Prunus fruit species, especially for their adaptation in a global warming context. Numerous quantitative trait loci (QTLs) have been identified and a major one was previously located on LG4. The objectives of this study were to fine-map this QTL in sweet cherry, to identify robust candidate genes by using the new sweet cherry genome sequence of the cultivar “Regina” and to define markers usable in marker-assisted selection (MAS). We performed QTL analyses on two populations derived from crosses using cultivars “Regina” and “Garnet” as parents. The first one (n = 117) was phenotyped over ten years, while the second one (n = 1386) was evaluated during three years. Kompetitive allele specific PCR (KASP) markers located within the QTL region on LG4 were developed and mapped within this region, consisting in the first fine mapping in sweet cherry. The QTL interval was narrowed from 380 kb to 68 kb and candidate genes were identified by using the genome sequence of “Regina”. Their expression was analyzed from bud dormancy period to flowering in cultivars “Regina” and “Garnet”. Several genes, such as PavBOI-E3, PavSR45a and PavSAUR71, were differentially expressed in these two cultivars and could be then considered as promising candidate genes. Two KASP markers were validated using a population derived from a cross between cultivars “Regina” and “Lapins” and two collections, including landraces and modern cultivars. Thanks to the high synteny within the Prunus genus, these results give new insights into the control of flowering date in Prunus species and pave the way for the development of molecular breeding strategies.Flowering dateis an important trait in  Prunus avium L.), breeding strategies for crop adaptation aim at the development of early or late blooming cultivars [In temperate fruit tree species, flowering date (FD) is a trait of main importance and highly dependent on the climate conditions of the production area. In sweet cherry  analyses on sweet cherry and other species belonging to is trait ,8–10. Mo regions . It is w regions ,8. Moreo regions , as welle almond , apricote almond  and peace almond . FD and  species .1 populations from crosses “Regina” × “Garnet” and “Regina” × “Lapins”, Castède et al. [1 populations of sour cherry (Prunus cerasus), among them two were located on LGs 1 and 4. This detection on sour cherry is relevant for QTL analyses in sweet cherry since half of the sour cherry genome is derived from sweet cherry, the sour cherry genome being divided in two subgenomes from two Prunus species, “avium” subgenome and “fruticosa” subgenome [1 and two F2), most of them obtained from the early blooming cultivar “Cristobalina”, with low CRs [Although QTLs for FD were detected on all linkage groups (LGs) in sweet cherry, major loci were located on LGs 1 and 4 ,9. Usinge et al.  detectede et al.  identifiubgenome . Calle e low CRs , and det low CRs .DORMANCY-ASSOCIATED MADS-box (DAM) genes. These genes, six in number (DAM1–6), were initially studied in the non-dormant evergrowing peach mutant (evg), which presents a deletion in this region and does not cease growth to enter dormancy despite dormancy-inducing conditions [DAM 5 and 6 were considered to be strong candidate genes for the QTLs for FD and CRs on LG1 [Candidate genes have been suggested for QTLs on LGs 1 and 4 –20. The nditions ,19. In ss on LG1 .Prunus species to create new cultivars well adapted to the future climatic conditions.The aims of this study were i) to detect stable QTLs across numerous years for FD, ii) to fine-map the major QTL on LG4, iii) to identify candidate genes within the reduced QTL interval and characterize their expression, and iiii) to develop markers usable for breeding selection. This work should contribute to increase the efficiency of breeding programs for sweet cherry and other Populations #1 and #2 , as wellFD was highly dependent on the year of evaluation . In Popust), “Regina”: 106 and 101 JDs)  .Flowering stages  were highly correlated in both populations (from 0.59 to 0.97 in Population #1 and from 0.66 to 0.94 in Population #2) . Correla2) for each flowering stage in both populations. In Population #1, H2 was equal to 0.96 for BF, FF and EF. In Population #2, heritabilities were lower: 0.90 for BF, 0.88 for FF  and 0.77 for EF .We calculated broad-sense heritability (H(<lmxll>), 446 heterozygous in “Garnet” (<nnxnp>), and 575 were heterozygous in both parents (<hkxhk>). As we constructed parental linkage maps and not a consensus map of both parents, we did not use the heterozygous markers in both parents. Genetic maps of each parent are described in New genetic maps of “Regina” and “Garnet” were constructed using a subset of Population #2 (454 R × G hybrids). After filtering SNPs according to their quality and low missing data, 1619 SNPs were retained. Among these, 598 SNPs were heterozygous in “Regina” QTL analyses were performed for BF, FF and EF in both populations, with year-by-year and multi-year approaches. Due to the high correlation between the three flowering stages, only the QTLs for FF are presented, as in Castède et al. , and aremqP-FD4.1), R7 (mqP-FD7.1), G1 (mqP-FD1.2) and G6 (mqP-FD6.2) explained more than 5% of the phenotypic variation in the multi-year analysis.Concerning Population #1, QTLs were found on all LGs of “Regina” and on LGs G1, G2, G3, G5, G6 and G8 of “Garnet” using year-by-year and multi-year analyses  and S6. mqP-FD4.1) and G1 (mqP-FD1.2) explained more than 5% of the phenotypic variation  were only detected with Population #1 and QTL on LG G7 (mqP-FD7.2) was only found with Population #2.With Population #2, QTLs were detected on almost all LGs of both parents: all LGs of “Regina” (R1 to R8) and LGs G1, G2, G3, G7 and G8 of “Garnet”  and S7. ariation . QTLs onmqP-FD4.1, explained the largest PVE  and less than 0.5 cM (378 518 bp) in Population #2  were genotyped with nine KASP markers: KASP_9.269, KASP_9.271, KASP_9.456, KASP_9.727, KASP_9.780, KASP_9.890, KASP_9.935, KASP_10.090 and KASP_10.159  and S4. Recombinant individuals were grouped into 12 distinct recombinant genotypes  depending on the position of the recombination events  and S8. Non-recombinant individuals homozygous for all the 17 KASP markers, as “Garnet”, presented a FD similar to “Garnet” itself, with FF lsmeans equal to 90.5 and 90.7 JDs, respectively . In compAmong the 41 recombinant individuals, eight presented intermediate phenotypes that could not be clearly assigned to early or late flowering classes and consequently could not be used to fine-map the QTL . These iSeventeen individuals with Rec #3 and Rec #4 genotypes indicated that the QTL was located downstream (after) KASP_9.456: individuals that were homozygous from KASP_9.727 to KASP_10.159 (Rec #3) were early flowering while individuals that were heterozygous (Rec #4) were late flowering. FDs of these two recombinant groups  were statistically different  and KASP_10.159 (10 158 864 bp). We identified 65 predicted genes within this region of less than 380 000 bp  and KASP_9.958 (9 957 756 bp), covering around 68 kb. Twelve candidate genes were located within this new interval (shaded in grey in ectively .PavBOI-E3 was slightly more expressed in “Regina” than in “Garnet” during endodormancy of both cultivars (prior February). However, after “Garnet” dormancy release, expression of this gene significantly increased in this cultivar. After the dormancy release of “Regina”, the gene remained more expressed in “Garnet”. Levels of expression of PavSR45a increased during dormancy and after “Garnet” dormancy release in both cultivars, and decreased in both cultivars after “Regina” dormancy release. The expression decline was more important in “Garnet”. PavSAUR71 was not expressed during endodormancy and its expression started to increase after dormancy release in both parental cultivars, the increase being more important in “Garnet”. For these three candidate genes, expression profiles obtained in both RNA-seq analyses were consistent .1 population derived from the cross between cultivars “Regina” and “Lapins” , accessions from the germplasm collection (n = 104) and a set of cultivars (n = 51)  were used to genotype an F(n = 51) .Markers KASP_9.814 and KASP_9.916 presented a very low level of polymorphism in the two sets of accessions. In the germplasm collection, 92 accessions out of 104 were homozygous “G:G” (as “Garnet”) for KASP_9.814, and 100 were homozygous “G:G” (as “Garnet”) for KASP_9.916. In the set of 51 cultivars, 49 were homozygous “G:G” for KASP_9.814 and all were homozygous “G:G” for KASP_9.814. Therefore, we could not use them to conduct statistical analyses. For this reason, only the results obtained with KASP markers KASP_9.936 and KASP_9.958 are presented .In the R × L population, heterozygous individuals (52% of the population) were flowering 2.9 and 3.0 days later than homozygous ones (48%) for KASP_9.936 and KASP_9.958, respectively  . When coIn both germplasm collection and set of cultivars, a few individuals presented a new homozygous genotype (G:G for KASP_9.936 and T:T for KASP_9.958) . Due to Concerning the germplasm collection, for KASP_9.936, 43% of the individuals were heterozygous and 44% were homozygous as “Garnet” (A:A) . For KASConcerning the panel of 51 cultivars, most individuals presented the same genotype as “Regina” and “Garnet” as well. For KASP_9.936, 59% of the individuals were heterozygous and 37% were homozygous as “Garnet” (A:A). For KASP_9.958, the heterozygous/homozygous distribution was 55% and 41% of the individuals . Again, In conclusion, for both KASP markers, significant differences were observed between heterozygous individuals as “Regina” and homozygous individuals as “Garnet” in all three panels.Within both populations, FD was not stable across years. This trait is highly dependent on temperatures and can show important inter-annual variations, resulting from the variability of the chilling and heat accumulations across years. It is known that advances in FD can be explained by an early fulfillment of the HRs induced by high temperatures in late winter . In our The linkage maps developed using the RosBREED 6 + 9 K SNP array were compared with the ones previously obtained with the RosBREED 6 K SNP array . Sizes o1 population. These maps respectively contained 910 SNPs covering 636.7 cM and 789 SNPs covering 666.0 cM. However, only 324 and 310 SNP markers mapped at unique genetic positions in “Vic” and “Cristobalina” parental maps, respectively [Recently, Calle et al.  presenteectively . In our ectively . TherefomqP-FD5.1 and mqP-FD8.1). With Population #2, confidence intervals were