Exosome-derived miR-146a-5p from decidual macrophages in preeclampsia inhibits the viability and invasive ability of trophoblast cells by targeting HIF1α

Objective:To investigate the effect of exosomes secreted by decidual macrophages on trophoblast cells and their molecular mechanism.Methods:The decidual tissues of patients with preeclampsia(PE)and normal-term pregnant women were collected.Macrophages were obtained by the density gradient method and then flow cell sorting,then the exosomes were extracted.The structure of the exosomes was observed by transmission electron microscope.The expression of CD63,a marker protein of the exocrine body,was detected by western blot,and the exosomes were identified.CCK-8 was used to detect the effect of exosomes on trophoblast cell viability.Transwell migration experiment was used to detect the influence on migration ability.The expression of miR-146a-5p in exosomes was detected by qPCR.The effect of exosomes on the expression of HIF1αprotein in trophoblasts was detected by western blot and detection of the binding site between miR-146a-5p and HIF1αby double luciferase reporter gene was conducted.Results:The exosomes of macrophages present a"cake"structure with a middle depression about 30-130 nm in diameter,and CD63 is highly expressed,which conforms to the characteristics of exosomes.Compared with the normal group,the exosomes of decidual macrophages in the PE group inhibited the activity and migration of trophoblast cells(P<0.001).The expression of miR-146a-5p in the exosomes of decidual macrophages in the PE decreased significantly,and after exosomes of PE decidual macrophages treating trophoblast cells,the protein expression of HIF1αin trophoblast cells was significantly increased.There are targeted binding sites between miR-146a-5p and HIF1α.Conclusion:PE decidual macrophage exosomes can inhibit the viability and migration of trophoblast cells,which may be related to the decreased expression of miR-146a-5p in exosomes,thus promoting HIF1αprotein expression of trophoblast cells.

Effect of IFN-γ/IL-10 on IL-10 Receptor Expression of Human Decidual Stromal Cells in Early Pregnancy

Objective To study the role of IFN-γ/IL-10 cytokines protein expression of human decidual stromal cells（DSC） vitro. on IL-10 receptor gene and in human early pregnancy in vitro. Methods Human DSC was isolated and cultured in vitro, and the expression of IL-10R1 and IL-10R2 gene was analyzed after cells had been treated with TH2-type cytokines IL-10 and TH1-type cytokines IFN-γ within 60 rain with semiquantitative reverse transcriptase-PCR, then the influence of IL-10 and IFN-γ on expression of IL-10R protein was examined by first trimester DSC using flow cytometry. In addition, the vitality of DSC was detected by MTT. Results IL-10R1 mRNA levels of DSC treated with IL-10 （10 ng/ml） reached the peak level within 15 rain, and were significantly lower at 30 rain, then were not detected at 45 min. The expression of IL-10R1 were induced to moderate level by IFN-γ（10 ng/ml） within 30 rain, and reduced to undetected levels at 60 min. There was no significant difference of IL-10R2 expression （P〉0.05） between treated and not with the abovementioned cytokines. The IL-10R protein expression and vitality of DSC were significantly enhanced by IL-10 （10 ng/ml） and IFN-γ （10 ng/ml） which treated DSC 48 h （P〈0.05）. Coneclusion IL-10 and IFN-γ may play an important role of biologic function in early pregnancy by influencing IL-10R expression of DSC.

Co-Culture of Early Embryo with Human Decidual Stromal Cells in vitro by Improvement of Early Embryo Development

Summary: An early embryo co-culture system with human decidual stromal cells was established to study its effect on early embryonic cleavage and growth in vitro. Three hundred and eight 2-cell mouse embryos were co-cultured with human decidual stromal cell monolayer in MEM+0. 4 % bovine serum albumin (BSA) and 163 embryos cultured in MEM+15 % FCS alone as control. Among the mouse 2-cell embryos co-cultured with human decidual stromal cells, 72.73 % developed to the morula stage and 67.21 % cavitated to blastocysts with 59. 74 % hatching, as compared with 61. 34 % to morula stage, 48. 47 % to blastocysts and none hatching in the controls, respectively. Co-cultured embryos cleaved slightly faster than controls and showed no or less fragmentation than those in the control. These results suggested that human decidual stromal cells can support early embryonic development and yield a reasonable number of embryos with good quality up to blastocyst stage.

