Appropriate rehabilitation of stroke patients at a very early phase results in favorable outcomes. However, the optimal strategy for very early rehabilitation is at present unclear due to the limited knowledge on the effects of very early initiation of rehabilitation based on voluntary exercise (VE). Environmental enrichment (EE) is a therapeutic paradigm for laboratory animals that involves complex combinations of physical, cognitive, and social stimuli, as well as VE. Few studies delineated the effect of EE on apoptosis in very early stroke in an experimental model. Although a minimal benefit of early rehabilitation in stroke models has been claimed in previous studies, these were based on a forced exercise paradigm. The aim of this study is to determine whether very early exposure to EE can effectively regulate Fas/FasL-mediated apoptosis following hypoxic–ischemic (HI) brain injury and improve neurobehavioral function. C57Bl/6 mice were housed for 2 weeks in either cages with EE or standard cages (SC) 3 h or 72 h after HI brain injury. Very early exposure to EE was associated with greater improvement in motor function and cognitive ability, reduced volume of the infarcted area, decreased mitochondria-mediated apoptosis, and decreased oxidative stress. Very early exposure to EE significantly downregulated Fas/FasL-mediated apoptosis, decreased expression of Fas, Fas-associated death domain, cleaved caspase-8/caspase-8, cleaved caspase-3/caspase-3, as well as Bax and Bcl-2, in the cerebral cortex and the hippocampus. Delayed exposure to EE, on the other hand, failed to inhibit the extrinsic pathway of apoptosis. This study demonstrates that very early exposure to EE is a potentially useful therapeutic translation for stroke rehabilitation through effective inhibition of the extrinsic and intrinsic apoptotic pathways. IschemiAcute brain ischemia triggers an “ischemic cascade” of pathophysiological events that includes impaired energy metabolism, excitotoxicity, oxidative stress, inflammation, and apoptosis; ultimately resulting in neuronal cell death. Ample evidence supports the critical role of apoptosis in the pathophysiology of acute brain ischemia, resulting in a significant loss of brain cells . WhereasIschemic stroke triggers two main apoptotic pathways: The intrinsic pathway is initiated by the disruption of mitochondria and the release of cytochrome c, which is mediated by members of the B cell lymphoma/leukemia-2 (Bcl-2) family such as anti-apoptotic protein Bcl-2 and the pro-apoptotic protein Bcl-2-associated X protein (Bax) . On the via the usual care protocol  brain injury and to compare them with those resulting from delayed exposure to EE in adult mice. We describe the effects of altered expression of apoptosis-related genes on neuronal survival and functional recovery, and explore novel neuroprotective rehabilitation strategies after stroke with a focus on the self-motivated nature of EE.2.2.1.via unilateral right carotid artery ligation under anesthesia with a mixture of Ketamine and Rompun. A mixture of Ketamine (100 mg/kg) and Rompun (10 mg/kg) was administered for each mouse based on their weight. Hypoxic brain injury (8% O2 for 30 min) following unilateral right carotid artery ligation was also generated as previously described (Ischemic brain damage was induced in 6-week-old C57BL/6 mice escribed . To ensu2.2.n = 35 per group) within 3 h of exposure to HI brain injury for 2 weeks (n = 15 per group), 3 days after exposure to HI brain injury for 2 weeks  or in EE cages .Mice assigned to EE were housed in a large cage (86 cm × 76 cm × 31 cm) containing novel objects such as tunnels, shelters, toys, and running wheels for VE and social interaction (12–15 mice/cage), whereas the control mice were housed in SCs (27 cm × 22.5 cm × 14 cm) without social interaction (4–5 mice/cage). All experiments were performed with C57BL/6 mice housed in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care and provided with food and water 2.3.n = 10 per group); for Open field test (n = 13 per group); for Ladder walking (n = 15 per group); for Y-maze test (n = 15 per group). All the subjects assigned to the delayed exposure model participated in the entire battery of behavioral tests.Mice assigned to both EE and SC conditions underwent cylinder tests, open field test, ladder walking tests, and Y-maze tests immediately after HI brain injury induction and 14 days after the induction. For the very early EE model, fourteen mice per group were subjected to the cylinder test, thirteen to the open field test, eight to the ladder walking test, and twelve to the Y-maze test. A total of mice was used for the delayed exposure model as follows: For Cylinder test . The 260/280 nm ratio is used to determine the purity of RNA, with values from 1.8 to 2.0 considered to reflect pure quality.Brain regions were dissected based on the gross anatomy of the mouse brain atlas. Total RNA from the cerebral cortex and hippocampus was prepared using the TRIzol reagent  according to the manufacturer’s protocols. For quality control, RNA quality and quantity were evaluated ® TruSeq™ RNA Sample Preparation Kit . The mRNA was transcribed into a library of templates. The successive cluster generation using the reagents was achieved using the Illumination Kit . We perfKyoto Encyclopedia of Genes and Genomes (KEGG) pathway mapping of the identified proteins was performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) software.2.5.Brain lysates were isolated from the cerebral cortex and the hippocampus. Total protein was extracted from the pooled lysates, dissolved in Invitrogen™ NuPAGE™ LDS Sample Buffer and separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis . The separated proteins were further transferred onto a 0.45 μm polyvinylidene difluoride (PVDF) membrane . The membrane was blocked in 5% bovine serum albumin with Tris-buffered saline containing 0.1% Tween 20 for 1 h at room temperature, followed by incubation with primary antibodies overnight at 4°C. Anti-Fas , anti-FADD , anti-Caspase-8 , Danvers, MA, United States), anti-Cleaved Caspase-8 (CST), anti-Caspase-3 (CST), anti-Cleaved Caspase-3 (CST), anti-Drp1 (Abcam), anti-Bax (Santa Cruz), anti-Bcl2 (Santa Cruz), anti-iNOS (CST), anti-COX2 (CST), and anti-β-actin (Santa Cruz) were the primary antibodies (1:1000). After three washes with Tris-buffered saline containing 0.1% Tween 20, all membranes were incubated with horseradish peroxidase-conjugated secondary antibodies  for 1 h at room temperature. Finally, the membrane signal was visualized using an Amersham ImageQuant 800 equipment (Amersham Pharmacia Biotech).2.6.The animals were sacrificed and perfused with 4% paraformaldehyde. The harvested brain tissue was cryo-sectioned at a 16 μm thickness along the coronal plane and stained with hematoxylin–eosin (H&E). H&E staining was performed in four sections, from the frontal pole to the midbrain, to measure the infarct volume. The sections were photographed using a digital camera and analyzed using ImageJ program. The infarct volumes of the lesion were expressed as a percentage of the volume of the structures from the contralateral hemisphere using the formula:Where VC is the volume of control hemisphere and VL denotes the volume of non-infarcted tissue in the lesioned hemisphere. The total volume of infarcted tissue in each brain was calculated as the sum of the volumes of infarcted tissue estimated for each of the four brain sections.2.7.via fluorescent microscopy (LSM700) and positive cell death (μm2) with respect to DAPI area (/μm2) was measured using ZEN Imaging Software version 2.1 . To validate Fas/FasL pathway-related apoptosis, endogenous expression of MAP-2 and FADD in the cerebral cortex and hippocampus was measured using an anti-MAP-2 antibody  and an anti-FADD antibody . The sections were incubated with Alexa Fluor® 488 goat anti-rabbit  and Alexa Fluor® 594 goat anti-mouse  secondary antibodies, and covered with Vectashield® mounting medium with 4C, 6-diamidino-2-phenylindole . For the quantification of the immunohistochemistry images, the average value obtained from three images from each animal was used for analysis. Images of apoptotic and FADD-positive (FADD+) cells were obtained using a fluorescent microscope (LSM700), and positive apoptotic cells (μm2) with respect to DAPI area (/μm2), and FADD+ cells (μm2) with respect to MAP2-positive (MAP2+) area (/μm2) were quantified using ZEN Imaging Software version 2.1 . Furthermore, 3D images of the apoptotic cells were acquired using the same software.Sections of 16 μm thickness were cut along the coronal and sagittal plane of the collected brains, and immunohistochemical staining of four sections was performed over a range of >128 μm. Three images per area were selected from each mouse for analysis. Fluorometric terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay  was conducted to analyze DNA fragmentation in cells from the cerebral cortex and hippocampus according to the manufacturer’s protocol. Images were acquired 2.8.p value <0.05 was considered statistically significant. All graphic artwork was produced using GraphPad Prism version 9 .All data were expressed as means ± SEM. The differences between the two groups were analyzed using Student’s t-test with SPSS statistical software . A 3.3.1.p < 0.001), indicating that the hyperactivity derived from HI brain injury was significantly alleviated by very early exposure to EE. In the open field test, the total distance traveled was decreased in the very early EE group (p < 0.001), indicating that the injury-induced hyperactivity and anxiety were significantly alleviated by very early exposure to EE. Moreover, the percentage of total slips was significantly reduced in the very early EE group (p < 0.05), indicating that fine motor impairment was significantly alleviated by very early exposure to EE. In the Y-maze test, the percentage of alterations was significantly increased in the very early EE group (p < 0.05) after the condition, indicating that cognitive function was significantly improved by very early exposure to EE.In the cylinder test, the number of rearings was significantly reduced in the very early EE group (p < 0.001), indicating that the hyperactivity derived from HI brain injury was significantly alleviated by delayed exposure to EE. In the open field test, the total distance traveled was significantly decreased in this group as well (p < 0.001) after the condition, indicating that injury-induced hyperactivity and anxiety were significantly alleviated by delayed exposure to EE. Moreover, the percentage of total slips was significantly reduced in the delayed EE group (p < 0.001), indicating that fine motor impairment was significantly alleviated by delayed exposure to EE. In the Y-maze test, the percentage of alterations was significantly increased in the delayed EE group (p < 0.05) after the condition, indicating that cognitive function was significantly improved by delayed exposure to EE.The delayed EE group showed a significant reduction in the number of rearings displayed during the cylinder test (3.2.p < 0.01) compared to that from the control group, indicating that very early exposure to EE might have induced neuroprotection (p < 0.01).The volume of the infarcted area was significantly decreased in the EE group . A total of 1,691 DEGs  in the cortex and 2,942 DEGs  in the hippocampus in enriched mice were identified in mice with very early exposure to EE  compared to the control group in the cerebral cortex (p < 0.05 and p < 0.05), Bcl-2 (p < 0.05 and p < 0.05), and Cleaved Caspase 3 (p < 0.01 and p < 0.01) were significantly altered in the very early EE group compared to the control group in both regions (p < 0.01) in the cerebral cortex (p < 0.001) (p < 0.01 and p < 0.05), Bcl-2 (p < 0.01 and p < 0.05), and Cleaved Caspase 3 (p < 0.05 and p < 0.05) showed similar changes as those observed in the very early EE group compared to the control group, again in both brain regions (The levels of dynamin related protein 1 Drp1), a key mediator of mitochondrial dynamics, were significantly decreased in the very early EE group  ; and the regions –H. These3.5.p < 0.05, p < 0.01 and p < 0.001; It is well known that HI brain injury increased mitochondrial reactive oxygen species (ROS) production . To inve3.6.