further reduced with the multi-year detection analysis. Working with a population containing more individuals increased the power and the accuracy of the detection. QTL analyses were very consistent in both populations: the large majority of the loci detected with Population #1 were significant with Population #2 as well. The QTL mqP-FD6.2 on LG G6 was an exception. This QTL was detected with Population #1 across a few years and showed PVE values up to 14.6%, while it was not found in any of the analyses performed with Population #2. Because it was not stable across years, the significance of this QTL could be due to specific climatic conditions and/or genotype × environment interactions (in 2011 and 2016), which were not found in the three years of measurements in Population #2.Few QTLs for FD were detected within Population #1 with the single-year analyses, three on average, which could be related to the reduced size of the population. In comparison, more QTLs were detected with the single-year analyses within Population #2, nine on average, and confidence intervals were smaller. The detection was highly improved in Population #1 when combining all years together through the multi-year analysis. QTLs were detected on almost all LGs (thirteen), with some of them accounting for a very small proportion of the phenotypic variance, and confidence intervals of the major QTLs detected with the single-year analysis were reduced. Prior to our study, Castède et al.  performemqP-FD1.2) and R4 (mqP-FD4.1) were the only loci with PVE values higher than 5% to be found in common in both populations. Both were firstly identified in sweet cherry by Dirlewanger et al. [Prunus species including peach, apricot, almond and sour cherry [DAM genes [Overall, the large number of QTLs detected confirmed that FD is a complex trait with a polygenic control. Most of these QTLs were also found in peach and apricot . QTLs onr cherry ,13,16,27r cherry  and Popur cherry . In bothr cherry  detectedr cherry . In our AM genes ,18,20,28mqP-FD4.1 on LG R4. The high significance of this locus was demonstrated in Dirlewanger et al. [mqP-FD4.1: this QTL alone was significant across all the growing seasons. In both single and multi-year analyses, the accuracy of the analyses for this major QTL was significantly improved with Population #2. While the QTL region spanned 2 126 110 bp in Population #1 , it covered around 380 000 bp in Population #2, consisting in a reduction of more than 1.7 Mb. This result could be also explained by the denser genetic maps of the R × G Population #2 (obtained with the RosBREED cherry 6 + 9 K SNP array) as compared to those of Population #1 (obtained with the RosBREED cherry 6 K SNP array) and by KASP markers added in the region of the QTL.The QTL that explained the highest percentage of phenotypic variation within both populations was r et al.  using boOur study is the first one presenting QTL analyses using two different experimental designs obtained from a same cross, “Regina” × “Garnet”. It provides important information from a methodological point of view, as we were able to compare QTL analyses performed on a small population of 117 individuals evaluated for FD during ten years (Population #1) and a large population of 454 individuals evaluated for FD during three years (Population #2). When using a small population, single-year QTL analyses allowed to detect few QTLs within large confidence intervals. Multi-year analyses improved detections; however, as showed when comparing our results with those presented by Castède et al. , a high D locus within a 100 kb region), maturity date [Tssd gene in a 500 kb region), powdery mildew resistance [Vrn3 gene in a 270 kb region) and skin fuzziness [G locus in a 481 kb interval).The first objective of fine mapping is to narrow a given QTL region in order to reduce the number of candidate genes. This strategy has already been used in peach for several traits, among them fruit acidity   was identified in LG4 [While several studies have confirmed that the QTL for FD on LG1 contains AM genes –20,27,37d in LG4  but no cd in LG4 . In sweeEMF2 (EMBRYONIC FLOWER2) and NUA (NUCLEAR PORE ANCHOR), were included in the new map and were found far from our QTL region  by two different ways: BOI binds to CONSTANS (CO) to inhibit its targeting to FT; or BOI targets FT via DELLA proteins [FT mRNA and inhibit flowering. Based on our transcriptomic experiments, PavBOI-E3 was more expressed in “Regina” than in “Garnet” during endodormancy. This could be related to a stronger flowering inhibition in “Regina”. However, after “Garnet” dormancy release, PavBOI-E3 expression importantly increased in “Garnet” and became higher than in “Regina”, which is not anymore in accordance with the predicted function of the gene and its effect on FD. PavSR45a was also differentially expressed between “Regina” and “Garnet” especially after the endodormancy release period. This gene encodes the serine/arginine-rich (SR) protein SR45a, a splicing factor (PavSR45a). In Arabidopsis thaliana, the loss-of-function mutant sr45–1 exhibits pleiotropic phenotypes, among them a late flowering phenotype and elevated levels of FLOWERING LOCUS C (FLC), a major flowering repressor [FLOWERING WAGENINGEN (FWA), another flowering-related gene [PavSR45a was slightly more expressed in “Regina”, especially during the late sampling dates of both transcriptomic analyses. Moreover, the expression drop was faster in “Garnet”. We could hypothesize that several flowering-related genes are down-regulated by SR45. Hence, the lower expression level observed in “Garnet” could lead to an earlier flowering as compared to “Regina”. PavSAUR71 is also a promising CG. SAUR genes constitute the largest family of early auxin-responsive genes and play crucial roles in plant growth and development control [PavSAUR71 expression levels increased more importantly in “Garnet” and could be related to its early flowering phenotype, compared to “Regina”. Several genes encoded a G-type lectin S-receptor-like serine threonine-kinase (GsSRK) that regulates both plant architecture and salt stress responses [We could reduce our 46 candidate genes list down to a set of twelve genes based on fine mapping. Several genes, differentially expressed in “Regina” and “Garnet”, could be considered as promising, such as proteins . Both meepressor  within tepressor ,42. SR45ted gene . SR45 prted gene  and to nted gene . Therefoesponses . A gene esponses . HoweverRosaceae species peach, apple and sweet cherry, NAC TFs have been reported as candidate genes for a maturity date QTL located in a distinct region of LG4 [Arabidopsis, JUB1 TF represses GA3ox1 and DWF4 genes involved in gibberellins and brassinosteroids biosynthesis pathways, leading to reduced levels of these hormones and the accumulation of DELLA proteins, restricting plant growth while promoting stress tolerance [2O2 levels and also enhances various abiotic stress tolerance responses by targeting DREB2A [ABA3, JUB1 and COL3 were found to be under positive selection during apricot domestication, most likely for selection on tree phenology and environment adaptation [Although they were not located within the fine mapping confidence interval, some genes with relevant functions were worthy to consider. PAV04_REGINAg0203151 encodes the CONSTANS-LIKE 3 (COL3) transcription factor (TF) from the CCT  family. To date, genes from the CCT family are manly described in cereal crops, where they are involved in the control of flowering time in response to the photoperiod and the circadian clock –51. In tn of LG4 ,35,55. Iolerance . JUB1 deg DREB2A . Recentlaptation .1 population. Phenotypic differences between heterozygous and homozygous individuals were much more important in the R × L population (three days) than in both panels (between one and two days). It is known that the QTL on LG4 is the major locus in this population as well [For two KASP markers, KASP_9.936 and KASP_9.958, we found that the heterozygous accessions (as “Regina”) were significantly later flowering than homozygous ones (as “Garnet”) using two validation panels and a FIn the diversity panels (germplasm collection and cultivars), some individuals had an additional homozygous genotype compared to the population. In the set of 51 cultivars, those individuals  presented a late flowering phenotype. They were slightly later flowering than heterozygous individuals for both KASPs. This was in accordance with what we expected from allele combinations: for instance for KASP_9.958, homozygous C:C are early FD, heterozygous T:C are late flowering, and homozygous T:T are further late flowering. Different results were found in the germplasm collection. Firstly, more individuals presented the new homozygous genotype (6% for KASP_9.958 and 12% for KASP_9.936), confirming that a larger genetic diversity is present in the collection. Interestingly, these individuals were flowering earlier than those having the other homozygous genotype (as “Garnet”). One explanation could be that these individuals have the early allelic combination at other FD QTLs. Moreover, in these genetic backgrounds, the QTL on LG4 might not be the one explaining the largest PVE.FD is a quantitative trait controlled by many genomic regions. The major QTL on LG4 is one of them, but other regions such as LG1 play an important role too . TherefoSR45a) and auxin-response (SAUR71). Further analyses based on transformation experiments on model species could be performed to validate the robust candidate genes we identified and might give new insights into the control of FD in Prunus species. Moreover, our study provides relevant information from a methodological point of view by using a same cross in two different experimental designs to compare QTL analyses.We report in this study the first fine mapping performed in sweet cherry, for the major FD QTL on LG4. The QTL on LG4 was highly stable in our plant material, which has relatively high CRs and is rather late blooming. Therefore, this region is of main interest in sweet cherry breeding programs as well as the QTL on LG1 associated to low chilling cultivars to create new cultivars well adapted to their growing area. Our results constitute the first step for the development of a set of markers within the LG4 QTL that could be used in MAS for FD in sweet cherry. We reduced the 380-kb region obtained with the new QTL analyses using a large population to an 68-kb region containing only twelve candidate genes. The most likely candidate genes, with interesting expression patterns, were related to splicing  [For the KASP marker validation, a population