The Effect of Gossypol and Misoprostol on Luteal and Decidual Cells in Vitro

Gossypol and Misoprostol could directly damage the luteal and decidual cells cultured in vitro. The LD50 of gossypol alone to luteal and decidual cells were 1.27±0.09 μg/ml and 3.06±0.23 μg/ml, respectively; however when combined with misoprostol (to luteal cells 5μg/ml, or to decidual cells 10μg/ml), the LD50 of gossypol signifcantly decreased to 0.89±0.25 μg/ml and 1.88±0.26 μg/ml, respectively. The LD50 of misoprostol alone to luteal and decidual cells were 14.29±1.29μg/ml and 24.37±4.49 μg/ml, respectively; but it decreased to 8.79±2.18 μg/mland 17.29±1.56 μg/ml, respectively when combined with gossypol (to luteal cells 0.5 μg/ml, or to decidual cells 1.0 μg/ml), also showing statistical difference. The results suggested that the combination of gossypol with misoprostol had synergistic effect on the degeneration of luteal and decidual cells in vitro.

Modulation of the Culture Supernatant of Decidual Cells with Exogenous Cytokines on Killing Activity of Natural Killer Cells in Early Pregnancy

To investigate the important function of cytokines in early pregnancy and to provide basic and experimental evidence for understanding the mechanism of their action. Methods Add interferon γ(IFN γ),interleukin 2(IL 2), interleukin 6(IL 6) and epidermal growth factor(EGF) to the confluent culturing decidual cells with three different concentrations and harvest the culture supernatant after 12, 24 and 48 h separately. Observe the effect of the supernatant on killing activity of NK cells with radioimmunological assay of 51 Cr immersion. Results The culture supernatant of decidual cells can promote the killing activity of NK cells in various degrees, and the effect is independent of the type, concentration and acting time of cytokines. Conclusion In normal pregnancy, decidual cytokine network is in a dynamic equilibrium. Exogenous cytokines would be harm to normal pregnancy by interfering the equilibrium state, but the exact mechanism needs further study.

Decidual Natural Killer Cells Are Essential for a Successful Pregnancy (Review)

Pregnancy is a complex physiological process involving several interconnected systems. Many researchers were concerned that the formation of a fetus with different genetic components may contradict the normal state of immunity, which attempts to reject and fight foreign bodies. This piqued the interest of biologists and immunologists, who set out to discover the immune system’s composition and mode of response in the uterus. According to several studies, natural killer (NK) cells are present in a significant percentage that differs from what is seen in peripheral blood. As a result, several scientific studies have been conducted on uterine NK cells, investigating their types, characteristics, receptors, secretions, and interactions with the surrounding environment. Research has also indicated the capacity of uterine NK cells to strike a balance between eradicating uterine infections and effectively contributing to different phases of pregnancy. Various studies have shown that NK cell activity is intimately related to the success or failure of pregnancy. In this review, we describe the uterine NK cell subtypes;decidual (dNK) cells and endometrial NK cells (eNK) cells and their important role during different phases of pregnancy.

Expression and molecular mechanism of PCNA,Caspase-3,IL-6 and Survivin proteins in chorionic villi and decidual tissue of early embryo damage