+ cells in the very early EE group was significantly decreased in the cerebral cortex (p < 0.01) and the hippocampus , FADD (p < 0.01 and p < 0.01), and Cleaved caspase-8 (p < 0.01 and p < 0.01). On the other hand, neither FAS nor FADD were significantly decreased in the delayed exposure to EE group compared to the control group, but Cleaved caspase-8 exhibited a significant decrease on its levels in both the cerebral cortex and the hippocampus  and the hippocampus (p < 0.05) of experimental subjects from the very early exposure to EE group  promotes functional recovery, reduces post-stroke complications, and allows for faster reintegration into society . Howevervia downregulation of both the extrinsic and intrinsic signaling pathways of apoptosis. A schematic diagram for the mechanism underlying the neuroprotection elicited by very early exposure to EE is shown in In contrast to previous studies, the current study focuses on the effects of exposure to EE on the inhibition of apoptosis during the very early phase of ischemic stroke. Very early exposure to EE significantly improved functional recovery and promoted neuronal survival. These neuroprotective effects were mediated via exploratory movements as provided by EE may have greater benefits  exposure to EE post-stroke suppresses both extrinsic and intrinsic pathways of apoptosis in the cerebral cortex and the hippocampus. This may be due to the higher sensitivity of these brain areas to therapies that suppress neuronal apoptosis .via downregulation of Fas/FasL-mediated signaling in both the cerebral cortex and the hippocampus, but delayed exposure to EE failed to have the same effect.In a rodent model of ischemic stroke, acute treatment with edaravone was neuroprotective in transient focal ischemia, and the mechanism involved suppression of the Fas/FasL signaling pathway . RecentlThe Fas/FasL system plays an important role in apoptosis during the acute phase in other neurological disorders, and our findings are in agreement with previous studies . In a moDrp1 is known as a pivotal factor of mitochondrial dynamics . Drp1 isThe mitochondrial DNA (mtDNA) and oxidative stress have been implicated in the pathogenesis of neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis and Huntington’s disease . MoreoveThis experiment was designed to use clinically relevant animal model for ischemic stroke . We expoThe HI brain injury model used in this study may also represent HI brain injury after cardiac arrest . Unlike n = 4–8 per group), which restricts their generalization. Moreover, the diverse potential mechanisms of neuronal cell death contributing to long-term neuroprotection and functional recovery after stroke were not investigated, given their inherent complexity. We have not used aged animals in our study. Since stroke primarily affects elderly patients, it would be highly desirable to investigate the effects of very early exposure to EE in an aged animal model with multiple comorbidities to demonstrate the clinical relevance of the present findings for stroke rehabilitation. Therefore, it is highly desirable to investigate the effects of very early exposure to EE in an aged animal model with multiple comorbidities to demonstrate the clinical relevance of the present findings for stroke rehabilitation.Another limitation arises from the small sample associated with the molecular and histological findings  of Yonsei University Health System (permit number: 2021-0182).HL: study design, data interpretation, manuscript drafting, and revision. S-YS and SP: study design, data acquisition, and analysis. JH, AB, DB, SK, and JP: manuscript drafting and revision, data acquisition, data analysis, and intellectual discussion. SC: data collection and analysis. S-RC: funding acquisition, study design, data interpretation, manuscript editing and final revision. All authors contributed to the article and approved the submitted version.This research was supported by the National Research Foundation (NRF-5-2017-A0154-00395) grant funded by the Ministry of Science and Technology, Republic of Korea, the Ministry of Land, Infrastructure and Transport (MOLIT) Research Fund (NTRH RF-2021005), Republic of Korea, the Korean Fund for Regenerative Medicine (KFRM) grant funded by the Korea government  (21A0202L1 and 21C0715L1) and the Korean Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (HI21C1314 and HI22C1588).SP was employed by Neuracle Science Co. Ltd.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Nectogale elegans, is one of the small mammal species most adapted to a semi‐aquatic lifestyle. The taxonomy of the genus Nectogale has received little attention due to difficulties in specimen collection. In this study, we sequenced one mitochondrial and eight nuclear genes to infer the phylogenetic relationship of Nectogale. Phylogenetic analyses revealed two large clades within Nectogale. One clade represented N. elegans, and the other was regarded as N. sikhimensis. The split between N. elegans and N. sikhimensis dated back to the early Pleistocene (2.15 million years ago [Ma]), which might be relevant to the Qinghai‐Tibet Plateau (QTP) uplift. The morphological comparison showed several distinguishing characters within Nectogale: the shape of the mastoids, the first lower unicuspid (a1), and the second upper molar (M2). Overall, the molecular and the morphological evidences supported that the genus Nectogale consists of two valid species: N. elegans and N. sikhimensis.The elegant water shrew,  Nectogale sikhimensis using molecular and morphometric analyses, and the genus Nectogale consists of two valid species, N. elegans and N. sikhimensis.We recover species status of  Subsequently, Winton (1899) examined two specimens of Nectogale from Sikkim and nominated them N. sikhimensis  based on morphological differences with N. elegans. He considered the two specimens browner in color without a clear boundary between dorsal and ventral pelage, and the shorter cusps of the first upper incisor. However, Allen believed that this should be considered a subspecies  were used as outgroups for phylogenetic analyses because of the close phylogenetic relationship between the genera Chimarrogale and Nectogale  and eight nuclear genes . These genes represented good genetic markers to solve the molecular phylogenetics of some mammals , 1× reaction buffer, 0.2 mM of each dNTP, 0.4 mM of each primer, and approximately 100–500 ng of genomic DNA. The primers used are listed in Table Total genomic DNA was extracted from the muscle or the liver tissue preserved in 95% ethanol using the phenol/proteinase K/sodium dodecyl sulfate method   and aligned using MEGA5  model in MEGA5, with 1000 bootstrap replications    TheWe used Network v4.5 to construct phylogenetic trees of each nuclear gene and mitochondrial gene CYT B. Before building the tree, we phased our data in DnaSP v5.10 .We explored the population genetic structure in STRUCTURE 2.3.1 , lower tooth row length (LTR), mandibular length (ML), and length of the lower incisor (i1L). The measuring methods were following Jiang and Hoffman , Sichuan Academy of Forestry (SAF), and Sichuan University (MSCU). Three external measurements— namely, head and body length (HB), tail length (TL), and hind foot length (HL) were measured in the field or recorded from original specimen labels. However, they were not used for morphological analyses because these measurements may show considerable inter‐observer variation . The overall variables were log10‐transformed before conducting the PCA, and a few missing values were replaced by means. On the basis of the results of our molecular analyses, we assigned two specimens of N. elegans from Tibet (Markam) (see Section 3).The numerical analyses were performed using SPSS v20.0   and the genus was divided into two clades (clade A and clade B). Clade A comprised eight specimens from southeast QTP . The clade B , including all sequences from Sichuan, Yunnan, Qinghai, and two sequences from Tibet (Markam), showed differentiated tree topologies in the BI tree and ML tree. Clade B was divided into two subclades (B1 and B2) in the BI tree but not in the ML tree .The K2P genetic distances of CYT B among the 29 samples in the genus 3.2Nectogale as the mitochondrial and nuclear gene tree . N. elegans and N. sikhimensis diverged from their common ancestors at approximately 2.15 million years ago (95% CI = 1.22–3.18 million years ago).The Bayesian analysis of the divergence time tree showed the same phylogenetic relationship of e Figure . The div3.3N. sikhimensis and N. elegans did not share haplotypes. However, haplotypes of the other nuclear genes  were shared between N. sikhimensis and N. elegans  mean values showed the highest probability of ln for K = 4, after which it stabilized  and ∆K, the best number of clusters in STRUCTURE was 2 and 4 .The plot for ∆9 Figure A. The L, the other nine comparisons were significantly different.The skull measurements are given in Table le Table . Except 2–M2). The first principal component was highly correlated with these characters, and therefore, this component reflected a size effect. The second principal component (PC2) accounted for 10.074% of the total variation and was highly positively correlated with the length of the lower incisor (i1L) (loading >0.6). On the PC1 and PC2 plot  accounted for 74.785% of the total variance and was positively correlated with all variables  is shorter than the later (26.17 mm). In addition, some characters that could distinguish these two clades were found. For example, the mastoid does not extend laterally in N. sikhimensis  were recently described (C. hoffmanni and C. dabieshanensis) , Chimarrogale leander , and Neomys milleri  were recommended species status  was suggested to be attributed to a new genus ] by Abramov, Bannikova, Lebedev, and Rozhnov  or four clusters . The results are very interesting, especially because this could indicate that cryptic diversity might be present. The results may be important to provide additional insights for future work.Within the genus N. elegans is in Baoxing, Sichuan, which was also found in Yunnan, Shaanxi, Gansu, and Qinghai, China. On the basis of our collection, it is also distributed in Markam, eastern Tibet of China . This species is mainly distributed in southeast QTP of China and was also found in Bhutan, Myanmar, and Nepal       and 5.97 million years ago . The difference in calibration information may be the main reason for the discrepancy. According to recent studies, the division of Soricinae and Crocidurinae occurred at 36 million years ago . Furthermore, several diagnostic characteristics were found in these two clades. Therefore, the molecular and morphological evidence supported that the genus Nectogale consists of two valid species: N. sikhimensis and N. elegans. In addition, our divergence time tree suggested that the split of Nectogale species might be relevant to the QTP uplift.In the present study, we used mitochondrial and nuclear genes to investigate the phylogenetic relationships and evolutionary history of the genus Ronghui Fan: Formal analysis (lead); investigation (lead); methodology (lead); software (lead); writing – original draft (lead); writing – review and editing . Keyi Tang: Data curation ; formal analysis ; methodology ; writing – review and editing (lead). Liang Dou: Data curation ; investigation ; resources . Changkun Fu: Formal analysis ; investigation ; methodology . Abu ul Hassan Faiz: Investigation ; resources . Xuming Wang: Formal analysis ; investigation . Yufan Wang: Data curation ; investigation ; resources . Shunde Chen: Formal analysis ; funding acquisition (lead); investigation ; methodology (lead); resources (lead); supervision ; writing – review and editing (lead). Shaoying Liu: Data curation ; formal analysis ; investigation ; methodology ; resources (lead); supervision (lead); writing – review and editing (lead).The authors declare that they have no conflict of interest.Figure S1Click here for additional data file.Figure S2Click here for additional data file.Table S1Click here for additional data file.Table S2Click here for additional data file.Table S3Click here for additional data file.Table S4Click here for additional data file.