derived from a cross using “Regina” and “Lapins” (R × L) and two sets of accessions were used . The R × France) . This pa France) . The secFor the RNA-seq analyses, flower bud samples were collected during two seasons from “Garnet” and “Regina” trees grown at the INRAE UEA in Toulenne (December 2009–March 2010) and Bourran (July 2015 – March 2016). As previously described in Vimont et al. , a mix oThree flowering stages were scored: beginning of flowering (BF), when approximately 10% of the floral buds reached full bloom; full flowering (FF), when 75% of the floral buds reached full bloom; end of flowering (EF), when more than 50% of the flowers were wilting. Trees were observed from three to four times a week during the season to score the different flowering stages in Julian days, JDs. Within Population #1, FD was evaluated during ten years from 2008 to 2017. The first five years of evaluation have been utilized in Castède et al. [For KASP validation, FD (BF stage) was scored during ten years in the R × L population, from 2006 to 2016 . The twoDaily temperatures were collected in the orchard located at Toulenne using an automatic data-logger  in order to characterize environmentally the years of evaluation.th 2015 to April 4th 2016 for “Garnet” and “Regina”. Branches were incubated in water pots placed in a growth chamber . The water was replaced every 3–4 days. After ten days under these forcing conditions, the percentage of bud break, i.e. flower buds at BBCH stage 53 [Measurements for the dormancy stages were performed on randomly chosen branches cut every two weeks from November 162), to measure the between-year stability of the flowering traits, was also estimated from the analysis of variance based on the following mixed model, as previously described in Dirlewanger et al. [Distribution, mean, minimum and maximum values of BF, FF and EF were estimated for each year. Additionally, Spearman correlation coefficients between the three flowering stages and between years were calculated. Analyses were performed using “ggplot2” and “psych” R packages. Broad-sense heritability . This linear mixed-effects model was fitted in R using the lme4 package [where Y package .Heritability was then estimated using the following equation:2 = HHeritabilities were calculated for both populations.<lm x ll > and < nn x np>). The independence LOD test (threshold = 15.0) and the regression mapping function were used for markers grouping and maps construction, respectively. SNPs with identical segregation in the population were included in the maps .Genomic DNA from parental lines and from hybrids from Population #2 was extracted from young leaves. A subset of 454 R × G hybrids (randomly chosen) from Population #2 was genotyped with the RosBREED cherry 6 + 9 K Illumina Infinium® SNP array . The strhttp://www.multiqtl.com). Detections were carried out separately for “Regina” and “Garnet” parental maps by using the “single QTL model” (one-QTL per linkage group). We tested the “two-linked QTLs model” but no consistent detections were observed (results not shown). Both single-year (or year-by-year) and multi-year models were utilized (multi-environment model available in MultiQTL). When performing multi-year analysis, a single position (in cM) and a single LOD value are given while values of percentage of variation explained (PVE) are estimated for each year. For ease of reading, the mean PVE value across years is presented. This study gives an update of the QTL analyses presented in Castède et al. [www.rosaceae.org for more details), they were called “qP-FD”.QTL analyses were based on linkage maps already available for Population #1  and on te et al.  by usinge et al. . Only QThttp://software.broadinstitute.org/software/igv/), and heterozygous markers with high RNA-seq coverage were selected. Well-distributed SNPs were then selected to be transformed into Kompetitive Allele Specific PCR (KASP) markers based on the dual Fluorescence Resonance Energy Transfer (FRET) method, in which the sample DNA is amplified with allele specific primers conjugated to fluorometric dyes HEX and FAM at their 5′ end. For each KASP, three primers were developed by the BioGEVES laboratory  and the reactions were performed as described in Bernard et al [SNPs located within the confidence interval of the FD QTL on LG4 were selected from different sources: i) SNPs within the RosBREED cherry 6 + 9 K Illumina Infinium® SNP array, based on their position on the sweet cherry physical map; ii) SNPs from GBS analyses described in a previous study ; and iiird et al .Fine mapping of the QTL region on LG R4 was carried out into two steps. Firstly, nine KASPs were used to genotype the complete Population #2 : KASP_9.269, KASP_9.271, KASP_9.456, KASP_9.727, KASP_9.780, KASP_9.890, KASP_9.935, KASP_10.090 and KASP_10.159 . Recombinant individuals detected in this region were then genotyped with eight new KASP markers, selected on IGV software and located within genes between KASP_9.780 and KASP_10.159, named KASP_9.781 (gene PAV04_REGINAg0203151), KASP_9.801 (gene PAV04_REGINAg0203181), KASP_9.814 (gene PAV04_REGINAg0203191), KASP_9.916 (gene PAV04_REGINAg0203361), KASP_9.933 (gene PAV04_REGINAg0203391), KASP_9.936 (gene PAV04_REGINAg0203391), KASP_9.958 (gene PAV04_REGINAg0203421) and KASP_9.970 (in the gene PAV04_REGINAg0203451). Least square means (lsmeans) of the three years of data  for FD were calculated for the parents, the non-recombinant genotypes and the groups of same recombinant genotypes. With this fine mapping of the QTL, our objective was to reduce the confidence interval of its position and, further, to reduce the number of putative candidate genes.in silico CG analysis was selected based on the QTL on LG4 detected with MultiQTL within Population #2 . Predicted cherry gene models and corresponding protein sequences of this genomic region were retrieved from “Regina” sweet cherry genome repository [https://doi.org/10.15454/KEW474). Data mining on the gene ontology terms associated with candidate genes was done using Blast2GO Version 1.4.4 [−6 before gene ontology mapping and annotation. When no functional annotation from Blast2GO was available, an annotation from the Arabidopsis database TAIR (The Arabidopsis Information Resource) was added.Chromosomal region for pository   performed in 2009/2010 and 2015/2016 on cultivars “Regina” and “Garnet”, in order to obtain the expression profiles of the candidate genes.www.bioinformatics.babraham.ac.uk/projects/fastqc/) and possible adaptor contaminations and low quality trailing sequences were removed using Trimmomatic [During the 2009/2010 sampling season, three dates were chosen for RNA sequencing: 3 December 2009, 1 February 2010 and 16 March 2010, associated with different stages of dormancy, respectively endodormancy, endodormancy release and ecodormancy. For both cultivars, three biological replicates were used at the three dates. Total RNA was extracted from 50 mg of frozen ground flower buds and sequenced on an Illumina® HiSeq 2000 (single read) by GATC Biotech  in 2011. For the 2015/2016 samples, total RNA was extracted and sequenced as described in Vimont et al. . Eleven mmomatic . Raw reammomatic  (v1.0) ummomatic . Raw coummomatic ,73.Four KASP markers from the set of the seventeen markers used for the fine mapping, were tested for validation on different genetic backgrounds. We selected markers among those included within the QTL interval established by our fine mapping approach, spanning from KASP_9.890 to KASP_9.958: KASP_9.916, KASP_9.936 and KASP_9.958. Moreover, although it was outside the interval, we also selected KASP_9.814 as several recombination events occurred between this marker and KASP_9.890 . Recombination events occurred between KASP_9.890 and KASP_9.916 for nine individuals, therefore, we also selected KASP_9.916. No recombination occurred between KASP_9.916 and KASP_9.933, so we did not select KASP_9.933. A recombination occurred between KASP_9.933 and KASP_9.935. However, as KASP_9.935 and KASP_9.936 were very close and no recombination event occurred between them, we decided to select KASP_9.936. Finally, one recombination event occurred between KASP_9.936 and KASP_9.958, so we selected KASP_9.958. In the end, KASP_9.814, KASP_9.916, KASP_9.936 and KASP_9.958 were tested for validation on the R × L population and on the two sets of accessions .In order to determine if FD was different between heterozygous and homozygous genotypes, analyses of variances (ANOVAs) were performed in R software. The following linear model was used:ij = μ + gi + yj + eij where Yij is the phenotypic value of the ith individual in the jth year, μ is the mean value of the trait, gi is the random genotypic effect of individual i, yj is the fixed effect of year j and eij is the residual of the model.YWeb_Material_uhac042Click here for additional data file.


F, are constructed and then diagonalized to give hyperfine-resolvedenergies and wave functions. Electric transition dipole moment curvescan then be used to generate a hyperfine-resolved line list by applyingrigorous selection rules. The algorithm is implemented in Duo, which is a general program for calculating spectra of diatomicmolecules. This approach is tested for NO and MgH, and the resultsare compared to experiment and shown to be consistent with those givenby the well-used effective Hamiltonian code PGOPHER.Analgorithm for the calculation of hyperfine structure and spectraof diatomic molecules based on the variational nuclear motion is presented.The hyperfine coupling terms considered are Fermi-contact, nuclearspin-electron spin dipole–dipole, nuclear spin–orbit,nuclear spin-rotation, and nuclear electric quadrupole interactions.Initial hyperfine-unresolved wave functions are obtained for a givenset of potential energy curves and associated couplings by a variationsolution of the nuclear-motion Schrödinger equation. Fullyhyperfine-resolved parity-conserved rovibronic Hamiltonian matricesfor a given final angular momentum,  The most immediate application isto reveal the properties of the molecules.7 Due to parity conservation inside the nuclei,only even electric and odd magnetic multipoles are nonvanishing. Althoughhigher multipole effects are observed in some experiments, the dominantcontributions to the hyperfine structure arise from magnetic dipoleand electric quadrupole interactions.In the absence of external fields, the rotational hyperfine structureresults from interactions between the electric and magnetic multipolemoments of the nuclei and their molecular environments.8 performed a pioneeringtheoretical study of the magnetic