Objective:This study aims to explore its role in embryo damage and the relationship between them by testing the expression of PCNA,Caspase-3,IL-6 and Survivin protein in the chorionic villi and decidual tissue of patients with unexplained early embryo damage and normal early pregnancy at the same time voluntarily requested uterine aspiration abortion in order to clarify the pathogenesis of early embryo damage from the cellular and molecular perspectives,and provide theoretical basis for the diagnosis and treatment of embryo damage.Methods:From July 2018 to July 2019,30 patients with unexplained embryo damage were selected as embryo damage group,thirty normal early pregnancy patients with aspiration abortion were selected as the normal early pregnancy group.The decidual tissue and chorionic villi of the two groups were collected to observe the structural and pathological changes.Immunohistochemical method was used to detect the distribution of PCNA,Caspase-3,IL-6 and Survivin in the chorionic villi and decidual tissue,as well as the expression changes of the above four proteins.Results:Compared with the normal early pregnancy group,the chorionic villi in the early embryo damage group were obviously dysplasia,degenerative changes,structural disorders,homogeneous destruction,obvious reduction or even disappearance of trophoblast cells,inflammatory cell infiltration,more neutrophils and lymphocytes,interstitial edema and cell atrophy,accompanied by hemorrhage;The decidual tissue of the intima was not good,most of the cells were spindle-shaped,the structure was disordered,the stroma has edema,inflammatory cells can be seen,and hemorrhage existed.The results of immunohistochemistry showed that the expression of PCNA protein in chorionic villi and decidual tissue of early embryo damage group decreased significantly(P<0.05),while the expression of Caspase-3 protein increased significantly(P<0.05).The expression of IL-6 protein decreased significantly(P<0.05),and the expression of Survivin protein decreased significantly(P<0.05).Conclusion:During early pregnancy,due to the down-regulation of the expression of proliferating cell nuclear antigen PCNA and apoptosis inhibitor protein Survivin in chorionic and decidual tissues,the balance between proliferation and apoptosis is broken,which has an increase in Caspase-3 expression and a decrease in IL-6 expression.It may be that the balance of Th1/Th2 in chorionic and decidual tissues inclines to Th1,which leads to excessive apoptosis of chorionic and decidual tissues and the cessation of early embryo damage.

A case of resection of giant decidual polyp in first trimester

A 29-years-old primiparous woman,presented at 9 weeks 2 days pregnant due to moderate vaginal bleeding with neoplasm prolapsed from vagina for two days.She have suffered from recurrent vaginal spotting for about 4 weeks,and took dydrogesterone more than 3 weeks following the doctor's instructions at the local hospital.She had diagnosed as an endometrial polyp of about 1.3×0.6 cm in size by ultrasound scan before pregnancy.At present,the color doppler ultrasound revealed that a giant decidual polyp occupied the whole cervix canal,with wide pedicle base arised from the middle and lower part of the uterine cavity,and the lower part prolapsed the outside of cervix.After hospital admission,the speculum examination showed that a dark purple neoplasm with a size of about 5×3×3cm in the vagina protruding out of the cervical canal,on which surface there was a spontaneous bleeding point.The blood test findings included a WBC count of 8.59×910/L and C-reactive protein level of 3.02ng/L.Her temperature was 36.2℃.Cefuroxime was given to prevent infection after admission.Compression was given to stop bleeding,but there was still active bleeding on the surface of the polyp the next day.Therefore,a ultrasound-guided torsion polypectomy was performed in emergency.In the therter,we twisted the neoplastic tissues by oval forceps taking a step-by-step approach,until 1cm above the outsise of cervix.At this point,ultrasound scan showed that the pregnant sac was obviously pulled by external forces.So,we stop the polypectomy,leaving a small portion of the giant polyp.Ceftrixone sodium and dydrogesterone were given after the operation and a small amount of vaginal bleeding lasted for 6 days without obvious abdominal pain.A medium to strong echo of about 1.7×0.6×0.9cm in the cervical canal was showed by color doppler ultrasound,which continued upward to the upper part of the cervical os.Histopathological examination revealed a decidual polyp.The patient had no abdominal pain and vaginal bleeding during pregnancy.She had a premature rupture of membranes and gave birth to a baby with the weight of 3300g through vaginal at 37 weeks and 1 day of gestation.

Stromal cell decidualization and embryo implantation: a vulnerable step leading to successful pregnancy

Endometrial stromal cell decidualization is a crucial step in endometrial remodeling during pregnancy.Decidualization is controlled by orchestrated ovarian hormones,followed by the activation of various downstream signaling pathways.Accumulating evidence has shown multiple functions of decidualized endometrial stromal cells during embryo implantation,including tissue remodeling,antioxidative stress,angiogenesis,and immune tolerance.The distinct secretomes of decidualized stromal cells also reveal their intensive interactions with epithelial,endothelial,and immune cells.However,aberrant decidualization leads to pregnancy failures,such as recurrent pregnancy loss and repeated implantation failure.This review aimed to provide an overview of the molecular mechanisms underlying the divergent functions of decidualized endometrial stromal cells and their potential clinical applications.Moreover,the use of single-cell RNA sequencing data further enhances our understanding of these biological processes.This review discusses decidualization-related signaling pathways that serve as potential therapeutic targets for treating implantation failure in in vitro fertilization and provides novel approaches to investigate the underlying causes of female infertility.