The ornamental crabapple is a multipurpose landscaping tree that bears brilliant fruit throughout the winter. However, whether or not its fruit persists after maturation is specifically correlated to cultivar characteristics. In this work, we screened two different types that display fruit-retention  and fruit-abscission (“Radiant” and “Flame”) in Northern China across the whole winter using multi-year successional records. Fruit-abscission was determined predominantly by the abscission zone established at the base of the pedicel, regardless of fruit size and pedicel length, according to the results of the comparative research. The primary physiological rationale was the accumulation of hydrolases activity . Comparative transcriptomics further identified a number of upregulated DEGs involved in the synthesis pathways of canonical phytohormones, such as ethylene, jasmonic acid, abscisic acid, and cytokinin, as well as 12 transcription factors linked in downstream signaling in fruit-abscission cultivars. Finally, a model incorporating multi-layered modulation was proposed for the fruit abscission of ornamental crabapple. This study will serve as the foundation for the development of fruit-viewing crabapples that have an extended ornamental lifetime. Malus crabapple) is a superb attractive tree owing to its vividly colored flowers, foliage, and fruits. Currently, the majority of researches are devoted to these features  were used to compare the two and explore the internal processes determining fruit abscission in ornamental crabapple. This study offered a framework for future research on the morphology, anatomy, and molecular biology of organ abscission in ornamental crabapple and provided the foundation for uncovering the molecular mechanism of organ abscission in ornamental crabapple and developing cultivars with a prolonged ornamental lifecycle.In 2011, ornamental crabapple cultivars including “Donald Wyman,” “Red Jewel,” “Sugar Tyme,” “Radiant,” and “Flame” were imported from the United States. The grafted seedlings were transplanted to the resource garden at Hebei Normal University of Science & Technology . Each cultivar was planted with five replications, as well as the block layout was completely random. After they were flowered in 2013, the fruit growth was monitored for 5 years, including the fruit ornamental period, the abscission period, and the abscission position on the dwarf shoot.Depending on whether the fruit falls off in autumn, these ornamental crabapples may be divided into two groups . “DonaldAccording to our previous investigations , the endThose samples fixed in FAA for 24 h initially were sectioned longitudinally and transversely to a thickness of approximately 8 μm using a microtome. Then, the slices were positioned on the slides. These slides were finally treated with the following reagents in sequence: 1.0% safranin solution, 50% ethanol, 70% ethanol, 85% ethanol, 0.1% solid green solution, 100% ethanol, and resin sealing. These were finally inspected with an Olympus DP 72 light microscope.g for 10 min at 4°C. The manufacturer’s kit outlines the methods available for the specific examination of each enzyme. The enzyme activity of the PE was examined using titration, while the activities of the other three enzymes were examined using an Enzyme-Linked Sorbent Assay (ELSA), with absorbance values recorded at 620, 540, and 400 nm, respectively. There were three replicates for each sample.Pectinesterase (PE), CX, PG, and β-glucosidase (BG) were assayed in accordance with the manufacturer’s instructions (Suzhou Keming Biotechnology). Initially, 0.05 g of frozen shoot and fruit pedicels were pulverised with 200 ml of a pretreatment solution in an ice-bath mortar. The supernatant containing the enzyme was then retrieved by centrifuging at 16,000 RNA extraction was performed using the RNAprep pure Plant Kit . RNA purity and integrity were examined using an Agilent 2100 Bioanalyzer. The cDNA was synthesized with a Thermo Frist Strand cDNA Synthesis Kit (Thermo Fisher Scientific). Using Illumina Truseq RNA Sample Prep Kits , five cDNA libraries each with roughly 130–150 bp insertion fragments were generated, and subjected to paired-end sequencing on an Illumina HiSeq platform owned by Beijing Biomarker Technologies Co., Ltd. All raw data were deposited to the Genome Sequence Archive (GSA) with the accession number CRA004670.http://www.biocloud.net/, Beijing Biomarker Technologies Co., Ltd). These unigenes served as reference repository to examine the variations in gene expression among five cultivars. In addition, these assembled non-redundant unigenes were functionally blasted against a number of databases, including Non-Redundant Protein Sequence (NR), Gene Ontology (GO), Clusters of Orthologous Groups for Eukaryotic Complete Genomes (KOG), Kyoto Encyclopedia of Genes and Genomes (KEGG), Clusters of Orthologous Groups (COG), and Swiss-Prot, among others. Furthermore, homology search for non-redundant unigenes against plant transcription factor databases (Plant TFDB) identified the transcription factor using iTAK software with a significant cut-off of E-value  value .McPME12 (Pectinesterase 12), McBGLU12 (β-glucosidase 12), McCEL1 (Cellulase 1), McPG , McbHLH75 (Basic helix–loop–helix domain 75), and McWRKY75 (WRKY transcription factor 75). The gene-specific PCR primers designed by Primer Premier 5 were presented in McActin gene was used as an internal reference gene. The relative expression levels of genes were quantified using ABI StepOne Plus equipment. Each sample comprises three biological replicates. Relative expression levels for each gene were analyzed using the 2^ (−∆∆Ct) method  method .All data were analyzed using the IBM SPSS Statistics 20.0 software . Significance and multiple comparisons in each cultivar were assessed using ANOVA and Tukey’s multiple range test with a significance threshold of 0.05.Our prior sequential field observations spanning 5 years suggested that the five cultivars’s fruit-shedding periods varied substantially. The fruit abscission phase of “Donald Wyman,” “Red Jewel,” and “Sugar Tyme” occurred normally in mid-March of the following year. “Radiant” dropped all of its fruit in early October, while “Flame” shed sooner, virtually all of its fruit by the end of September. Since fruit and fruit pedicel characteristics are major determinants of fruit drop, we evaluated the fluctuations in these two related parameters on September 25 for each cultivar. The changes in the two assessed fruit-related variables were showed in Fruit-retention cultivars, including “Donald Wyman,” “Red Jewel,” and “Sugar Tyme,” featured fruit length, width, and weight, respectively, of 10.33–12.37 and 9.99–11.42 mm, and 0.82–1.06 g, while fruit-abscission cultivars, “Radiant” and “Flame,” with 11.43–18.73 and 11.47–16.83 mm, and 1.05–3.42 g, respectively . “Flame”Abscission describes the detachment of plant organs from their parent plant. This procedure frequently occurs at the intersection of two distinct organs in one or more AZ-specific cell layers . A microThe attachment of fruit pedicel to the dwarf shoot pith  was confIt was further revealed that in fruit-abscission cultivars, the cortical layer at the junction of the fruit pedicel and dwarf shoot had conspicuous microscopic parenchyma cell , but in We measured the amounts of four enzymes  in five ornamental crabapple cultivars’s dwarf shoot and fruit pedicel . These rUsing RNA-seq, we sequenced five ornamental crabapple cultivars to explore the transcriptional processes underlying the establishment of the AZ, a determinant in fruit abscission. Transcriptome sequencing results for fruit-retention and fruit-abscission cultivars of ornamental crabapple were shown in After quality control and data collection, the total clean data were about 51.59 G. with each cultivar ranging between 6.7 and 10.7 Gb. The GC content ranged from 46.39 to 47.84%, while the Q30 content ranged from 91.79 to 94.60%. A total of 44,830 consensus non-redundant unigenes were generated after assembly, of which 55,196 were longer than 1,000 bp and N50 was 1,807 bp. The total length of all unigenes was 51,673,246 bp. The majority of the unigenes were between 300 and 500 bp in length, accounting for 32.15% of the total. The proportions of lengths between 500 and 1,000 bp, 1,000–2,000 bp, and more than 2,000 bp are 27.75, 23.44, and 16.67%, respectively .The annotation of unigenes derived from ornamental crabapples was shown in 2(Fold Change) ≥ 1 and FRD < 0.01 was identified between fruit-abscission cultivars and fruit-retention cultivars. Of these, 442 DEGs were upregulated whereas 325 were downregulated.We used in RNA-seq analysis to study the changes in gene expression during fruit shedding in different ornamental crabapple cultivars, and then revealed the effects of hormones, transcription factors and cell wall hydrolases on fruit abscission. A total of 767 DEGs satisfying the criteria of logHormones govern fruit abscission directly and play a crucial function as a signaling component in the fruit abscission process. To identify potential regulatory mechanisms that leading to the fruit abscission of ornamental crabapple, we compared the transcript levels of key enzymes involved in hormone biosynthesis across fruit-retention and -abscission cultivars . The resMcACS1 and McACOs in the ethylene biosynthesis pathway. 