interactions between nuclei andelectron spins in diatomic molecules based on the Dirac equation,see discussion by Brown and Carrington.9 Bardeen and Townes10 provided the firstextensive discussion of the electric quadrupole interactions.Frosch and Foley13 although onemust still pay attention to anomalous commutation relationships whencoupling angular momenta.15 Standard practice isto use these matrix elements to solve problems for which hyperfinestructure is important using effective Hamiltonians which implicitlyuse a perturbation-theory-based representation of the problem.6 The effective Hamiltonian of a fine or hyperfine problem is usuallyconstructed within a particular vibrational state, and the rotationalcoupling terms are treated as perturbations. The assumptions implicitin this approach are usually valid because the splitting of the energy levels due to hyperfine effects are generally small comparedto the separation between electronic or vibrational states. However,this assumption can fail, such as for example, for Rydberg statesof molecules.17 The B 2Π–C 2Π avoided crossing structure in NO is another exampleof strong electronic state interaction. The perturbative treatmentof this vibronic coupling is difficult: it requires a lot of parameters,18 and is not very accurate. The interaction betweendifferent states leads to significant complications which are difficultto model using the standard effective Hamiltonian approach.The application of irreducible spherical tensor operators facilitatesthe evaluation of effective hyperfine Hamiltonian matrix elements,19 proposeda compact solution for the problem based on the use of a variationalmethod to treat the nuclear motion. In our approach, which is basedon the use of potential energy curves and appropriate couplings, itwas only necessary to introduce one potential energy coupling curvebetween the coupled B 2Π and C 2Π electronic states; this gave an accurate rovibronicline list for NO.20 These calculationsused a general program for the calculation of spectra of diatomicmolecules, Duo.21In contrast, our recent work on a spectroscopic model for the fourlowest electronic states of NODuo is a variational nuclear motion program developedfor the calculation of rovibronic spectra of diatomic molecules aspart of the ExoMol project.22 It providesexplicit treatment of spin–orbit and other coupling terms andcan generate high-accuracy fine-structure diatomic line lists. Duo has been used to generate many line lists including thosefor AlO,23 CaO,24 VO,25 TiO,26 YO,27 and SiO,28 which are provided via the ExoMol database.29Duo was also recently employed to calculate temperature-dependentphotodissociation cross sections and rates.30Duo has also been adapted to treat ultralow energy collisionsas the inner region in an R-matrix formalism;31 hyperfine effects are very important in such collisions. Recently,a new module treating electric quadrupole transitions has been addedto Duo,32 which makes it capableof predicting spectra for diatomic molecules with no electric dipolemoment, for example O2 and N2. However, up untilnow Duo had not treated hyperfine effects. In this contextwe note that hyperfine coupling is particularly strong for VO,34 meaning that the current ExoMol VO line list, VOMYT25 which is not hyperfine resolved, is unsuitable for highresolution work, such as the study of exoplanets using high-resolutionDoppler-shift spectroscopy.35Duo. In general, the most challenging partof solving quantum mechanical problems using a variational methodis finding good variational basis sets. We show below that Duo gives appropriate basis sets thanks to its well-designed calculationhierarchy and algorithm. Numerical tests indicates that the algorithmproposed here can achieve high accuracy for the calculation of hyperfinestructure.Herewe present a variational procedure for calculating hyperfine-resolvedspectra of diatomic molecules. The new algorithm we design is implementedas new modules in 2In this section, we outlineour algorithm so that the readers caneasily follow the details given in the following sections. Duo originally used to give fine structureresolved solutionsfor diatomic molecules, and We write the Hamiltonian for the problem as2.1Duo has well-developedmodules, surrounded by black rectangles in Duo to obtain solutions for Vstate(R), to give vibrational energy levels, Evstate,, and wave functions, ψvstate,:R is the internucleardistance, μ is the reduced mass of the molecule, “state” and v indicate the electronic state andvibrational quantum numbers. Duo employs contracted vibrationalbasis sets given by ψvstate, = |state, v⟩ to define a finite-dimension space.The computationalprocedure used by J, and parity, τ,is constructed using a Hund’s case (a) basis set:36v, Λ, S, Σ, J, Ω, and MJ, correspond to the electronic state, the vibrationaleigenstate, the projection of the electron orbital angular momentum L on the molecular axis, the projection ofthe electron spin angular momentum S on the molecular axis, the projection of J on the molecular axis, and the projection of J on the space-fixed Z-axis, respectively.Note that, Duo calculates the spectra of diatomic moleculesin field-free environments. Thus, we do not really use MJ to construct the basis set, as theleft-hand side of In thesecond step, a rovibronic Hamiltonian matrix, correspondingto C(R) can be either a diagonal couplingcurve for a particular electronicstate or an off-diagonal coupling curve between two states. Supportedcouplings include electron spin–orbit, electron spin–spin,electron spin–rotation etc.36When evaluating the matrix elements using the basis functionsof Duo uses linearcombinationsof them to define parity-conserved basis functions:s = 1 for Σ– states and s = 0 for all other states.Note that, the parity is independent of MJ. Each matrix of J and parity τ. Let |ϕmJτ,⟩ be the mth eigenfunctioncorresponding to a given J and parity τ, wehaveEmJτ, is the mth eigenvalue.The basis functions of Thanks to the use of complete angular basissets and the variationalmethod, the final energies are independent of the coupling schemeused. If there is enough vibrational basis (determined by the users’setup), the choice of Hund’s case (a) will give correct resultseven for cases for which other coupling schemes provide a better zeroth-orderapproximation.2.2Duo modules to accomplish the functions denoted by thered rectangles in I, is coupled with J to givetotal angular momentum, F, that is,I and F are quantizedto thespace-fixed axes; J is quantized toboth the space-fixed and the body-fixed axes; L and S are quantized to thebody-fixed axes. Without an external field, MF can be omitted:Duo and thus, can be simply denoted ask is a countingnumberfor the basis functions associated with a given J. It is an equivalent representation of k, J⟩ is short for We programnew J, I, and F, satisfy the triangle inequality:β),8 and is illustratedin The quantum numbers, mJτ,⟩,to define the basis functions:k, J|ϕmJτ,⟩,have been obtained when calculating rovibronic fine structure by solvingfor To obtain a parity-conservedbasis set, we rely on the symmetrizationprocedure given in In practice, we first construct the matrix elements of 37 are integratedover the vibrational wave functions. Duo uses these vibrationalmatrix elements to compute the hyperfine matrix elements within aHund’s case (aβ) basis set, F. Next,the matrix, corresponding to Duo constructs thecomplete Hamiltonian matrix, corresponding to F and τ. Diagonalizing thismatrix gives the hyperfine-resolvedenergy levels and corresponding wave functions in the representationof β) representation of As a first step, the hyperfine coupling curves, such as the Fermicontact interaction curves,313We investigate the field-free hyperfinestructure of diatomic moleculesin which only one of the nuclei possesses nuclear spin, and considerfive nuclear hyperfine terms in this work:e, gS, μB, gN, μN, and μ0, are the elementary charge, the free electronspin g-factor, the electron Bohr magneton, the nuclearspin g-factor, the nuclear magneton, and the vacuumpermeability,respectively. I is the spin of the nucleusof interest (defined as nucleus 1), ri1 is the relative position between the ith electron and nucleus 1, Si is the spin of the ith electron, Li is the orbit angular momentum of the ithelectron, and δ(·) is the Dirac delta function. In cI(R), which is a function of internucleardistance. Section 8.2.2(d) of Brown and Carrington9 and Miani and Tennyson38 definethe nuclear spin-rotation tensor and how it can be reduced to a constantfor a diatomic molecule. In Cp(2) is the modified rank-2 sphericalharmonic:Yp(2)is the standard spherical harmonic;  and  are the positions of the ith electronand the nth proton, respectively.The constants, 12 for the order of magnitude ofthe hyperfine terms.The firstfour hyperfine Hamiltonians, given by 9k(·)indicates a rank-k tensor. All the tensors here aredefined in space-fixed frame. The two tensors in To aid the evaluation of matrix elements,the hyperfine Hamiltonianscan be written as scalar products of irreducible tensor operators:44.1In this section, primitive matrix elements of thehyperfine structure are initially evaluated in the representationof F = J + I, we caninitially decouple the representation of |J, I, F, MF⟩ in 39 fora formal definition and irreducible spherical tensor operators. Takingthe Fermi contact term as an example, the nonvanishing matrix elementon the primitive basis functions for MF = M′F isj symbol.As 1(I) isq:si is the spin ofthe ith electron in body-fixedsystem, j symbol.The electron tensor operators, Tq1(si), do not directly act on the electronicpart of Hund’s case (a) basis. We may replace the electronspin operators with an effective one:S is thetotal spin. Requiring Σ = Σ′, the Fermicontact interaction curve can be defined as3701(si)/Σ representsthe projection operator foreach electron i (see eq (7.152) of Brown and Carrington9). On the basis of The nuclear spinis quantized to the space-fixed axes, and thus,the reduced matrix element of T±11(L) are not consideredhere. The diagonal nuclear spin–orbit interaction curve isdefined as37li is the orbital angular momentumof the ith electron defined in the body-fixed frame.For the nuclear spin–orbit term, we areonly interestedin the diagonal matrix elements of Λ9)ri1, θi1,, ϕi1,) are the sphericalpolar coordinates of electron i relative to nucleus1, we shall give two kinds of matrixelements. For the term diagonal in Λ, that is, q2 = 0 and q = q1:37q2 = ∓2, that is, q1 = ±1 and q = ∓1,we have37The nuclear spin-electron spin dipole–dipole interactionis somewhat complicated. With the