by a pulse wave reflection42 and N-terminal pro BNP levels in patients with peripheral artery disease43. We also observed the positive correlation between AIx75 and BNP  in this study. A possible mechanism explaining this relationship is that arterial stiffness has an impact on diastolic dysfunction44. The progression of arterial stiffness increases pulse wave velocity generated by left ventricular contraction, which enhances the early return of wave reflection to the heart and the augmentation of central aortic pressure. As a result, the cardiac afterload increases and then may cause diastolic dysfunction and/or left ventricular hypertrophy42. Considering that the prevalence of left ventricular diastolic dysfunction in patient with T2DM is significantly high (35%)45, the relationship among HGS, arterial stiffness, and BNP that is a prognostic marker for heart failure should be further investigated. Both HGS and BNP are predictive for CV events in patients with T2DM46. As mentioned above, skeletal muscle has been recognized as an endocrine organ that secretes myokines with a favorable effect on metabolism47. Irisin increases myocardial cell metabolism48. Fibroblast growth factor 21 increases adiponectin levels, which has a cardioprotective effect, and attenuates cardiac remodeling after myocardial infarction49. Myokines are promising for cardioprotective effects and may be the key to reveal the effect of skeletal muscle on arterial stiffness.The inverse correlation between HGS and BNP levels is intriguing. Previous studies showed that AIx was positively associated with the elevation of BNP levels in patients with paroxysmal atrial fibrillation50 reported that AIx was 77 ± 12% in men and 86 ± 12% in women, which is approximately equal to the result of the present study. The authors argued that the change in sex hormones in postmenopausal status might affect arterial stiffening in women. Indeed, the mean age of female subjects in this study was 63.7 years, and 81.6% of the women were 50 years and older. The higher AIx75 in women than men can be explained by the effect of postmenopausal status on arterial stiffness51.The gender difference in AIx was consistent with the finding of a previous study. Rosenbaum et al.52. In the present study, AIx75 was positively correlated to age ; however, AIx75 was inversely associated with the duration of diabetes in the multiple regression models. As such, we divided study subjects by the median value of the duration of diabetes and reanalyzed the differences in clinical data between groups. Then, we found that the subjects with a long (≥ 8 years) duration of diabetes had more exercise habits  and lower BMI  compared with subjects with a short duration of diabetes (< 8 years). No significant difference between groups in glycemic control, lipid profile, blood pressure, and the smoking habit was identified. The indicators of obesity, including BMI, have a positive relationship with arterial stiffness53. Excess adipose tissue is a key determinant of the development of hypertension, endothelial dysfunction, and arterial stiffness54. Moreover, regular exercise improves arterial stiffness measured by carotid intima-media thickness and PWV in patients with T2DM55. Even short-term, aerobic exercise effectively decreases arterial stiffness in radial, femoral, and carotid arteries in patients with T2DM57. Therefore, regular exercise habits and less body fat in subjects with longer duration of diabetes could contribute to a decrease AIx in this study population.Generally, arterial stiffness measured by AIx progresses with aging58 and Duran et al.59 reported that skeletal muscle mass was inversely correlated with HDL-C levels, on the other hand, higher HGS was related to higher HDL-C levels in a middle-aged population60. In this study, we observed inverse correlations of HGS with HDL-C levels and LDL-C/HDL-C. Excess adiposity is responsible for low HDL-C levels61; thus, obesity could probably explain this inconsistency. HGS is a useful tool for evaluating overall muscle mass; however, there is yet no consensus on how HGS should be used in clinical practice62. For example, there have been studies showing that not absolute but relative HGS such as HGS/BMI that takes the influence of obesity into consideration might be a better indicator for muscle weakness and the risk of cardiometabolic diseases64. Then, we re-analyzed the associations between HGS/BMI and clinical data. The