1-aminocyclopropane-1-carboxylate oxidase (ACO) is a significant rate-limiting enzyme that plays an essential role in ET biosynthesis regulation , WRKY , bHLH , NAC , and TGA (McTGA10 c243460.graph_c1), were upregulated in our research. Furthermore, McWRKY19 (c261806.graph_c1) was considerably down-regulated in fruit-abscission cultivars. This finding highlighted the function of TFs in the fruit abscission of ornamental crabapple and shed light on the underlying molecular process.Numerous studies have shown that the aforementioned transcription factor families are connected with the shedding of plant organs. The AP2/ERF family of transcription factors,  hybrida . After m hybrida . CitbHLHT and JA , may alsT and JA . LikewisT and JA . In addif petals . SeveralMcPMEs , McPG (c245127.graph_c0), McCEL1 (c241958.graph_c0), and McBGLUs  were significant expressed in the AZ pectin and cellulose metabolic pathway. Other enzyme-encoded genes were not significantly varied among fruit-retention and -abscission cultivars. The findings confirmed that the buildup of PE and CX in the AZ facilitated the fruit abscission of ornamental crabapple.Pectin and cellulose are the main components of plant cell walls, they sustain the entire cell structure and prevent its degeneration. Pectin in the abscission layer was dissolved during fruit abscission by hydrolases such as CX and PG, which weakens the cell wall and result in fruit abscission . To furtMcPME12, McCEL1, McBGLU12, and McPG) and two TFs . The elevated expression levels of McPME12, McCEL1, McBGLU12, and McPG in fruit-abscission cultivars relative to fruit-retention cultivars indicate the importance of cell wall hydrolases in fruit abscission. In addition, the increased expression of McbHLH75 and McWRKY75 in fruit-abscission cultivars provides more evidence for the function of TFs in fruit abscission. The identification of expression levels further confirmed the reliability of the transcriptome data  are closely related to shedding  was detected in fruit separations  and LcACS1/4/7, LcACO2/3 (ether biosynthesis genes) through interaction with promoters to affecting the process of fruit shedding  can affect moss’s internode growth by interacting with IAA. Furthermore, MeJA can induce the formation of secondary AZs in bryophytes by altering endogenous levels of auxin and JA-related compounds . SimultaMcNCED1 and McAAO, two ABA synthase genes, were identified to be up-regulated in fruit-abscission cultivars of ornamental crabapple. Previous research also showed that LgAAO3 and LgNCED genes were upregulation during longan fruit drop, which resulted from the accumulation of ABA in the AZ  acts as a central TF to activating CELs and PGs  and NAC  transcription factors were different in ornamental crabapple with distinct in fruit-abscission cultivars. It is also worth noting that McTGA10 is a member of the bZIP transcription factor. TGA transcription factor can bind to downstream genes specifically to regulate fruit shedding of Phaseolus vulgaris . Subsequently, the activity of cell wall hydrolases  was enhanced to finally degrade the cells in the AZ, resulting in the abscission of ornamental crabapple fruit.Following the conclusion of the preceding research, we put forth a hypothesized model in an effort to regulate the fruit abscission of ornamental crabapple . In thisThe data presented in the study are deposited in the Genome Sequence Archive repository, accession number CRA004670.GZ and XW conceived the study. XW, SY, XS, HL, BC, and XX collected materials and performed experiments. XW, YW, and ZW analyzed the RNA-seq data and drafted the manuscript. XW, YW, ZW, and GZ revised the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the Hebei Provincial Department of Science and Technology Project (21326301D), National “Twelfth Five-Year” Plan for Science and Technology Support (2012AA102002-5) and Hebei University Horticultural Crop Breeding Application Technology R & D Center (YF201404). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.https://www.frontiersin.org/articles/10.3389/fpls.2022.1013263/full#supplementary-materialThe Supplementary material for this article can be found online at: SUPPLEMENTARY FIGURE S1(A) Show the changes of fruit growth status of five cultivars of ornamental crabapple from August to November. Fruit abscission fell off completely in October (B). The location of fruit-retention cultivars and the location of fruit-abscission cultivars.Comparison of the different period of ornamental crabapple fruit. Click here for additional data file.Click here for additional data file.


The roles of methyl jasmonate (MeJA) and prohydrojasmon (PDJ) in postharvest color development and anthocyanins biosynthesis in the skin of peach fruit remain unclear. In this study, peach fruit were infiltrated with MeJA (200 μM) or PDJ (40 μM) and stored at 22°C for 7 days. The results showed that treatment with MeJA or PDJ had a positive effect on red color formation in peach fruits due to anthocyanins accumulation (∼120% increase). This was attributed to increased enzyme activities, and enhanced transcript abundance of the genes associated with anthocyanins biosynthesis, induced by MeJA or PDJ. Both MeJA and PDJ promoted sucrose biosynthesis, and the subsequently elevated levels of the sucrose during storage were positively correlated with anthocyanins accumulation (0.49) and the activities of key biosynthesis enzymes (0.42–0.79). Based on these findings, we proposed that MeJA or PDJ treatments promote anthocyanins biosynthesis by regulating sucrose metabolism during the postharvest storage of peach fruit. Prunus Persica L.) is one of the most popular fruit crops due to its attractive color, sweet taste and high nutritional content  Batsch cv. Baifeng] were picked at commercial maturity (approximately 100 d after full bloom) from a commercial orchard in Xinyi City, Jiangsu Province, China. The fruit were placed at 4 ± 2°C for 12 h (precooling). After screening, approximately 500 damage free fruits of uniform size and appearance were selected and randomly divided into three groups for the following treatments: (i) Immersion in a solution of MeJA (200 μM in 0.1% ethanol v/v) for 15 min; (ii) immersion in a solution of PDJ (40 μM in 0.1% ethanol v/v) for 15 min; (iii) immersion in ethanol solution (0.1% v/v) for 15 min (control). Jasmonate treatment concentrations were obtained from preliminary experiments to determine the effects of MeJA (0–800 μM) and PDJ (0–400 μM) solutions on the color formation of peach skin [Prunus persica (L.) Batsch cv. Chunmei]. After treatment, samples were sealed in polyethylene bags , each containing four 2 cm diameter holes. All bags were stored at 22 ± 2°C and a light intensity of 300–400 lux, and three bags from each treatment were taken randomly for determination of the key fruit physiological indices after 0, 1, 3, 5, and 7 days of storage. Each determination comprised a minimum of three measurements. Post color evaluation, the peach skins (∼1 mm) from each treatment group were removed with a paring knife, combined, snap-frozen in liquid nitrogen, and stored at –80°C until required for further analysis.Preharvest bagged (double-layer fruit bags) peach fruit . Final results were expressed as cyanidin-3-O-glucoside equivalents (mg/kg) sample weight basis.Phenylalanine ammonia lyase (PAL) activity was determined using the method by Manganaris et al.  with sliThe activities of chalcone synthase (CHS), chalcone isomerase (CHI), flavanone-3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), and UDP-glucose-flavonoid glycosyltransferase (UFGT) were measured using the corresponding ELISA Detection Kits obtained from Jingmei Bio  according to the manufacturer’s instruction. The results were expressed as U/g protein.Finely powdered samples (0.25 g) were homogenized with ultrapure water (1 mL), centrifuged at 4°C , and the supernatant was passed through a 0.45 μm filter membrane prior to analysis. Prepared samples (10 μL) were separated on an Hi-Plex Ca column  maintained at 80°C  using deionized water at a flow rate 0.6 mL/min on a 1260 HPLC system fitted with a refractive index detector . The concentration of each sugar (g/kg) was quantified by the external standard method using corresponding standard solutions of each sugar.®Plant Total RNA Isolation Kit . For each sample, DNA-free RNA  was reverse-transcribed using the HiScript® III RT SuperMix for qPCR (+gDNA wiper)  according to the protocol supplied by the manufacturer. The expression of 10 genes  involved in anthocyanins synthesis , as well as genes involved in sugar metabolism , were monitored. Real-time quantitative reverse transcription PCR (qRT-PCR) was carried out with the ChamQ Universal SYBR qPCR Master Mix kit  and a CFX Connect™ Real-Time PCR Thermal Cycler . Relative changes in gene expression were obtained by 2Ct–ΔΔ method . The primer sequences are listed in Actin and TEF2 was used as the internal reference gene .All data were analyzed and plotted using IBM SPSS Statistics for Windows, Version 26.0  and Prism 8.4.2 . Experimental data obtained from each sample were reported as the mean ± SD. Differences between group means were assessed using one-way ANOVA: The visual images in a*, b*, CIRG, and L* values were observed in all groups  for all groups increased with increasing storage time although the rate of increase was significantly greater in the treated fruits. At day three, the a* value of the MeJA-treated fruits was 1.25 time higher than that of the PDJ group, otherwise both groups followed a similar trend. These observations also agreed with the visual changes shown in a* values  revealed significant differences between the treated groups and the control during the late stages of storage, indicating a yellowish color in untreated fruits  decreased with increasing storage. Compared with the