Decidualization and Related Pregnancy Complications

Decidualization is the differentiation of endometrial stromal cells into secretory decidual stromal cells.Human decidualization involves some amount of signaling molecules and pathways as well as genetic reprogramming,which is driven by the postovulatory rise in progesterone levels and local cyclic adenosine monophosphate production.Decidualization extends from the primary decidual zone to the secondary decidual zone,and then exits through apoptosis.Evidences support that decidual fibroblasts function as the pool of decidual stromal cells during pregnancy.Decidualization undergoes an acute inflammatory phase,an anti-inflammatory secretory phase to the final recession phase.The decidualization of the inner layer of endometrium,termed decidua,is the most critical determinant of pregnancy success,which can promote placenta formation,modulate immune tolerance,foster resistance to oxidative stress,sense embryo quality,and control labor.Failure to adequate decidualization in terms of hormones,biochemistry,and immunology leads to adverse pregnancy outcomes,including diseases such as preeclampsia,miscarriage,premature labor,repeated implantation failures,and some age-related decline in reproductive capacity.The development of animal models and in vitro culture systems combined with emerging technologies provides a powerful system to explore the mechanism of decidualization.However,decidualization is a dynamic,multi-step process,and translating of current research progress into disease predictions and interventions for pregnancy complications remains to be achieved.The study of periodic regeneration and spontaneous decidualization of the endometrium will be beneficial to the diagnosis and treatment of pregnancy diseases.

Decidual stromal cells recruit Th 17 cells into decidua to promote proliferation and invasion of human trophoblast cells by secreting IL-17

T helper 17 （Th17） cells have both regulatory and protective roles in physiological conditions. The Th17 subset and the cytokine interleukin-17A （IL-17A） have been implicated in the pathogenesis of certain autoimmune diseases, several types of cancer and allograft rejection. However, the role of Th17 cells at the maternal/fetal interface remains unknown. Here, we demonstrate that Th17 cells are present in decidua and are increased in the peripheral blood of 10 clinically normal pregnancies based on intracellular cytokine analysis. Our results suggest a potential role of Th17 cells in sustaining pregnancy in humans. Furthermore, we demonstrate that decidual stromal cells （DSCs） but not trophoblast cells recruit peripheral Th17 cells into the decidua by secreting CCL2. The recruited Th17 cells promote proliferation and invasion and inhibit the apoptosis of human trophoblast cells by secreting IL-17 during the first trimester of pregnancy. These findings indicate a novel role for Th17 cells in controlling the maternal-fetal relationship and placenta development.

CircRNA expression profiles in decidual tissue of patients with early recurrent miscarriage

Circular RNAs(circRNAs)are a novel class of endogenous noncoding RNAs that play important roles in gene expression regulation.This study aimed to evaluate the potential role of circRNAs in decidual tissue of patients with early recurrent miscarriage(RM).We constructed circRNA expression profiles in decidual tissue using microarray data.A total of 123 differentially expressed circRNAs,including 78 upregulated and 45 downregulated circRNAs were detected in the early RM group compared with the control group(P<0.05).Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis also revealed the enrichment of specific circRNAs.The verified circRNA-targeted miRNA-mRNA network was constructed,most of the circRNAs harbored miRNA binding sites.The network involved 3 circRNAs,27 microRNAs and 82 mRNAs.Hsa_circRNA_103092-miR-224-PRLR network was selected to verify by qRT-PCR.These results showed that circRNAs are aberrantly expressed in the decidual tissue in early RM patients and play potential roles in the development of early RM.It gives new insights into the mechanism of recurrent miscarriage.