definition (see Appendix 8.2 ofBrown and Carrington1(J) to the body-fixed axis system:I ≥ 1; it can be evaluatedaseQ is the nuclear electricquadrupole moment; see Cook and De Lucia7 or Appendix 8.4 of Brown and Carrington.9 The values of Q for various atoms were collectedby Pyykkö.40 The reduced matrixelement of the gradient of electric field isR-dependent constants of the gradientof electric field are respectivelydefined as (see eqs (7.159) and (7.163) of Brown and Carrington9):q0 is denoted as q1, see for example,eq (2.3.76a) of Hirota.41 We follow theconvention of Brown and Carrington9 andpreserve the variable q1 for the nuclearelectric quadrupole coupling constant between different electronicstates arising from T±12(∇E) whichwill be the subject of future work. Finally, the diagonal matrix elementsof nuclear electric quadrupole coupling ared(R) in q2(R) in A-tensor.42 When the second-order contributionsof paramagnetic spin orbit (PSO) interaction are considered, the hyperfinecoupling constants defined in this paper can be further revised bythe PSO terms and determined by the matrix elements of the total hyperfine A-tensor.43The case of the nuclear spin-rotation interaction is muchsimpler,as it is not necessary to rotate T4.2β) basis in k, J, I, F⟩ and the basis functions wedefined in mJτ,, I, F⟩;the hyperfine matrix elements under the basis set can be expandedask′, J′, I, F|ϕm′J′τ,, I, F⟩ arethe matrix elements of Hhfsτ,F, HhfsF, and ΦFτ, , respectively, and,Recall the short notation of Hund’scase (a4.3HF(0),τ, is the matrix of F results in the energies and wavefunctions of the hyperfine structure:UFτ, is represented in theparity-conserved rovibronic basis set defined in EmFτ, is the correspondingeigenvalue of |ψmFτ,⟩.The final Hamiltonianwhich is constructed from summation of therovibronic and hyperfine matricesF, τ, and counting number m are goodquantum numbers.The basis transformation procedures from 57FDF′ and Fτ, DF′τ′,  are the reduced transition dipole momentmatrices in the representation of FDF′, that is, ⟨k, J, I, F∥T1(μ)∥k′, J′, I, F⟩.In the absence of an external field, the linestrength of a nuclearspin resolved rovibronic transition is defined byF = J + I and T1(μ) commutes with I,v, Λ, S, Σ|Tq1(μ)|state′, v′, Λ′, S′, Σ′⟩ is the same as the oneused for the calculation of rovibronic transition intensities excludingnuclear spin in Duo,21q(R) is the electric dipolemoment curve representedin the body-fixed frame which can be obtained from ab initio calculation.As F comesfrom the Wigner-6j symbol of J; as a result, electric dipole transition“forbidden” lines with |ΔJ| >1 are observable. For example, when I = 1/2, we can observe electric dipole transitions of O and S branches (ΔJ = ±2), even if they might be much weaker than the transitionsof P, Q, and R branches.For dipole moment transitions, parity has tobe changed and thusfollows the selection rule:614N16O and 24Mg1H. While both 16O and 24Mg have nuclearspin zero; 14N has I = 1 and 1H has I = 1/2 which allowsus to test different coupling mechanisms. For this purpose we comparethe results of our Duo calculations with that of PGOPHER44 using the same model for each calculation. PGOPHERobtains the energy levels and spectra from effective Hamiltoniansgiven appropriate spectral constants. In contrast, Duo takesin coupling curves and performs variational calculations. To get consistentinputs between the two codes it was necessary to simplify the treatmentused by Duo.To illustrate and validate ournew hyperfine modules, we calculatehyperfine-resolved rotational spectra for electronic and vibrationalground state of 14N16O we approximatethe Duo solution by using only one contracted vibrationalbasis function,that is; |X 2Π, v = 0⟩which ensures that we avoid any hyperfine-induced interaction betweendifferent vibrational states. In PGOPHER, we used values for the rotationalconstant, B0, and spin–orbit couplingconstant matrix, A0, computed using Duo:14N16Oand CSO(R) is the spin–orbitcoupling curve. Note that, forspin–orbit interaction, the coupling curve, CSO(R), describes the coupling energies,while the constant, A, is defined by the splittingenergies. Thus, A is defined by twice the matrixelement. The NO X 2Π potential energy curveused by Duo was taken from Wong et al.45CSO(R) wasassigned an artificial constant CSO(R)= 60 cm–1 and the transition dipole moment curvewas set to 1 D. Our adopted values for B0 and A0 are given in For b, defined by Froschand Foley,8 rather than bF. They are related by the dipole–dipole constant, c,Duo achieves excellent agreementwith PGOPHER for the calculation of both the line positions νand line strengths S. The slight differences aredue to rounding error. As we did not include Λ-doubling termsin our calculation the wavenumbers corresponding to bF, a, eQq0, and cI in the firstand second columns of the same F = 0.5  of F in thefirst and third columns (or in the second and fourth columns). Incontrast, the wavenumbers obtained with eQq2 or d included are different from each other evenfor the same values of F due to the hyperfine contributionto both Λ-doubling and hyperfine splitting.For this analysis, the hyperfinecoupling was chosen using artificialcurves much greater than experimental values. By including only onehyperfine constant at a time, we test the affects of a particularhyperfine interaction. The results are compared in I = 1/2 case by calculating pure rotational transitions withinthe v = 0, X 2Σ+ state of 24MgH, again using a unit electric dipole momentcurve. This is a rather realistic case, as the input spectral constantsto PGOPHER listed in 46 As for the input to Duo, the potential energycurve was shifted from an empirically determined one48 to reproduce the B0 constant given in D0 is not allowedfor when only one contracted basis functionis used in Duo. Just like the Bv constant, Duo does not use rotational constants, Dv, Hv, etc., either, and introduction of these centrifugaldistortion would require manipulation of the potential energy curveswhich are beyond the scope of this work. Nevertheless, Duo still gives hyperfine splittings which are consistent with PGOPHER,see the comparison in D0 uniformly shiftsthe hyperfine energy levels within the same N rotationallevels, where N is the quantum number correspondingto N which is defined asWe also tested the code for an Duo by increasingthe contracted vibration bases to five functions,that is, |X 2Σ+, v = 0, 1, 2, 3,4⟩. As shown in v = 0 stateand improves the accuracy of the calculation, compared with the lowerrotational levels in We then allowedfor the effect of vibrational coupling in S branch (ΔJ = 2) transitions in the second and fourth rowsof R branch(ΔJ = 1) transitions in the first and thirdrows.Finally,we list two calculated 7Duo.21We demonstrate an algorithm for the calculationof the hyperfinestructure of diatomic molecules based on a variational treatment ofnuclear motion. Nuclear magnetic dipole coupling terms including Fermi-contact,nuclear spin-electron spin dipole–dipole interaction, nuclearspin–orbit, nuclear spin–rotation, and nuclear electricquadrupole interaction terms are considered in our calculation. Newmodules for the hyperfine structure calculation are added to the flexiblevariational nuclear-motion package J, a parity-conserved rovibronicHamiltonian matrixof particular total angular momentum, F, is constructed and diagonalized. The hyperfine wave functions arefinally represented using a Hund’s case (aβ) basis set. Hyperfine-resolved line lists for diatomic moleculescan be computed depending on the hyperfine energy levels and wavefunctions. To test the new module, we calculate the hyperfine structureof the v = 0, X 2Σ+ stateof 24 MgH. The results of Duo and PGOPHER showexcellent agreement for both line positions and line strengths. The Duo code and the input file used for 14N16O and 24MgH are available at https://github.com/ExoMol/Duo.On the basis of the eigenfunctions and eigenvaluesof 50 and CFOUR.51 It is also possibleto evaluate them manually after obtaining electronic wavefunctions.37 We will discuss the ab initio calculation of hyperfine coupling constants in future work.Our newly developed methodology builds a bridge between calculationsof electronic motion and nucleus motion of diatomic molecules whichmakes it possible to calculate nuclear magnetic dipole and electricquadruple hyperfine structure effects from first principles. Somehyperfine coupling constants considered in this work may be calculatedby quantum chemistry programs, for example, DALTON35Cl, Slotterback et al. also included the hyperfine coupling termsbetween X 1Σ+ and A 3Π states.52 Implementingthis effect in Duo would require some further work on thematrix elements but should not be a major undertaking. Treating thecase where both atoms possess a nuclear spin introduces another sourceof angular momentum, and the interaction between the two nuclei alsointroduces new matrix elements.12 Herethere are two possibilities, homonuclear systems, such as 1H2 or 14N2, can be treated by generalizingthe scheme given in this paper. Heteronuclear systems, such as 1H14N, are a little more complicated as they giverise to different possible coupling schemes.13 Our plan is to gradually update Duo for each of these casesas the need arises.The current implementation only allows for nuclear spin effectson one atom and neglects coupling between electronic states. The hyperfinecoupling between two electronic states is known to be important forsome molecules. For instance, to analyze the spectrum of I


Translational Psychiatry 10.1038/s41398-022-01847-8, published online 14 March 2022Correction to: The original version of this article unfortunately contained mistakes. To ensure the accuracy of their article the authors would like to make the following corrections: Correction #1 Page 5, paragraph 1, line 5: Text currently reads “136”. This is incorrect and the authors are requesting to change it to “149”. Correction #2 Page 7, paragraph 4, line 17: Text currently reads “mild and moderately depressed patients”. This is incorrect and the authors are requesting to change it to: “patients with no or mild depression”. Correction #3 Supplementary Materials, Supplementary Table 2: The following Antipsychotic medications are incorrectly listed under Antidepressants: Olanzapine, Paliperidone, Perphenazine, Quetiapine, Risperidone, Thiothixene, Ziprasidone. The authors are requesting to update Supplementary Table 2 to include the above Antipsychotic medications in the Antipsychotic medication section of the table. The authors apologize for the errors. The original article has been corrected accordingly.