control, the rate of decrease was greater in the MeJA- and PDJ-treated peaches, which agreed with formation of the deep red color hue (reduced brightness).In agreement with visual changes in color, significant variations in l groups . Figure * values . Inspectd fruits . As showThese findings were similar to previous studies using apple , raspberTPC in the skin of all peach samples increased over the first 5 days, decreasing slightly thereafter . CompareThe changes in total anthocyanins showed a similar trend. Although anthocyanins in the PDJ treated samples were lower than those of MeJA treated fruits at days one and three, they shared similar levels in the late stages of storage, and the overall increase (∼ 1.2 times) was similar to that of the TPC.Thevetia peruviana and cotton  were affected by MeJA and PDJ to different extents , further confirming the key roles of these enzymes in peach anthocyanins biosynthesis in response to the jasmonate treatments.Arabidopsis (in vitro experiments (in vivo in our lab by the application of exogenous sucrose (200 mM) to peach fruits, leading to significant increases in endogenous sucrose and hence increases in anthocyanins contents of up to 1.85 times in the skin tissues (Sucrose content showed a significant association with anthocyanins content (0.49) as well as the activities of most biosynthesis enzymes including PAL, DFR, and UFGT (0.42–0.79). Glucose and fructose were also found to stimulate the expression of anthocyanins biosynthesis genes in bidopsis , radish bidopsis , and grabidopsis . Howevereriments . This wa tissues . In this tissues .This study has demonstrated that postharvest applications of MeJA or PDJ could improve the appearance of peaches by inducing red color development in the skin. The red color was attributed to anthocyanins accumulation, which correlated with the increased activities of PAL, DFR, and UFGT, while expression of the related structural genes and TFs was also upregulated. Both jasmonate treatments ptomoted the biosynthesis of sucrose. The resulting increased concentrations of sucrose during storage were positively correlated with anthocyanins accumulation and the activities of the related enzymes PAL, DFR, and UFGT. Hence, we proposed that MeJA or PDJ treatments could promote anthocyanins biosynthesis by increasing amounts of the signal molecule sucrose during postharvest storage. Notably, PDJ exhibited similar activity to MeJA at one fifth of its concentration, although there was an initial delayed effect, and further studies are required to understand these differences. The results presented here demonstrate that the application of these two hormones, especially PDJ could provide a simple strategy to improve the commercial value of peach.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.TT: lab. experiment and writing. HZ: conceptualization, design, and data curation. LW, JL, and WH: methodology. JZ and LM: lab. experiment. GL: project administration. PL: funding acquisition and supervision. YZ: investigation, reviewing, and editing. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Retrospective cohorts have suggested that levosimendan may facilitate the weaning of veno-arterial extracorporeal membrane oxygenation (VA-ECMO). We therefore studied this clinical question by emulating a randomized trial with observational data.All patients with refractory postcardiotomy cardiogenic shock and assisted with VA-ECMO, admitted to a surgical intensive care unit at La Pitié-Salpêtrière Hospital between 2016 and 2019, were eligible. To avoid immortal-time bias, we emulated a target trial sequentially comparing levosimendan administration versus no levosimendan administration in patients treated with VA-ECMO. The primary outcome was time to successful ECMO weaning. The secondary outcomes were 30-day and 1-year mortality. We performed a multivariable analysis to adjust for confounding at baseline.p = 0.659 in multivariable analysis), or 30-day mortality  and 1-year mortality .Two hundred and thirty-nine patients were included in the study allowing building a nested trials cohort of 1434 copies of patients. No association of levosimendan treatment and VA-ECMO weaning was found (HR = 0.91, , Using the emulated target trial framework, this study did not find any association of levosimendan treatment and ECMO weaning success after postcardiotomy cardiogenic shock. However, the population of interest remains heterogeneous and subgroups might benefit from levosimendan.The online version contains supplementary material available at 10.1186/s13054-023-04328-6. Veno-arterial extracorporeal membrane oxygenation (VA-ECMO) is the most widely used temporary mechanical circulatory support to restore adequate blood perfusion in patients with refractory cardiogenic shock –3. AlthoThis is a single-center cohort study with retrospective data collection. Patients included in this study received standard of care without any change related to the research. Patient data were handled in accordance with the French regulations (MR004 reference methodology). The study protocol was approved by the Research Ethics Committee of the French Society of Anesthesiology and Critical Care Medicine (CERAR), registered as IRB number 00010254-2021-124. The reporting of this study complies with the STROBE statement . The STRWe enrolled all patients admitted to the surgical intensive care unit (ICU) at the Cardiology Institute of La Pitié-Salpêtrière University Hospital  between January 2016 and December 2019. The main inclusion criterion was the presence of mechanical circulatory support with VA-ECMO in the setting of cardiovascular surgery. We excluded patients who were less than 18 years old, those who had other types of mechanical circulatory support, those who had ECMO in a non-surgical context, and those who started ECMO support before surgery.ECMO was implanted either in the operating room, as a relay of cardiopulmonary bypass when patients presented low cardiac output refractory to adrenergic agonists, or postoperatively in ICU when patients presented a refractory cardiogenic shock. Implantation was always performed by cardiac surgeons. The initial ECMO flow rate was set between 80 and 100% of patient’s theoretical cardiac output to ensure perfusion while maintaining cardiac ejection.Patients were weaned from VA-ECMO according to a local protocol previously reported . BrieflyAll patients in the levosimendan group received a continuous infusion of 0.2 µg/kg/min over 24 h  administered as a single dose without an initial bolus of levosimendan according to local standard protocol. Administrated levosimendan was diluted in 0.9% NaCl at a concentration of 0.25 mg/mL, according to the manufacturer recommendations. In this study, none of the patients who received levosimendan exhibited adverse effects requiring cessation of the drug administration before completion of the standard dose.The primary outcome was time to successful weaning from VA-ECMO within 30 days. Successful ECMO weaning was defined as follows: ECMO removal within 30 days in a patient being alive and without one of the following events occurring in the 30 days after ECMO removal: death, or need for repeated ECMO support or another mechanical circulatory device or heart transplantation . The secondary outcomes were 30-day mortality and 1-year mortality.Baseline covariates and follow-up of patients were collected retrospectively by medical record consultation and computer extraction of biological data. The following covariates were collected at baseline: age, height, weight , sex, Simplified Acute Physiology Score (SAPS) II at ICU admission, history of chronic left heart failure, hypertension, previous sternotomy, chronic kidney disease, diabetes mellitus, indication for surgery, and type of acute heart failure at beginning of ECMO support. The following covariates were collected daily during the ICU stay: creatinine, total bilirubin, platelet count, ECMO output, ongoing renal replacement therapy (RRT), lactate, total epinephrine dose, total norepinephrine dose, and total dobutamine dose. If multiple values were available for a covariate, the maximum value for the day was retained, except for platelet count, for which the minimum value was retained.The daily biological SOFA score was calculated from the daily values of creatinine, platelet count, and total bilirubin, as described elsewhere . SimilarIn this study, we aimed to evaluate whether levosimendan administration in patients with VA-ECMO support shortened the time to successful ECMO weaning and reduced mortality at 30-day and at 1-year. We thus emulated a hypothetical target trial in which patients were randomly assigned to levosimendan administration or no levosimendan administration, and for which eligibility criteria, ECMO procedures (implantation and weaning), and outcomes would be the same for its emulation with observational data –23. See The target trial was emulated from our cohort each time the comparison between the two arms of the target trial were possible during the duration of ECMO support, i.e., when there was at least one patient eligible to the study who received levosimendan. Patients were eligible multiple times during follow-up; thus, a copy of a patient was created each time a patient could participate to a nested emulated trial. Patients eligible at a given time point were alive, still receiving ECMO, and did not receive levosimendan at previous time points (new-users design). At baseline of each emulated trial, patients were classified as receiving levosimendan if they started levosimendan on the ECMO day or not receiving