Research Progress on Human Endometrium Decidualization In vitro Cell Models

Decidualization is a special type of differentiation of endometrial stromal cells into secretory decidualized cells,which is closely related to the occurrence of menstruation and establishment of pregnancy.Decidualization abnormalities can cause female infertility and abortion,and the decidualization modelin vitro is an important tool for studying relevant mechanisms.This article summarizes severalin vitro decidualization models in recent research from three aspects,including the selection of model cells and culture systems,evaluation of decidualization markers,and induction schemes.These models can be appropriately selected and applied in specific endometrium-related disease models,such as endometriosis,recurrent pregnancy loss,and preeclampsia.

Cyclooxygenase-2 and Decidual Immune Cells

Cyclooxygenase-2(COX-2)is a rate-limiting enzyme in arachidonic acid(AA)metabolism.COX-2 and its products(prostanoids)serve versatile biological functions during pregnancy.Numerous evidences demonstrate special reprogramming of COX-2-catalyzing AA metabolism in decidual immune cells(DICs),particularly in decidual macrophages,corresponding to special gestational phases.This review summarizes the reprogramming of COX-2-catalyzing AA metabolism in DICs as well as the immunoregulation of diverse COX-2-generating prostanoids in DICs during the different phases of gestation.

A Defective CXCL16/CXCR6 Axis Increases the Risk of Pregnancy Loss via the Abnormal Crosstalk between Decidual γδ T Cells and Trophoblasts

Objective::The maternal-fetal interface undergoes dynamic changes to allow the fetus to grow and develop in the uterus.The interaction between decidualγδT cells and trophoblasts plays a pivotal role during successful pregnancy;however,their physiological functions in early-term human pregnancy are still not completely illustrated.This study was undertaken to illustrate the functional roles of CXCL16/CXCR6 to prevent pregnancy loss via the crosstalk between decidualγδT cells and HTR8/SVneo trophoblast cells.Methods::The percentile of CXCR6+γδT cells in the peripheral blood from normal female and recurrent spontaneous abortion(RSA)patients was analyzed by flow cytometry.The expression of CXCR6 was detected in decidual immune cells via flow cytometry,and the expression of CXCL16 was analyzed in HTR8/SVneo trophoblast cells and lentivirus(LV)-HTR8/SVneo trophoblast cells via enzyme-linked immunosorbent assay.Reverse transcriptase-polymerase chain reaction was used to verify the expression of the CXCL16 gene in LV-HTR8/SVneo trophoblast cells.Expression of granzyme B and cytokines and proliferation of decidualγδT cocultured with HTR8/SVneo trophoblast cells were analyzed by flow cytometry.Invasion of HTR8/SVneo trophoblast cells was assessed via Matrigel transwell assay.Adoptive transfer was induced in vivo further to illustrate that the normal expression of CXCL16/CXCR6 could prevent pregnancy loss.Results::The percentile of CXCR6+γδT cells in the peripheral blood from RSA patients was lower than normal pregnancies.The expression of CXCR6 was highest in the decidualγδT cells among decidual immune cells,and the expression of CXCL16 increased as the amount of HTR8/SVneo trophoblast cells increased.Expression of granzyme B in the decidualγδT cells was downregulated by cocultured with HTR8/SVneo cells dependent of CXCL16,and HTR8/SVneo trophoblast cells induced the Th2 cytokines production in the decidualγδT cells.Both the expression of CXCR6 in the decidualγδT cells and proliferation of the decidualγδT cells were promoted by HTR8/SVneo trophoblast cells.On the other hand,decidualγδT cells enhanced the invasion of HTR8/SVneo trophoblast cells and thus promoted embryo implantation.In vivo study was taken further and shown that low expression of CXCL16/CXCR6 results in pregnancy loss because of dialog disorder between decidualγδT cells and trophoblasts.Conclusions::Low expression of CXCL16/CXCR6 results in pregnancy loss because of the dialog disorder between decidualγδT cells and trophoblasts,and it showed a light on the effective strategy of adoptive transfer of CXCR6+γδT cells on the treatment of RSA.This observation provides a scientific basis on which a potential strategy can be applied to the early-detect and treatment of RSA.