associations of HGS/BMI with HDL-C  and LDL-C/HDL/C  were not significant. In addition, the adjusted associations between HGS/BMI and AIx75 were as follows: model 1, β = − 0.140, p = 0.025; model 2, β = − 0.201, p = 0.009; model 3, β = − 0.223, p = 0.022, respectively. There were still significant and inverse associations between HGS/BMI and AIx75 in model 1, 2, and 3. Meanwhile, the ability of HGS to predict health outcomes may not be different between absolute HGS and relative HGS65. How HGS should be used in clinical studies has been disputed8, and further validation studies will be needed.The relationship between skeletal muscle mass/strength and HDL-C levels is controversial. Pietrobelli et al.75 may not prove the presence of large artery stiffness. However, Betge et al.66 reported that measuring AIx was suitable for detecting atherosclerosis as compared to undergoing coronary angiography. The lower HGS in subjects with a history of CV in the present study and the strong evidence that HGS was associated with an increased risk of CV events8 suggests that HGS could be a non-invasive indicator for arterial stiffness. Additionally, König et al.27 recently showed that low HGS and increased arterial stiffness are closely tied to each other. HGS could still be a precise measurement for arterial stiffness.There are several limitations need to be addressed. First, this study is a single-center study, and the results may not be generalized to other populations. Second, the cross-sectional design cannot be used to infer causality. A prospective study is warranted to clarify the mechanisms regarding the relationship between HGS and the progression of arterial stiffness. Third, medications used to treat T2DM, hypertension, and dyslipidemia may intricately influence AIx and cardiovascular parameters. The possibility that medications affect the relationship between HGS and AIx cannot be excluded. Fourth, AIx is a surrogate marker for systemic arterial stiffness; thus, the inverse relationship between HGS and AIxIn conclusion, HGS was independently and inversely associated with arterial stiffness represented as AIx in patients with T2DM. This finding will shed a light on the mechanism behind the increased CV mortality in T2DM patients who have lower skeletal muscle mass/strength compared with healthy individuals. Although, there is yet no standard method for using HGS in clinical practice, T2DM patients who have lower HGS could be targeted by an intensive exercise therapy to reduce the risk of arterial stiffness and CV.


Triglyceride-glucose (TyG) index has been proposed as a simple and credible surrogate for insulin resistance and an independent predictor of cardiovascular outcomes. Due to lack of data on TyG index in stroke, we aimed to evaluate the predictive value of the index for recurrent vascular event risk among stroke patients.This was a prospective observational study, in which 866 patients  with ischemic stroke (n = 781) or transient ischemic attack (n = 85) within 1 week of onset were consecutively enrolled and followed up for 1 year. The TyG index was calculated as ln (fasting triglycerides [mg/dL] × fasting glucose [mg/dL]/2). Patients were divided into 3 groups according to the tertile of TyG index levels: tertile 1, < 8.48; tertile 2, 8.48–9.01; and tertile 3, > 9.01. The primary outcome was a composite of major adverse cardiovascular events (MACE), including nonfatal stroke, nonfatal acute coronary syndrome, and vascular death.P = 0.032) and intracranial (P = 0.003) atherosclerotic stenosis. There were significant differences in the MACE risk between the three groups . After multivariable adjustments, the TyG index remains to be a significant predictor of MACE, with an adjusted hazard ratio for tertile 3 versus tertile 1 groups  of 2.01 (1.16–3.47). Similar results were also found for the risk of recurrent stroke.The median TyG index was 8.74 . Higher levels of TyG index were significantly associated with increased prevalence of ipsilateral extracranial carotid (TyG index is associated with cervicocerebral atherosclerosis and the MACE risk after a stroke, suggesting the potential value of TyG index to optimize the risk stratification of stroke patients.Trial registration URL: https://upload.umin.ac.jp. Unique identifier: UMIN000031913.The online version contains supplementary material available at 10.1186/s12933-022-01695-2. Insulin resistance (IR), a hallmark of metabolic syndrome, is an important