levosimendan otherwise, and potential confounding was measured. The day zero (first follow-up) was defined as the day of beginning of ECMO support. Patients (and their respective copies) were followed from baseline of each emulated trial until death or 1-year after ICU admission . This methodological strategy makes coincide the time of eligibility criteria assessment, treatment assignment and starting of follow-up, thus removing immortal-time bias related to the delay between ECMO initiation and levosimendan administration . For morFor each analytical population, patient selection process was represented by a flow diagram, and characteristics at inclusion were described as percentage for categorical variables, median with interquartile range (IQR), or mean with standard deviation (SD) for continuous variables, as appropriate. Characteristics were stratified according to whether levosimendan was administered for weaning from ECMO. The two groups were compared using adequate tests according to the type of variables and their distribution .The time to successful weaning from VA-ECMO was assessed for each copy of patient. Follow-up started at beginning of ECMO support and ended at first ECMO removal or death. The 30-day period after ECMO removal was only used to qualify ECMO weaning success but not included in the calculation of the time-to-event. Patients that were still on ECMO support at 30 days were censored at 30 days. As the primary objective of the study was to estimate the etiologic effect of levosimendan on ECMO weaning success, we first took into account competing risk of death and of ECMO weaning failure after ECMO removal by using a cause-specific hazard model, as proposed by Austin, Lee, and Fine . In the The effect of levosimendan on the primary outcome was then estimated on pooled nested trials data using a univariable and a multivariable Cox model to adjust for confounders measured at baseline of each nested trial, as the decision to prescribe levosimendan was at the discretion of the medical team in this cohort, and some confounders may be imbalanced between the two treatment groups at baseline of nested trials. Covariates entered into the model were as follows: age, sex, SAPS II at ICU admission, history of chronic heart failure, previous sternotomy, hypertension, diabetes mellitus, type of acute heart failure, indication for surgery, chronic kidney disease, BMI, SOFA score, lactatemia, ECMO blood flow rate indexed to body surface area, and VIS. SOFA score, ECMO output, lactatemia, and VIS were measured at baseline of each nested trial, all other covariates were measured at ICU admission or at beginning of ECMO support. All confounders were entered into the model based on clinical expertise and prior knowledge , 29–31. .Mortality at 30 days and at 1 year after assignment to the nested trials was estimated in a univariable analysis by calculating the odds ratio and its confidence interval. Multivariable analyses with logistic regression were then performed to adjust for confounders. See Additional file First, we performed a sensitivity analysis excluding patients from 2016, as only four patients received levosimendan this particular year , whereas the proportion of patients receiving levosimendan in 2017, 2018, and 2019 was quite stable (comprised between 26 and 38%), and to assess whether change in clinical practice may have affected the per-protocol estimate of effect of levosimendan on the primary outcome. Then, we used Fine and Gray’s competing risks model to estimate the subdistribution hazard ratio for ECMO weaning success (sHR) . The mulSeveral subgroups were explored for the primary outcome, with tests for treatment-by-subgroup interaction. The interaction terms tested were: time to treatment assignment, or the effect of an early levosimendan administration versus a later one after beginning of VA-ECMO support (until day 2 or after day 2), time of ECMO implantation (in the operating room or in the ICU), type of acute heart failure (right or left ventricular heart failure or right and left ventricular heart failure), indication for surgery (heart transplantation versus others), and renal replacement therapy (RRT) at baseline . The interaction was tested with the Wald test.Missing data for all covariates were handled with an approach that considers the time-dependence of the covariates in the imputation model , 34; seehttp://cran.rproject.org) using the following packages: survival  to account for the fact that observations of patients have been duplicated in the analysis. Significance level was set at 0.05. Analyses were performed using R software, version 4.1.2 , mice , and cmv3.2–13, ).p = 0.015), higher SAPS 2 score and Euroscore at admission , more often isolated left ventricular failure at ECMO implantation and less often biventricular failure (22/65 (33.8%) vs. 28/174 (16.1%) and 35/65 (53.8%) vs. 126/174 (72.4%), respectively, p = 0.008), and received higher doses of daily dose of dobutamine at beginning of ECMO support . During follow-up, patients who received levosimendan had more strokes (13/65 (20.0%) vs. 13/174 (7.5%), p = 0.006) and less acute mesenteric ischemia (5/65 (7.8%) vs. 37/174 (21.5%), p = 0.014). Population’s characteristics are summarized in Table Between January 1, 2016, and December 31, 2019, 239 patients were included in the study, among whom 65 received levosimendan , as well as the length of the ICU stay . Patients were more often successfully weaned from ECMO in the levosimendan group (39/65 (60.0%) versus 81/174 (46.6%)) at the end of follow-up. Death within 30 days following ICU admission occurred in 19/65 patients (29.3%) in the levosimendan group and 79/174 (45.4%) in the control group. Death within one year following ICU admission occurred in 27/60 patients (45.0%) in the levosimendan group and 94/172 (54.7%) in the control group. After one year, seven patients were lost to follow-up.A total of 65 patients were included in the levosimendan group and 1369 copies of patients in the control group. The description of the target trial emulation population construction is shown in the flowchart in Fig. p = 0.011), more often isolated left ventricular failure and less frequently biventricular failure (22/65 (33.9%) versus 237/1369 (17.3%) and 35/65 (53.9%) versus 984/1369 (71.9%), respectively, p = 0.002), and higher SAPS 2 on admission . They had a lower daily biological SOFA , a lower lactate , lower doses of epinephrine  15.09 versus 1.35 mg, SD 6.19, p = 0.02) and dobutamine , and lower VIS score . During follow-up, the population of the levosimendan group had less acute mesenteric ischemia (5/64 (7.8%) versus 258/1350 (19.1%), p = 0.023), acute kidney injury (44/65 (67.7%) versus 1117/1361 (82.1%), p = 0.004), see Additional file In the nested trials population, patients receiving levosimendan had lower BMI  in the cause-specific hazard model for competing risks. Likewise, in multivariable analysis, levosimendan was not associated with successful ECMO weaning , see Additional file p = 0.014) that was not significant anymore after adjustment on confounding factors . Cumulative incidences curves for ECMO weaning success and competing risks of death and ECMO weaning failure are shown in Fig. p = 0.889).Regarding the time to successful ECMO weaning, no statistically significant association with levosimendan treatment was found in univariable analysis , single or biventricular failure, ECMO implantation timing (in the operating room or in the ICU), indication for surgery, and RRT showed no significant difference between patients receiving levosimendan or not Table .Table 2Sp = 0.064) nor multivariable analysis . Likewise, there was no significant association of levosimendan and 1-year mortality in univariable  nor multivariable analysis .For the secondary outcomes, there was no significant association of levosimendan and 30-day mortality in univariable . Second, missingness was present. Nonetheless, the proportion of missing data was low, and we imputed these data with multiple imputation, in a way that takes advantage of longitudinal collection of the data . Third, Fifth, the dosage of levosimendan administered in our study (0.2 µg/kg/min) is in accordance with the label dosage (maximum dosage). To our knowledge, the only pharmacological study of levosimendan under ECMO by Sangalli et al.  found a Sixth, the main cause of refractory postcardiotomy cardiogenic shock in our study was acute primary graft dysfunction. Therefore, the results of our study should be cautiously applied to other tertiary centers with more prevalent subpopulations at higher risk of failure of ECMO weaning  than patients after heart transplantation .Finally, we could not control timing of administration of levosimendan as in a randomized trial, and the time between ECMO implantation and levosimendan administration was a median of 5 days –6. This The positive inotropic effect of levosimendan is prolonged because of 80-h half-life of the active metabolite OR-1896 . PatientOur study did not find an association of levosimendan administration and VA-ECMO-weaning or mortality in patients with refractory postcardiotomy cardiogenic shock. These results issued from one of the largest cohorts studied to date and the only one using the emulated target trial approach might cast some doubt on the efficacy of levosimendan. The comparison with the results of an ongoing interventional study  for the same clinical question may help to resolve the current discrepancies in observational data.Additional file 1. Supplemental tables and figure. Additional information for statistical methods.