This systemic review aimed to pool data on the safety and outcomes of thrombolysis use in patients with PE and platelet count less than 150 × 103/mm3. Patients’ demographics, clinical characteristics, management, type of thrombolytic therapy, and outcomes were extracted and analyzed. Of 283 articles identified through the systematic search, 11 case reports fulfilled the inclusion criteria. The mean age of the patients was 52.27 years, and 54.5% were women. The median platelet level before thrombolysis was 65.50 × 103/mm3. Before thrombolysis was initiated, the lowest and highest platelet levels were 29 × 103/mm3 and 105 × 103/mm3, respectively. Alteplase was used in 10 patients and urokinase in one patient. One patient who had a massive PE died of aspiration pneumonia. Interestingly, no thrombocytopenia-related complications were reported. This systematic review highlights the potential benefits and safety of thrombolysis in patients with acute PE in the context of thrombocytopenia. Nevertheless, data available in the literature concerning this topic are scarce and limited to case reports. More extensive studies on the use of thrombolysis in patients with PE and thrombocytopenia are desperately needed.Thrombolysis is an established therapeutic modality for patients with high-risk (and some selected intermediate-risk) pulmonary embolism (PE) with hemodynamic instability. Physicians sometimes experience cases where both a high-risk PE and thrombocytopenia coexist. Although thrombocytopenia of <  100 × 10Systematic review registration: The protocol has been registered in the International Prospective Register of Systematic Reviews (PROSPERO): CRD42021286415. Pulmonary embolism (PE) is a severe emergency subset of VTE that warrants urgent diagnosis and management. The primary mechanism by which an acute PE can cause mortality lies in pulmonary vascular obstruction and constriction, which leads to right ventricular dysfunction. This may manifest as fatal arrhythmias and obstructive shock. Extensive research has been conducted on the management of acute PE. Established treatment modalities are anticoagulation (AC), thrombolysis, and surgical or percutaneous thrombectomy. Whether AC alone or AC with reperfusion therapy would suffice mainly depends on the risk stratification of the PE. Recent PE clinical guidelines stratify the risks and severity of acute PE based on its clinical presentation and hemodynamic effects. High-risk PE is defined by the presence of hemodynamic instability that includes one of the following clinical presentations: cardiac arrest, obstructive shock , or persistent hypotension . Intermediate-risk PE is defined by the presence of right ventricular dysfunction (on echocardiography/radiologic imaging) and elevated cardiac biomarker levels in the circulation (particularly a positive cardiac troponin test). Low-risk PE is defined by the absence of high- or intermediate-risk features. Furthermore, the treatment modality depends on the expertise of the physician and treating center, patient presentation, and PE severity. Patients with hemodynamic instability secondary to PE usually require aggressive management with urgent thrombolysis with or without surgical management. A recent Cochrane review  revealed that thrombolysis reduced the mortality in such patients compared with heparin alone (odds ratio (OR) 0.57, 95% confidence interval (CI) 0.37–0.87).Venous thromboembolism (VTE) remains a major contributor to worldwide mortality with a rising incidence. The current incidence of VTE is 1–2 cases/1000 persons per year in Europe and the USA. The primary concern when using thrombolytic therapy in intermediate- and high-risk PE is the risk of significant bleeding. Thrombolysis is increasingly being used because of its well-established efficacy in mortality reduction regarding intermediate–high to high-risk PE and easy availability of thrombolysis compared with other aggressive measures such as surgical removal or percutaneous thrombectomy. Methods used to perform thrombolysis in patients with PE include systemic thrombolysis, catheter-guided thrombolysis, and ultrasound-accelerated catheter-thrombolysis. Drugs approved for thrombolysis in patients with PE include streptokinase, urokinase, tenecteplase, reteplase, and alteplase. Among these, streptokinase and urokinase require loading doses, whereas others are given as boluses. Additionally, doses of alteplase lower than the recommended have been used successfully. Although thrombocytopenia of < 100 × 103/mm3 is generally considered a contraindication in patients with ischemic stroke, some evidence suggests the safe use of thrombolysis with lower platelet levels in acute ischemic stroke. Furthermore, physicians sometimes experience cases where a high-risk PE and thrombocytopenia coexist, and surgical or catheter-induced thrombolysis is not feasible in their facility. To the best of our knowledge, the safety and outcomes of thrombolysis in the setting of acute PE and thrombocytopenia have not been addressed in a previous systematic review. In this review, we searched and pooled data from the available articles describing the use of thrombolysis in patients with PE who had thrombocytopenia, focusing on patients’ demographics, methods of thrombolysis used, complications, and outcomes.Although still controversial, thrombolysis has shown mortality benefits in patients with intermediate-risk PE (without hypotension or shock but with right ventricular dysfunction).We searched systematically the databases of (from any date up to 20 October 2021) PubMed, Scopus, and Google Scholar for any English language articles pertinent to our research question using the following search terms:“thrombocytopaenia”[All Fields] OR “thrombocytopenia”[MeSH Terms] OR “thrombocytopenia”[All Fields] OR “thrombocytopenias”[All Fields] OR (“low”[All Fields] AND (“blood platelets”[MeSH Terms] OR (“blood”[All Fields] AND “platelets”[All Fields]) OR “blood platelets”[All Fields] OR “platelet”[All Fields] OR “platelets”[All Fields] OR “platelet s”[All Fields] OR “plateletes”[All Fields])) AND Search: pulmonary embolism Sort by: Most Recent“pulmonary embolism”[MeSH Terms] OR (“pulmonary”[All Fields] AND “embolism”[All Fields]) OR “pulmonary embolism”[All Fields] AND Search: ((((thrombolysis) OR ) OR (reteplase)) OR (Tpa)) OR (rtpa) Sort by: Most Recent “thrombolysis”[All Fields] OR (“tissue plasminogen activator”[MeSH Terms] OR (“tissue”[All Fields] AND “plasminogen”[All Fields] AND “activator”[All Fields]) OR “tissue plasminogen activator”[All Fields] OR “alteplase”[All Fields]) OR (“reteplase”[Supplementary Concept] OR “reteplase”[All Fields]) OR “Tpa”[All Fields] OR “rtpa”[All Fields].Our predefined PECO question was as follows:3/mm3) who underwent thrombolysis.Patients of any age with a radiologically confirmed diagnosis of PE and laboratory-confirmed thrombocytopenia  were excluded.Articles published in languages other than English, articles on PE without radiologic confirmation, and articles on PE without laboratory confirmation of thrombocytopenia  and categorical variables (presented as numbers with percentages) were reported as appropriate.Only 11 articles  that met the inclusion criteria of thrombolysis use in the context of PE and thrombocytopenia were found. One abstract that reported 17 patients with upper and lower limb deep vein thrombosis (DVT) and thrombocytopenia who underwent catheter-directed thrombolysis without thrombolysis-related complications was excluded because it did not include patients with PE and the required details about the cases. The mean age at presentation was 52.27 ± 13.22 years, and 54.5% were women. The most commonly reported clinical findings were orthopnea  and lower limb pain . Chest pain  and hemodynamic instability with features of obstructive shock  were less common. Only one patient with massive PE died from aspiration pneumonia. No thrombocytopenia-related mortality was reported.A concomitant peripheral DVT was observed in 3 (27.3%) patients; one (9.1%) patient reported bilateral DVT, one (9.1%) distal, and one (9.1%) proximal. A history of previous DVT and PE was reported in 18.2% and 9.1% of the patients, respectively. The most common reported provoking factor for DVT/PE was malignancy (27.3%), followed by heparin-induced thrombocytopenia (HIT) (18.2%). Other less common provoking factors were also reported .Of all 11 cases, chronic lung disease and hypertension were reported in 2 (18.2%) and 1 (9.1%) cases, respectively. A previous history of PE was reported in only 1 (9.1%) patient. A history of cardiovascular disease (including coronary artery disease and heart failure) was found in 2 (18.2%) patients. A patent foramen ovale was found in 1 (9.1%) case, and atrial fibrillation was reported in 1 (9.1%) patient.3/mm3 at presentation, four (36.4%) had levels between 50 and 99 × 103/mm3, and two (18.2%) had levels between 20 and 49 × 103/mm3 at presentation. A 56-year-old man who developed HIT had the lowest platelet level . A 13-day-old patient with bilateral PE and acute cor pulmonale had the highest platelet count just before thrombolysis (105 × 103/mm3). Both patients completely recovered without thrombolysis-related complications.Computed tomography pulmonary angiogram (CTPA) was the most commonly used diagnostic test , whereas a ventilation–perfusion (V/Q) scan was performed in 1 (9.1%) case. About half of the patients  had features of right heart strain on echocardiography. Electrocardiographic (ECG) findings were reported in 6 cases, with right ventricular strain in 4 (36.4%) cases. PE shown on CTPA were solitary clots in 4 (36.4%) cases, two clots in 3 (27.3%), and >3 clots in 3 (27.3%). They were segmental in 3 (27.3%) cases and subsegmental in 1 (9.1%). Four (36.4%) cases were reported as massive PE on CT scans.3/mm3). She was planned for catheter-guided or surgical embolectomy; however, due to the nonavailability of the former, thrombolysis was decided. The second patient was a 60-year-old man with pancreatic cancer. He developed thrombocytopenia due to HIT (77 × 103/mm3). He was deemed unfit for surgery because of his advanced cancer stage; thus, thrombolysis was performed. Overall, the most common thrombolysis method was systemic , whereas catheter-guided thrombolysis was used in 4 (36.4%) patients. The most common thrombolytic agents used were alteplase in 10 (90.9%) patients and urokinase in 1 (9.1%). One patient (9.1%) reported administration of thrombolysis as a bolus, whereas others reported infusion ranging from 2 hours , 12 hours , 24 hours , or >48 hours . None of the included articles reported platelet transfusion to optimize platelet count before thrombolysis.All 11 patients received thrombolysis, whereas 10 (90.9) were reported to have also received AC. The most commonly used AC method was heparin initially, followed by warfarin in 2 (18.2%) patients or heparin only in 2 (18.2%). Other patients reported the use of argatroban , bivalirudin, and fondaparinux , initial heparin then switched to argatroban , or phenprocoumon  due to HIT. The reason for the initiation of thrombolysis despite thrombocytopenia was not reported in the majority of the cases. A clear reason was reported in only two cases. The first patient was a 65-year-old woman with metastatic breast cancer who had thrombocytopenia due to chemotherapy (60 × 10 Four cases (36.4%) required intensive care unit admissions, and 3 (27.3%) reported endotracheal intubation. Clot resolution was documented in 5 (45.5%) patients. Interestingly, no thrombocytopenia-related complications were reported. Reported complications were early infection in 1 (9.1%) case and aspiration pneumonia in another (9.1%), which occurred late. Follow-up was conducted on 9 (81.8%) patients. The median hospital stay was 49 days, with a mean of 36.67 ± 25.81 days.Among the 11 included cases, 1 (9.1%) reported death as the final outcome. This patient had a massive PE and received alteplase. The cause of death was reported as aspiration pneumonia, which complicated his course of hospitalization. Nevertheless, the authors reported an initial improvement in the patient's condition after alteplase initiation. There are many situations in which thrombocytopenia and PE can coexist in clinical practice. Examples of such situations are patients with malignancies, thrombotic thrombocytopenic purpura (TTP), disseminated intravascular coagulation, autoimmune and connective tissue disorders. Cancer is a significant risk factor for PE and can cause thrombocytopenia because of the associated bone marrow suppression and adverse effects of chemotherapy. In the present review, 3 (27.2%) patients had malignancies. The first patient was a women with breast cancer. Her thrombocytopenia (60 × 103/mm3) was attributed to chemotherapy. She received