risk factor for stroke . The curRecently, a novel parameter named triglyceride-glucose (TyG) index, derived from fasting triglyceride (TG) and glucose levels, has been proposed as a simple and reliable surrogate marker of IR . Prior shttps://upload.umin.ac.jp). The data supporting the findings of this study are available from the corresponding author upon reasonable request.The Tokyo Women’s Medical University (TWMU) Stroke Registry is an ongoing, prospective, single-center, observational study that enrolled patients with acute ischemic stroke or transient ischemic attack (TIA) within one week of onset, hospitalized at our center , 17. TheOf 882 patients consecutively assessed for eligibility between December 2013 and January 2020, ten were excluded as their final diagnosis was a stroke mimic, more than one week had elapsed after stroke onset, or duplicate registrations were found. In addition, we excluded six patients who had no lipid or glucose profile data at baseline, leaving 866 patients  in the sample , management , and the occurrence of clinical events after the qualifying event, using a structured case report form. Upon admission, neurological symptoms were assessed using the National Institutes of Health Stroke Scale (NIHSS) score.The TyG index was calculated as ln (fasting TG [mg/dL] × fasting glucose [mg/dL]/2) [The TG and glucose levels were assessed after overnight fasting within 48 h of admission. Because the normal range of TyG index has not been well defined, we divided the patients into three groups according to tertiles of the TyG index: tertile 1, < 8.48; tertile 2, 8.48–9.01; and tertile 3, > 9.01.Intracranial arteries were examined using time-of-flight magnetic resonance angiography (n = 823) and/or computed tomography angiography (n = 183). The narrowest diameter of each stenosed vessel was measured and divided by the diameter of the normal vessel proximal to the lesion, or distal to the lesion if the proximal artery was diseased. Significant intracranial artery stenosis (ICAS) was defined as ≥ 50% stenosis or occlusion. ICAS was considered symptomatic if the stenosis was ipsilateral to the index stroke/TIA.Extracranial carotid atherosclerosis was examined using ultrasonography (n = 813), and/or computed tomography angiography (n = 105), and/or time-of-flight magnetic resonance angiography (n = 78). We defined significant extracranial artery stenosis (ECAS) as the presence of atherosclerotic stenosis of ≥ 50% or occlusion according to the European Carotid Surgery Trial criteria . ECAS waAortic atherosclerosis was assessed using transesophageal echocardiography (n = 260). Mobile plaques were defined as mobile components seen swinging on their peduncles. An ulcerative plaque was diagnosed as a discrete indentation of the luminal surface of the plaque with a base width and maximum depth of at least 2 mm each. Complex aortic atheroma were defined as any plaque ≥ 4 mm in thickness or a plaque with ulceration or mobile components .Cerebral small vessel disease was assessed using fluid-attenuated inversion recovery images acquired using a 1.5 T magnetic resonance imaging scanner  (n = 821). Degrees of periventricular hyperintensity and deep white matter hyperintensity were rated according to a published definition (0–4 for each hemisphere) . The higAn etiologic subtype of the index event was assigned to each patient using the atherosclerosis, small vessel disease, cardiac pathology, other definite causes, and dissection (ASCOD) grading system . The ASCFollow-up visits were scheduled at three months, one year, and three years after enrollment. The present study reports the one-year outcomes. At each follow-up visit, treatments, occurrence of clinical events, and modified Rankin Scale scores were recorded. If the patient could not be contacted for follow-up, a relative or caregiver was interviewed via telephone. The primary outcome was a composite of major adverse cardiovascular events (MACE), including nonfatal stroke (either ischemic or hemorrhagic), nonfatal acute coronary syndrome, major peripheral artery disease, and vascular death. Vascular death was defined as fatal acute coronary syndrome, fatal stroke, and other cardiovascular deaths including pulmonary embolism and sudden cardiac death. The secondary outcomes included the presence of cervicocerebral artery stenosis and the occurrence of any stroke (either ischemic or hemorrhagic) and all-cause death.P < 0.10 in univariable