N = 476, Mage = 16.24, 76.1% female, 88.7% Dutch; cohort 2: N = 814, Mage = 18.45, 82.8% female, 97.2% Dutch). ESM were used to measure daily positive and negative affect and coping . The results showed that, on average, adolescents decreased in daily positive affect and adaptive coping, and increased in their experienced negative affect. A positive relation between adaptive coping and positive affect was found, although independent of the CBT-based challenges. Latent class analysis identified two heterogeneous trajectories for both positive and negative affect, indicating that the majority of participants with low to moderate-risk on developing mental health problems were likely to benefit from the Grow It! app.Adolescents are at increased risk for developing mental health problems. The Grow It! app is an mHealth intervention aimed at preventing mental health problems through improving coping by cognitive behavioral therapy (CBT)-inspired challenges as well as self-monitoring of emotions through Experience Sampling Methods (ESM). Yet, little is known about daily changes in well-being and coping during a stressful period, like the COVID-19 pandemic. The current study aimed to elucidate daily changes in positive and negative affect, and adaptive coping, and to better understand the within-person’s mechanisms of the Grow It! app. The sample consisted of 12–25-year old Dutch adolescents in two independent cohorts (cohort 1:  Adolescence refers to the transition from childhood to adulthood, which is typically from the onset of puberty to guardian independence (12–25 years)  in an mHealth setting  is considered the gold standard for testing interventions, creating a group-based comparison that controls for differences between individuals by random assignment to a treatment, N = 1 designs have gained popularity  to prevent mental health problems using two key components  users played the Grow It! app for six weeks, and in cohort 2 (second lockdown), users played for three weeks , schools started teaching remotely, sports clubs were closed, and people were encouraged to stay at home and minimize the number of visitors. During the second lockdown (December 2020-March 2021), strict measures were maintained, including online learning, closure of non-essential shops, sport clubs and curfews. Over the course of this first year of the pandemic, studies have now established a decrease in functioning  where they were asked to sign an online informed consent form. For participants aged 12–16 years, their parents were also asked to sign an online informed consent form. The Grow It! app study has been approved by the Ethics Committee of the Erasmus Medical Center (registration number: MEC2020-0287). This longitudinal study used both baseline questionnaires and ESM data to estimate the changes in positive and negative affect, as well as in adaptive coping. After an online baseline questionnaire of one-two minutes, participants received a text message with a unique code to login on the Grow It! app on their smartphones. All measures can be found online. The current study includes a selection of items related to specific research questions.Participants were recruited via advertisements on  media and through online announcements by schoolteachers. Inclusion criteria were owning a smartphone, between 12 and 25 years, and being able to understand Dutch. First, participants registered via the website . Reminders were sent after 40 min. Participants did receive ESM questionnaires during school hours, but they were able to mute their phones. The total 90 min time to complete the questionnaire also ensured that it did not interfere with the class and could be completed during school breaks. A total of 210 ESM questionnaires (5 ESM/day*42 days) were sent out in cohort 1, and 105 (5 ESM/day*21 days) in cohort 2. A detailed description of the ESM questionnaires can be found online. As a reward system, a random group of participants, with at least 70% compliance, was drawn monthly to win a gift voucher worth €75, €50, €30 or €15 or Apple Airpods. The mean ESM compliance rate, which is defined as the percentage of ESM questionnaires that has been filled out, was 23.9% (± 22.7) in cohort 1, and 34.6% (± 25.6) in cohort 2 , age (years), ethnicity (Dutch/Non-Dutch/Mixed), whether participants received psychological care or were on a waiting list, and their educational level, including primary school, low = (preparatory school for) technical and vocational training, middle = (preparatory school for) professional education, high = (preparatory school for) university).Adolescent depressive symptoms were assessed in the baseline online questionnaire. Items were based on the Children’s Depression Inventory Kovacs, . A totalIn the baseline questionnaire, anxiety was assessed using nine items based on the Screen for Child Anxiety Related Disorders (SCARED)  to “very” (7)  /five (cohort 2) items that were derived from validated questionnaires  to “very” (7))   were conducted in R (Version 4.0.2). LMM analyses were done using the lme4 package  were generated as preregistered. The main predictor was time . Models were controlled for age, sex, COVID stringency index, and weather. Because the duration of the intervention and the period in which the study started differed between the two cohorts, the analysis were independently performed in each cohort.A stepwise approach per outcome  was used. First, an intercept-only model was conducted to assess the relative amount of between- and within-person variance in the outcome. Second, an unconditional growth model with time as predictor was specified (random intercept and random slope). Third, the fixed effects of time-varying covariates and interaction terms between the time-varying covariates and time were added.n = 50 iterations). To compare which model would best describe the underlying patterns in our data, multiple statistical fit indices were used, including Akaike Information Criteria (AIC), Bayesian Information Criteria (BIC), sample size adjusted Bayesian Information Criteria (ssaBIC). To assess classification accuracy, posterior probability, and entropy values were inspected, a full explanation of this technique see . Finally, it was tested whether coping could explain within-person associations between challenges and positive affect (mediating effect through coping), by estimating causal mediation effects.In addition to these preregistered models, sensitivity analyses were conducted to test the robustness of our findings. As the age range of users was quite broad (12–25 years) age stratification  was performed to estimate the within-person associations with positive affect, negative affect, and coping. It was tested whether a significant difference in terms of engagement in the Grow It! app would exist between younger and older adolescents, including the measures for compliance of ESM surveys and CBT-based challenges.p = 0.936; cohort 2: χ2 = 2501, DF = 2, p = 0.286). Therefore, all available data was included in the models. A Student’s t-test was performed to determine whether there is a statistical difference between the baseline sample and the current sample, including age, sex, depressive symptoms, anxiety symptoms, and well-being. For cohort 1, the baseline sample was slightly older than our participants , and there were fewer girls on average at baseline compared to the individuals in the current study . No differences were observed in other variables. For cohort 2, there were no statistical differences between the baseline and the current sample. Finally, Student’s t-test was performed to test the difference between participants with available ESM data  and the ones that surpass the ESM compliance and played CBT-based challenges criteria of the current study  (see Supplementary Tables 1-2). Only in the first cohort was sex significantly different between these two groups (p = 0.014), indicating that it is unlikely that bias was introduced by applying the exclusion criteria.As in any intensive longitudinal study, missing data existed , which did not confirm our hypotheses that participants would increase in daily positive affect over the course of the study.Regarding changes in positive affect Table , while cn = 308 (64.7%); cohort 2: n = 586 (72.0%)) were classified as having a slight increase in positive affect over the course of the study as indicated by the estimated effect coefficients of 0.06 (p < 0.0001) for the first cohort and 0.10 (P < 0.001) for the second cohort. The other class (cohort 1: n = 168 (35.3%); cohort 2: n = 228 (28.0%)) was characterized by decreases in positive affect  for the first cohort and −0.52 (p < 0.0001) for the second cohort. Background characteristics of the classes are shown in Fig. To assess for how many adolescents positive affect would increase or decrease, differences between individuals in their positive affect trajectories were explored. Latent class growth models indicated that a 2-class model had a better fit , compared to a 1-class model Table S3). This indicates significant heterogeneity in the population, and the presence of distinct subclasses. Most adolescents ; cohort 2: n = 664 (81.6%)). Given the estimated effect coefficients, of −0.03 (p < 0.0001) for the first cohort and −0.10 (p < 0.0001) for the second cohort, most individuals within this class display a significant decrease in negative affect while playing the Grow It! app. The other less prevalent class (cohort 1: n = 83 (17.4%); cohort 2: n = 150 (18.4%)) showed a significant increase in daily negative affect, with an estimated effect coefficient of 0.32 (p < 0.0001) in the first cohort and 0.47 (p < 0.0001) in the second cohort ) vs n = 115 (17.3%)) and had significantly lower ESM compliance in the first cohort (18.8% vs. 25.0%). In other words, adolescents who improved most in their well-being over the course of the study were characterized by lower levels of psychological problems, and were more engaged in terms of their ESM compliance.Again, exploring effect heterogeneity between individuals in their negative affect trajectory, two unique classes were identified Table S5). The largest class was characterized by a decreasing negative affect over the course of the study .Grow it contains CBT-based challenges, which aim to promote more adaptive cooping. However, as indicated in Table p < 0.0001, cohort 2: B = 0.17, p < 0.0001). This indicates that on days when individuals employ more coping strategies , they experienced a more positive affective well-being compared to other days and vice versa. However, whether individuals had played a challenge that day, was independent to their positive affect , nor with adaptive coping . Taking into account the baseline coping levels, the association between playing a challenge and daily adaptive coping remained statistically insignificant. Following the criteria for a variable to be defined as a mediator , it was not able to test the hypothesized meditative association between daily CBT-based challenges on positive affect via within-person changes in adaptive coping.At the same time, a positive association was found between within-person changes in adaptive coping and within-person changes in positive affect , however there was no difference observed in the number of played challenges .In cohort 2, younger participants had an average of 33.4 ESM responses against 37.2 ESM in the older group. Based on the Student’s t-test this was not significantly different (p = 0.078). Similarly, there was no significant difference in the engagement of played challenges .Because the sample had a large age range, sensitivity analyses were conducted with two distinct age groups. Younger adolescents were defined as younger than 16 years, whereas older adolescents were defined as 16 years or older. In cohort 1, there were 237 participants older than 16 years (49.8% of the ESM sample). In the first cohort, 20.6% of Grow It! users were enrolled in college. In cohort 2, there were 613 participants 16 years or older (75.3% of the ESM sample). In the second cohort, 49.5% of the Grow It! users were enrolled in college. In cohort 1, younger participants completed on average less ESM surveys compared to the older participants , as well as older participants . For negative affect, analysis revealed that younger participants significantly increased in mean levels of negative affect , whereas older participants demonstrated no significant change in mean levels of negative affect . For adaptive coping, we found that younger participants experienced a decrease in mean levels of adaptive coping in both cohorts , and older participants experienced decrease in mean levels of adaptive coping in cohort 2 only .Furthermore, it was tested whether the distinct age groups experienced differences in benefits of the app. Regarding daily positive affect, analysis revealed that younger participants decreased in mean levels of positive affect , and those with higher adherence in ESM and challenges increased in positive affect (cohort 1). However, the