thrombolysis as PE treatment following deterioration of her condition. She received alteplase (100 mg over 2 hours) with half-dose heparin and recovered successfully without treatment complications. The second patient, a man with metastatic pancreatic cancer, developed HIT when he was diagnosed and managed for a PE. His platelet level dropped from 150 × 103/mm3 to 77 × 103/mm3. However, due to worsening PE, he was re-perfused via thrombolysis . He received alteplase at 0.5 mg/h for 20 hours. Although the patient's symptoms improved and oxygen demands decreased post thrombolysis, he ultimately died of aspiration pneumonia on day 6. The third patient had gastric carcinoma complicated by PE and HIT . He received 30 mg of alteplase as reperfusion therapy. The reason for thrombolysis, despite thrombocytopenia, was not mentioned in this case. Other than solid malignancies, patients with hematologic cancers are also at dual risk of thrombocytopenia (bone marrow involvement and cytotoxic effects of therapy) and thrombosis . Very few studies have evaluated the effects of AC or thrombolysis in such patients. In one study, of 82 patients with malignancy and VTE who had anticoagulation, 31 (37.8%) developed bleeding. Nevertheless, significant bleeding was observed in 10.9%. Experts recommend full-dose AC in these patients when platelet levels are more than 50 × 103/mm3 and reduced dose AC when less than 50 × 103/mm3. AC is not an absolute contraindication in either case, and thrombocytopenia should be managed concomitantly if needed. Thrombocytopenia in the setting of an otherwise pro-thrombotic state may not be a significant bleeding diathesis, as it would be in the absence of a pro-thrombotic state such as malignancy. In the CLOT trial , 31 (4.5%) patients had a major bleeding event. Of these 31 patients, only two had thrombocytopenia.Although bleeding diathesis and active bleeding are absolute contraindications for thrombolysis, there is no agreement on the level of platelet count to consider an absolute contraindication. Pishgahi et al. reported a case of high-risk PE with contraindications to thrombolysis . The patient could not undergo surgery (due to her deteriorating condition) and received thrombolysis instead, with complete recovery from PE and no bleeding complications. Valencia et al. discussed the case of a male infant who was referred for treatment of PE and right heart failure. His platelet count was 105 × 103/mm3. The multidisciplinary team recommended a tPA for thrombolysis rather than a thrombectomy. The tPA therapy resulted in recovery without any bleeding complications. Wu et al. reported a case of systemic lupus erythematosus-linked protein-losing enteropathy that presented as PE with evidence of right ventricular failure and pulmonary hypertension. The thrombus was defragmented with a percutaneous catheter-based technique and lysed with alteplase. He was managed successfully and experienced no complications, despite a platelet count of 98 × 103/mm3.In the present review, we could also find situations  in which patients had PE along with thrombocytopenia and an immediate thrombectomy was not feasible, or the patient was not suitable for surgical management. In these cases, authors used thrombolysis as a last resort in patients with hemodynamically deteriorating condition and massive PE. Zhu et al. described a patient with TTP who experienced an acute PE attack. The patient received thrombolysis during the resuscitation, as he suffered a cardiac arrest secondary to PE. The patient responded well to thrombolytic therapy with no further neurological complications.These reports further emphasize that thrombolytic therapy can be life-saving and safe in patients with deteriorating condition, high-risk PE, and thrombocytopenia. The feasibility and wide availability of thrombolysis compared with surgical or percutaneous thrombectomy have also made it an attractive mode of therapy in patients with worsening PE and thrombocytopenia. Nevertheless, the need and necessity for thrombolysis amid contraindications requires an individualized case assessment with a benefit–risk consideration from the treating physician. Bhalla et al. and Schmidt et al. reported cases of patients who had undergone major surgery. They suffered from PE and had iatrogenic low platelet count with heparin therapy. In both cases, heparin administration was stopped, argatroban was started, and thrombolysis was used as rescue therapy to evade life-threatening hemodynamic instability. The follow-up revealed no further thrombosis or bleeding complications. Interestingly, the current coronavirus disease 2019 (COVID-19) pandemic appears to impose a considerable risk for thrombocytopenia and VTE (including PE). Recent evidence suggests that the severe acute respiratory syndrome coronavirus 2 interacts with different cellular molecules, including enzymes, endothelial cells, platelets, neutrophil extracellular traps, thrombin, and histones, resulting in endothelial damage, and formation of microthrombi. The increasing prevalence of HIT during the pandemic caused by increased heparin use as a VTE prophylaxis is also an issue. AC appears to be nonprotective in preventing VTE in these patients. Apart from COVID-19, all the mainstream mRNA vaccines against the virus  have been reported to cause thrombosis at one end and thrombocytopenia at the other . Given that the pandemic continues and mass vaccinations have been used, a considerable population is currently at risk of thrombosis and thrombocytopenia. Studying the safety of thrombolysis in such a population is interesting in formulating guidelines for this patient group.In the present review, some patients with HIT and PE underwent thrombolysis. HIT is a coagulation disorder that may complicate heparin therapy (more commonly with unfractionated heparin). The procoagulant state may occur before thrombocytopenia. Patients can develop serious thrombotic complications, especially in HIT type 2.The main strength of our review lies in its novelty and a detailed literature search  to find cases in which thrombolysis was used in patients with PE who had thrombocytopenia. Our review has some limitations inherent for systematic reviews of rare conditions. First, all the included studies are case reports. Despite the extensive search, we could not find any higher level of evidence, resulting in a small sample size. However, we did not conduct any correlation statistics to maintain data accuracy. Second, publication bias is possible, as we could only add published cases. Third, we acknowledge that case reports based on adverse effects of treatment may focus only on significant side effects and ignore minor events. In view of this, the results of this review should be taken with caution until a higher level of evidence is generated.This systematic review highlights the potential benefits and safety of thrombolysis use in patients with acute PE in the context of thrombocytopenia, as both the initial treatment and last resort when other treatment options are not feasible. Nevertheless, data available in the literature concerning this topic are scarce and limited to case reports. More extensive studies on patients with PE and thrombocytopenia are urgently required to refine guidelines concerning thrombolysis as a preferred treatment or as a second-line option.Private information from individuals will not be published. This systematic review does not involve endangering participant rights. Ethical approval is not required for this systematic review as only a secondary analysis of data already available in the electronic databases was conducted.Not applicableData sharing not applicable.The authors declare that they have no competing interests.This study was not funded.None.FA: Methodology, data collection, analysis and interpretation, literature review, manuscript preparation, critical review, and revisions in the manuscript.WI: Conceptualization, supervision, methodology, critical review, and revisions in the manuscript.MA: Literature review, data collection, data analysis, manuscript writing.HK: Literature review, manuscript writing.HY, ZM, SM, BD: Literature review, data collection.All the authors reviewed and approved the final version of the manuscript.


The effects of stress on surgical residents and how stress management training can prepare residents to effectively manage stressful situations is a relevant topic. This systematic review aimed to analyze the literature regarding (1) the current stress monitoring tools and their use in surgical environments, (2) the current methods in surgical stress management training, and (3) how stress affects surgical performance.A search strategy was implemented to retrieve relevant articles from Web of Science, Scopus, and PubMed. The 787 initially retrieved articles were reviewed for further evaluation according to the inclusion/exclusion criteria (Prospero registration number CRD42021252682).Sixty-one articles were included in the review. The stress monitoring methods found in the articles showed heart rate analysis as the most used monitoring tool for physiological parameters while the STAI-6 scale was preferred for psychological parameters. The stress management methods found in the articles were mental-, simulation- and feedback-based training, with the mental-based training showing clear positive effects on participants. The studies analyzing the effects of stress on surgical performance showed both negative and positive effects on technical and non-technical performance.The impact of stress responses presents an important factor in surgical environments, affecting residents’ training and performance. This study identified the main methods used for monitoring stress parameters in surgical educational environments. The applied surgical stress management training methods were diverse and demonstrated positive effects on surgeons’ stress levels and performance. There were negative and positive effects of stress on surgical performance, although a collective pattern on their effects was not clear.The online version contains supplementary material available at 10.1186/s12909-022-03841-6. Stress associated with surgery and surgical education represents an important field of research , 2. The Stress can be defined as the psychophysical response to emotional, cognitive or social tasks perceived to be excessive . In physAn aspect to studying the effects of stress in surgical performance is to monitor stress states in surgical-educational contexts. Thereby allowing a better understanding of surgical stress response as well as to acknowledge stress as an important aspect of skills training. Validated scales have been widely used in surgical environments to measure psychological stress states of surgeons, such as the shortened form of the State-Trait Anxiety Inventory (STAI), the STAI-6 , 12, or This study is a systematic review of the literature on stress in surgical environments from the last 10 years. A previous review in this area focused on the available methods of stress monitoring in surgical environments . InterveThis study addresses three main objectives: (1) the current stress monitoring tools and how they have been used in surgical environments  for surgeons, (2) the current methods in surgical stress management training to help reduce stress in the operating room, and (3) how stress affects technical and non-technical surgical performance.A systematic literature search was carried out according to the guidelines of the PRISMA statement , 26 and Main terms : “stress response”, “physiological stress”, “mental stress”, “stress management”, “intraoperative stress”, “intraoperative workload”, “subjective stress experience”, “psychological stress”, “acute stress”.Application terms : “Minimally Invasive Surgery”, “Surgery”, “Surgeon”, “Resident”, “Laparosc*”, “Endosc*”, “Endovascular”, “Arthrosc*”, “Robotic surgery”, “Surgical trainee”, “Robot-assisted surgery”.Environment terms : “Educ*”, “Train*”, “Learn*”, “Eval*”, “Assess*”, “Monitor*”, “Measur*”, “Simulat*”, “Operating Room”, “nontechnical skill”, “non-technical skill”, “surgical skill”.The main, application and environment terms were combined. Exclusion terms were applied to the resulting search output string to avoid including articles related to cellular or mechanical stress, mental illnesses and COVID-derived stress: “Urinary”, “bone”, “replacement”, “cartilage”, “ligament”, “molecular”, “cell*”, “oxidative”, “genet*”, “animal*”, “gender*”, “mental illness”, “mental disorder”, “psychiatric disorder”, “anesthe*”, “dexmedetomidine”, “*mechanic*”, “traumatic”, “injury”, “COVID”.The specific terms and words used for this review are based on the following search strategy , reviews and conference reviews were excluded from the review.The first screening