analysis  were included in the multivariable adjustments. Data for patients with no information at one year were censored at the time of the last available follow-up. For a given outcome, patients who died from causes other than the outcome were censored at the time of death. The Gray’s test was applied in the analysis of the cumulative incidence by treating death as a competing risk. Sensitivity analyses were conducted to assess the effect of TyG on vascular risk according to the etiological subtype of the index event. The area under the receiver operating characteristic curve (AUC) was used to describe the predictive value of the TyG index for MACE. The optimal cut-off was determined using the Youden index. For all analyses, statistical significance was set at P < 0.05. Statistical analysis was performed with JMP Pro version 16  and EZR version 1.61 .Continuous variables are expressed as mean (standard deviation) in cases of normal distribution, or median (interquartile range). Categorical variables are expressed as frequencies (percentages). Comparisons were made between multiple groups using the Jonckheere-Terpstra test for continuous variables, the Cochrane-Armitage test for categorical variables, and the log-rank test for censored variables. Event rates were estimated using the Kaplan–Meier method. Cox proportional hazard regression models were used to evaluate the associations between the TyG index and risk of recurrent vascular events by calculating hazard ratios and 95% confidence intervals (CIs). Age and all variables with A total of 866 patients  were included in the analysis. The median TyG index was 8.74 . Patients were classified into three groups according to tertiles of the TyG index . The mean (standard deviation) TyG index levels of the three groups were 8.11 (0.33), 8.74 (0.15), and 9.41 (0.49), respectively. Table Figure The treatments at discharge are presented in Additional file P = 0.001) and stroke (P = 0.004). The optimal TyG index cut-off for predicting MACE was 8.98 . There was no difference in the risk of all-cause death among the three groups. When patients were classified according to the etiologic subtype of the index event , whereas no differences were observed among those with small-vessel disease (log-rank P = 0.23) or cardioembolic stroke (log-rank P = 0.20).Among the 866 patients, 105 had at least one vascular event within one year, resulting in an event rate of 12.5% (95% CI 10.4–14.9%). As shown in Fig. In this prospective observational registry study involving Japanese patients with recent stroke or TIA, the TyG index was significantly associated with prevalence of intra- and extracranial artery atherosclerosis and was an independent predictor of future MACE. The prognostic value of the TyG index differed between the etiologic subtypes of stroke; a significant correlation between the TyG index and MACE risk was observed in atherothrombotic stroke but not in lacunar or cardioembolic stroke. Our results suggest the usefulness of TyG index as a simple risk predictor, allowing for more targeted secondary prevention.It is plausible that IR contributes to the development of vascular events through multiple mechanisms. The biological consequences of IR include hypertension, dyslipidemia, hyperglycemia, hyperinsulinemia, systemic inflammation, and vascular endothelial dysfunction, all of which can accelerate atherosclerosis . IR alsoThe TyG index can be easily and inexpensively obtained in clinical practice using routine blood tests. A previous study showed that the TyG index is a highly sensitive 96.5%) and specific 85.0%) method to detect IR, compared to the euglycemic hyperinsulinemic technique . In addi% and spe5.0% methThis study has several limitations. First, because the study participants were from a single-center cohort of Japanese patients, our results may not be generalizable to other populations. The annual MACE rate of 12.5% was higher than that reported in previous clinical trials , 29. ThiThe present study indicated independent associations of the TyG index, a surrogate for IR, with the risk of MACE and recurrent stroke in patients with recent stroke or TIA. The TyG index may be useful as a convenient marker for optimizing risk stratification for stroke or TIA.Additional file 1: Table S1. Examination and investigation findings.Additional file 2: Figure S1. Prevalence of atherosclerotic diseases.Additional file 3: Table S2. Medication use at discharge.