low entropy score (0.41 and 0.54) indicates that the model did not fit optimally and these findings should be interpreted with caution.Even though on average negative affect increased, 81.6–82.6% of the Grow It! users were classified into a trajectory of decreasing negative affect. Several significant baseline predictors that could partly explain the identified clusters and were associated with a decrease in negative affect, including being a boy (cohort 2), lower depression and anxiety scores, higher affective well-being score, higher coping score, receiving less psychological care (cohort 2), experience less COVID-19 related symptoms (cohort 2), higher ESM compliance (cohort 1). The rest (~18% of users) followed a sharp decline in daily well-being. Given the severity of their emotional problems and care use at baseline, this more seriously affected group seems as expected to benefit less from the prevention-oriented Grow It! app and may need more embedded clinical support. Because of the COVID-19 related necessary recruitment strategy, minimal exclusion criteria were applied.n = 932) found that nearly half of their sample reported changes that could contribute to lower well-being  . Furthermore, highly motivated individuals are especially willing to participate in such research. This suggestion is confirmed because only less than half of the subjects who completed the baseline questionnaire were included in the current study. Nevertheless, missing data was at random and we saw no difference in baseline characteristics between participants excluded due to low compliance and those included (Table S1 and Table S2), indicating that there was no evidence for induced selection bias. Furthermore, the Grow It! app is a study with scientific research purposes, including an extensive informed consent procedure, which raises a threshold for participation. In addition, the compliance of this ESM study was lower compared to other scientific studies in adolescent samples (van Roekel et al., Previous studies have suggested that the Grow It! app is beneficial in promoting overall well-being by integrating CBT-based challenges in adolescents at the population level (Dietvorst et al. Supplementary information


We aimed to compare the clinical and perinatal outcomes of patients with polycystic ovary syndrome (PCOS) undergoing in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) treatment with either an early-follicular long-acting gonadotropin-releasing hormone agonist (GnRH-a) long protocol (EFLL) or a midluteal short-acting GnRH-a long protocol (MLSL).This single–center, retrospective study, included patients with PCOS who underwent IVF/ICSI from January 2013 to June 2019 at the First Affiliated Hospital of Zhengzhou University, Zhengzhou, China. Patients underwent either MLSL (1179 cycles) or EFLL (2390 cycles). The primary outcomes were pregnancy outcomes, perinatal and maternal complications.P = 0.038), clinical pregnancy , and live birth rates  were higher in the EFLL group. However, the proportion of patients “freezing all" for high risk of ovarian hyperstimulation syndrome (OHSS)  and ectopic pregnancy  were lower in the EFLL group than in the MLSL group. The incidence of gestational diabetes was higher in the EFLL group than in the MLSL group  1.474–9.360, P = 0.003). There were no significant differences in the incidence of hypertension, premature rupture of membranes, placenta previa, congenital heart disease, or neonatal weight between the two groups. Logistic regression results showed that age , treatments , and endometrial thickness on trigger day  were correlated with clinical pregnancy. Pre-pregnancy BMI , fasting plasma glucose (FPG) , and treatments  were correlated with gestational diabetes mellitus (GDM). Treatments  and endometrial thickness on trigger day  were correlated with ectopic pregnancy.Fresh embryo transfer  pregnancy assistance program for patients with PCOS, but obese patients should be encouraged to lose weight before ART treatments to reduce the risk of GDM.The online version contains supplementary material available at 10.1186/s12958-022-01007-z. Polycystic ovary syndrome (PCOS) is a common reproductive, endocrine, and metabolic disease, with an incidence of approximately 9%–18% in women of reproductive age . An incrThis retrospective cohort study was approved by the Ethics Review Committee of the First Affiliated Hospital of Zhengzhou University. Written informed consent was waived due to the retrospective nature of the study. This analysis was conducted on patients with PCOS who underwent the first cycle of IVF/ICSI-assisted pregnancy treatment using either EFLL or MLSL at the Center of Reproduction Medicine of the First Affiliated Hospital of Zhengzhou University from January 2013 to June 2019. The inclusion criteria were as follows: ① aged 20–40 years and ② diagnosed with PCOS (in line with Rotterdam PCOS diagnostic criteria in 2003). The exclusion criteria were: ① untreated hydrosalpinx; ② endocrine diseases such as hyperprolactinemia and diabetes mellitus; ③ uterine malformation; ④ preimplantation genetic diagnosis/preimplantation genetic screening; ⑤ uterine fibroids with a diameter of > 3 cm and compression of the endometrium; ⑥ patients with endometriosis/adenomyosis; ⑦ intrauterine adhesion; ⑧ cervical insufficiency; and ⑨ history of tuberculosis.Patients were administered 3.75 mg of the long-acting gonadotropin-releasing hormone agonist (GnRH-a), triptorelin , on the 2nd to 3rd day of spontaneous menstruation or menstruation after oral administration of Dydrogesterone . Serum follicle-stimulating hormone (FSH), luteinizing hormone (LH), estrogen (E2), and progesterone (P) were measured 28 days after injection. Follicle size was monitored using transvaginal ultrasound.st or 22nd day of spontaneous menstruation or the 16th day of oral administration of drospirenone and ethinylestradiol tablets  at a dose of 1 tablet per day. Serum FSH, LH, E2, and P were measured after injection, meanwhile follicular size was monitored by vaginal ultrasound.A short-acting GnRH-a, tripraline , was administered subcutaneously at a dose of 0.1 mg per day for 14 to 16 days, starting on the 21Gonadotropin (Gn) was administered for controlled ovarian hyperstimulation when the downregulation standards were reached . The dosage of Gn in both protocols depended on factors such as the patients’ age, basal antral follicle count (AFC), body mass index (BMI), and basal hormone level. The dosage was adjusted according to the size of the follicles and the levels of FSH, LH, E2, and P.When at least 60% of the follicles were > 16 mm in diameter or when at least three dominant follicles were ≥ 18 mm, 250ug of Azer  and 2000 IU of human chorionic gonadotrophin (hCG)  were subcutaneously injected on the same night to trigger ovulation. About 36–37 h after the trigger, transvaginal ultrasound-guided puncture was performed for oocyte retrieval. The method of fertilization, IVF/ICSI, was based on semen quality. Fresh embryo transfers were performed 3–5 days after oocyte retrieval under ultrasound guidance based on embryo quality and patients’ overall and endometrial conditions. The transfer was cancelled if patients were deemed to be at high risk for OHSS or a uterine effusion was demonstrated. The luteal supplementation protocol was started from the day of oocyte retrieval. Progesterone sustained-release vaginal gel  and dydrogesterone tablets  were added at the dosage of 90 mg per day and 20 mg po BID respectively.To follow up with the pregnancy outcomes, we drew serum B-hCG levels 14 days after embryo transfer. Clinical pregnancy was confirmed when a gestational sac could be seen on ultrasound examination at 35 days after transplantation. Fetal nuchal translucency examinations were performed 9–10 weeks after embryo transfer. During the perinatal period, trained nurses provided follow-up via telephone. Standardized questionnaires were used to collect information on perinatal complications, gestational age, mode of delivery, neonatal sex, birth weight, diseases among newborns, and treatments. The follow-up information was recorded in detail and stored in the electronic medical records. The research data were extracted from the electronic database of our hospital.General patient information, days and total dosage of Gn, number of embryos transferred, hormone levels on day of trigger, incidence of OHSS, clinical pregnancy rate, spontaneous abortion rate, ectopic pregnancy rate, and perinatal maternal and infant adverse outcomes rates were recorded. The following metrics were also assessed: The grading criteria of high-quality embryos were based on previous publications of our center . High-qu2) test was used to assess differences in the proportion of categorical variables between two or more groups. Logistic regression analysis was conducted on the factors affecting the occurrence of pregnancy complications. Statistical significance was established at P < 0.05.All data in this study were analyzed and calculated using SPSS. Continuous variables are expressed as mean ± standard deviation (x ± s) and categorical variables are expressed as percentages. The t-test was used to determine the significance of the difference between the mean values of two continuous variables. The Chi-square , while the rate of fresh embryo transfer cycles (59.12% vs. 55.47%), the rates of biochemical pregnancy (79.23% vs. 58.87%), clinical pregnancy (75.23% vs. 53.82%), live birth (63.27% vs. 42.05%) and full-term birth (50.88% vs. 32.87%) were higher than in the MLSL group (P < 0.05). The proportion of patients “freezing all” for high risk of OHSS (24.27% vs. 32.06%) was lower than in the MLSL group. There were no statistically significant differences in the abortions, premature births, or incidence of moderate to severe OHSS between the two groups  and the high-quality embryo rate (54.23% vs. 59.37%) in the EFLL group were lower than in the MLSL group (P < 0.05), while the ectopic pregnancy rate was lower than that in the MLSL group (P < 0.05). There were no statistical differences in obstetric complications such as preeclampsia, multiple birth rate, placenta previa, premature rupture of membranes, and neonatal birth between the two groups  and the clinical pregnancy rate (53.92% vs. 40.00%) were significantly increased in EFLL compared with the GnRH antagonist protocol, and there was no significant difference in the incidence of OHSS . The incPatients with PCOS often have reduced endometrial receptivity due to their impaired glucose metabolism and abnormal hormone levels –18. The This study showed that the ectopic pregnancy rate was lower in the EFLL group than in the MLSL group. Logistic regression suggested that ovulation induction protocols and endometrial thickness on the day of trigger were independent factors for ectopic pregnancy. Endometrial thickness reflects the receptivity of the endometrium , and theThe results of the logistic regression in this study suggest that in addition to pre-pregnancy BMI and fasting plasma glucose, which are important risk factors of GDM in patients with PCOS , the ovuThis study comprehensively analyzed the clinical outcomes, perinatal and maternal complications, and fetal outcomes of patients with PCOS using EFLL and MLSL. We found that EFLL is superior to MLSL in terms of fresh embryo transfer rate and clinical pregnancy. Ovulation induction protocol is an independent factor of ectopic pregnancy and GDM. The ectopic pregnancy rate in EFLL is lower than that in MLSL. The incidence of GDM is higher than that in MLSL. However, there are no significant differences in the incidence of preeclampsia, premature rupture of membranes, placenta previa, fetal malformation, neonatal congenital heart disease, and neonatal weight between the two groups.The strength of this study lies in the comprehensive analysis of perinatal and maternal complications and fetal outcome measures and the analysis of data from a large sample adjusted for potential confounders. At the same time, our study also has some limitations. Our study is a retrospective study and does not consider all confounding factors. Second, our study population only included women with PCOS, who already have a higher risk of gestational diabetes and pregnancy-related complications than the normal population. Therefore, it is still necessary to perform a large, multicenter, prospective study in other populations to verify our conclusion.In conclusion, there was a higher fresh embryo transfer rate, clinical pregnancy rate, and live birth rate and lower ectopic pregnancy rate for patients with PCOS who underwent EFLL compared to those who underwent MLSL. This indicates that EFLL can be used as an ideal ART therapy for PCOS patients, but for obese patients, weight loss prior to assisted reproduction should be encouraged to reduce the risk of GDM.Additional file 1: Supplementary Table 1. Univariate logistic regression performed for GDM. Supplementary Figure 1. Forest plot: multivariate logistic regression performed for GDM.