process (based on the title and abstract) was carried out independently by two of the authors. Any disagreements were resolved by all authors and a final decision was made accordingly. Then, all authors independently assessed their assigned articles which had passed the initial screening. The final selection of articles was agreed upon after consensus by all authors. No additional articles were included.The results were structured according to the three main objectives of our review: (1) stress monitoring tools, including training set-ups used when monitoring stress parameters, (2) methods in surgical stress management training, and (3) effect of stress on performance, including measures of technical and non-technical performance.Additionally, we analyzed the levels of evidence of the studies to evaluate the results of training and learning according to Kirkpatrick’s model , 28 and Reaction: assesses learners’ satisfaction and perception of the training method.Learning: assesses learners’ acquisition of knowledge, techniques and skills involved in the training method. We further categorized this level into: (2a) acquired knowledge and (2b) in vitro performance .Behavior: assesses the impact of training on learners’ performance on the job. It can be associated to in vivo performance with animal models .Results: assesses the impact of changes in the operational performance and organization behavior attributable to the educational program .Kirkpatrick’s model with four levels of evidence:Content: Represents the relevance of the training method with its intended use .Response process : Represents “the data integrity and the extent to which the understanding and performance of those assessed aligns with the expectations and interpretations of whomever or whatever is making the assessment” .Internal structure : Relates to reliability  and reproducibility of the tested entity .Relations with other variables: Analyses statistically associated assessment scores with specified theoretical relationships. This validity evidence is in consonance with the construct and criterion validity types of the 1985 standards.Consequences of the assessment: It “explores whether desired results have been achieved and unintended effects avoided” .Messick’s validity framework with five sources of validity evidence:The initial search identified 787 articles, of which 673 articles were included after removing duplicates. Of those, 589 were excluded after title and abstract screening was applied, leaving a total of 84 articles. Out of those, 14 were excluded for not being related to minimally invasive surgical (MIS) areas –47, and An extensive review of the included articles is described in Table S3  were used to monitor stress responses in 36 articles , 58–88. Hormone-based technologies using analysis of cortisol, alpha-amylase or testosterone as indicators of stress was found in 10 articles , 90–92. Electrodermal Skin Response (EDA) or Galvanic Skin Response (GSR) monitoring technologies were found in 6 articles , 93–95. Heat-based monitoring technologies which include the analysis of thermal imaging, skin temperature, heat flux and perinasal thermal imaging were used in 6 articles , 96–98. Posture-based monitoring technologies include the analysis of posture patterns, muscle tone and body movements were used as indicators of stress in 4 articles –65, 94. Brain-related monitoring technologies including the use of electroencephalogram (EEG), and brain spectroscopy were used in 5 articles , 92, 99.Eye tracking methodologies were employed to monitor stress responses in 4 articles , 101. ThOther monitoring technologies were used in 6 articles. Specifically, monitoring of respiration frequency –65, 73, STAI is a commonly used scale to measure trait and state anxiety. It is often used in research as an indicator of subjective stress , 102. ItA six-item short form of the STAI, the STAI-6 was developed for use in circumstances where the full-form is inappropriate. The STAI-6 produces scores similar to those obtained using the full-form, but the STAI-6 focuses on the state anxiety only . The STANASA-TLX is a multidimensional assessment tool for perceived workload and task effectiveness. It consists of six domains designed to capture the mental response to a given task , 106;. TThe Surgery-TLX (Surg-TLX) is the NASA-TLX counterpart for surgical environments . The SurThe Perceived Stress Scale (PSS) is a stress assessment tool aimed at understanding how different situations affect subjects’ feelings and perceived stress . The queThe Pre/post Dundee Stress State Questionnaire (DSSQ) is based on a factor model that differentiates dimensions of task engagement, distress and worry . It analOther stress scales were used in 4 articles. Specifically, the Short Stress State Questionnaire SSSQ , 114), t, t114]),Box trainers were used in 25 articles , 120. MoReal interventions were described in 16 articles , 118. ToVR simulators were used in 13 articles , 107. StRobotic surgical simulators were used in 4 articles , 113. StOther methods were used in 10 articles. Specifically, studies using navigation aid systems , 64, manMental training methods were investigated in 13 articles. Mental training methods including coaching , 118, meSimulation-based training methods for stress management were employed in 5 articles and included laparoscopic training programs , 90, repStress feedback methods for stress management were employed in one article . LemaireThe results of validity analysis are found in Table S3  was analyzed in 31 studies , 119. ThEffect of stress on operative performance was analyzed during operative performances in 6 studies , 95, 105Effect of stress on non-technical performance. The effect of mental training on non-technical performance was assessed in 6 articles , 86, 92.For the studies which focused on the effect of stress on performance, the performance was assessed as technical or non-technical performance.The measures of technical performance included error measures which are the number of errors and critical mistakes made during the procedure or task, and time measures such as total time to complete a procedure or task. Several measures of technical performance linked to laparoscopic simulators were used. In addition, measures of performance in surgical skills such as knot tying, suture and cutting were employed in the studies. The measures of technical performance applied in the reviewed studies are presented in Table The non-technical measures included comprehensive questionnaires, written attention tests, scale-based self-reporting questionnaires, and psychometric evaluation tools that captured teamwork and interactions of the participants. The measures of non-technical performance applied in the reviewed studies are presented in Table This review analyzes the literature on effects of stress in surgical educational environments from 2010 to 2021. Specifically, current stress parameter monitoring tools, psychological and physiological, as well as the settings where they were used in educational and surgical contexts. In addition, surgical stress management methods were identified, and mental training, simulation-based training and stress feedback training methods were found. Finally, articles for the effect of stress on surgical performance and training were reviewed.n = 32). Specifically, HRV was used as a tool to measure the sympathetic and parasympathetic function of the autonomous nervous system [n = 21) [The most frequently used monitoring technologies to measure stress in the reviewed studies were based on HR and HRV (s system . HRV tens system . In addi[n = 21) , 81–89.n = 9) [n = 5) [n = 4) [Time metrics derived from HRV analysis include the SDNN (n = 9) , 81, 86, [n = 5) , 79, 86  [n = 4) , 76, 78. [n = 4) , 76, 86. [n = 4) .n = 11) [Frequency-domain metrics derived from HRV analysis, include low frequency (LF) (range 0.05–0.15 Hz) , 136 andn = 11) , 83, 86.n = 11) , 83, 86,n = 10). However, because hormone levels are rather long-term parameters, they are less accurate for measurements of acute stress; and not optimal when assessing acute surgical related stress [The second most used method for measuring stress was hormone-based analysis . In two articles, the correlation between STAI-6 and physiological stress was successfully demonstrated for LF/HF [n = 10), correlated to HR-based monitoring technologies in two articles (HR and LF/HF) [STAI-6 was found to be the most frequently used validated scale for stress measurement (or LF/HF  and EDA or LF/HF . The secd LF/HF) , 74. Thed LF/HF) , 139.In the reviewed articles, box trainers were most frequently used as a training set-up to assess stress. Box trainers are accessible, easy to use, less expensive and allow for multiple tasks with varied complexity . The tasThe second most frequently used method was interventions with patients. Interventions with patients provide authentic stressors and generate information of how surgeons cope with stress during an actual surgical procedure. Studies applying real-life operations showed that stress levels were high in participants when performing an operation , 105. ThRobotic surgical systems have been available in surgical environments for over a decade . In the n = 12). Mental training methods involved cognitive training and the activation of neural pathways, which may require time to develop [A variety of mental training methods were used for stress management in surgical environments  and error measures (n = 11)  Table . Total tMeasures of non-technical performance used in the reviewed articles Table  were maiIn the reviewed studies, surgical performance was used both as a stressor, i.e., complex procedures and as a setting, or in-situ operations, in which to validate novel methods to measure intraoperative stress or to compare different groups. Higher stress levels were measured among residents compared to experienced surgeons during real-life operations , and incIn the simulation-based study by Moawad et al. , gynecolIn the studies which employed mental training methods, improvement in technical performance was shown –71, 90. Analysis of gaze behaviors showed superior visual attentional control and performance when participants evaluated the surgical task as a challenge and not a threat. A challenge, as opposed to a threat, is associated with lower stress levels. Causer et al. , demonstIn the reviewed studies, a coherent association between surgical experience and stress levels was not found. Some studies demonstrated higher stress levels among novice surgeons during laparoscopic simulation compared to experienced surgeons , 97. In Studies employing mental training methods in their study design showed lower mean stress scores in the intervention group , 92, andA specific search strategy was applied for this review, and the articles retrieved were systematically analyzed. However, the scope of this review with several main topics could be considered too broad. This was evident when reviewing the effects of stress on performance, making comparisons of the included studies more difficult. By limiting the search to a specific surgical specialty could have reduced the number of included articles.The impact of stress responses presents an important factor in surgical environments, affecting residents’ surgical training and performance. To be able to measure the stress response and its effects, a wide range of monitoring techniques is needed. The results of the review of 61 articles from the past 10 years on stress in the surgical educational environments identified the main methods used for monitoring stress parameters to be heart rate-based analysis and subjective stress scales. Box trainers were the most used set-up to create stress-triggering tasks. Interventions that employ mental training methods appear in general to have beneficial effects on surgeons’ stress levels and their performances. However, the effects of stress on performance were found to be unclear as both negative and positive impacts were demonstrated in the reviewed articles. Further investigation into this should be the focus of future studies.Additional file 1: Table S1. Search queries for Scopus, Web of Science, and PubMed. Table containing information about the search queries for Scopus, Web of Science, and PubMed.Additional file 2: Table S2. The Cochrane bias test for the articles included in the review. The Cochrane bias test analysis of the reviewed articles.Additional file 3: Table S3. Characteristics of the studies, environment and training set-ups for monitoring stress parameters, measures of stress parameters and performance, results of intervention, and validation according to the Kirkpatrick level of evidence and Messick’s validity framework. The detailed evidence synthesis of the reviewed articles.