Role of Decidual Natural Killer Cells at the Maternal-Fetal Interface during Pregnancy

Pregnancy is a complicated process with intricate cell-to-cell crosstalk and immune regulation.Decidual natural killer(NK)cells account for 50%-70% of decidual immune cells in early pregnancy,suggesting that they play important roles in various events,such as embryo implantation and vascular remodeling.Many studies have shown that decidual NK cells interact with other cells either through direct contact or the secretion factors such as cytokines and chemokines.Hence,this review aimed to present the phenotypic characteristics,classification,and functions of decidual NK cells at the maternal-fetal interface during pregnancy.

Developmental Aspect of Decidual Patterning

In clinical practice,early pregnancy loss has afflicted approximately 15%–25%women of reproductive age worldwide,which is partially attributed to defects associated with the endometrium.During pregnancy,the endometrial stromal cells experience remarkable tissue remodeling and transformation,termed as decidualization,to support embryonic development,placental formation,and the maintenance of normal pregnancy in both mice and human.During this process,a series of dynamic developmental events,including rapid stromal proliferation,increased stromal cell size,enhanced angiogenesis,taken place in a highly-ordered manner under the precise regulation of steroid hormones.Meanwhile,diverse molecules exhibit spatiotemporal-specific expression pattern,implying their unique roles in decidual development.To achieve a more comprehensive understanding of these biological events and explore the underlying causes of early pregnancy disorders,this review emphasizes on the detailed developmental progression of decidual transformation and patterning as well as related pregnancy complications at the early stage of pregnancy.

Unique and independent role of the GABAB1 subunit in embryo implantation and uterine decidualization in mice

Embryo implantation and decidualization are crucial for successful pregnancy,which include multiple genes and signaling pathways,while the precise mechanism regarding embryo implantation and decidualization has yet to be explored.The GABA which activates GABA_(A)or GABA_(B)receptors has been found playing an important role in early pregnancy.Here we seek to investigate whether GABA_(B)receptors participate in embryo implantation in mice.This study first characterized the spatiotemporal expression pattern of GABA_(B)receptors in the uterus during the peri-implantation period and found that GABA_(B1)expression was drastically upregulated in stromal cells on days 4e6,a period of embryo implantation and early stages of decidualization.Embryo delayed implantation and oil-induced decidualization models were further used to confirm that the GABA_(B1)was associated with embryo implantation and decidualization.We also found estrogen or progesterone had no directly effect on expression of GABA_(B1)in ovariectomized model.Because we were unable to detect significant GABA_(B2)which couples with GABA_(B1)to form whole GABA_(B)receptors,and the agonist and antagonist of whole GABA_(B)receptors had weak effect on the proliferation and differentiation of stromal cells as well,we excluded the possibility whole GABA_(B)receptors function,and concluded it should be non-classical signals of GABA_(B1)involving in embryo implantation and decidualization.Future studies should focus on investigating the roles and mechanisms of GABA_(B1)during embryo implantation and decidualization.

Evaluation of the Effectiveness and Safety of One-Time Endodontics in the Treatment of Chronic Apical Periodontitis with Sinus Tract in Pediatric Deciduous Teeth

Objective:To observe the effectiveness and safety of one-time endodontics in the treatment of chronic apical periodontitis with sinus tract in pediatric deciduous teeth.Methods:109 cases of children with chronic apical periodontitis with sinus tract in the deciduous teeth treated in our hospital from January 2022 to December 2023 were selected and grouped by the randomized numerical table method,with 54 cases in the experimental group and 55 cases in the control group.The experimental group was treated with one-time endodontics and the control group was treated with conventional endodontics.Results:After the treatment,the total effective rate of treatment was higher in the experimental group than in the control group(P<0.05);the incidence of adverse events was lower in the experimental group than in the control group(P<0.05);the satisfaction of the children's family members was higher in the experimental group than in the control group(P<0.05);the pain duration was lower in the experimental group than in the control group(P<0.05).Conclusion:In the experimental group,children with chronic apical periodontitis with sinus tract of the deciduous teeth were given one-time endodontic treatment,and the results of its implementation were relatively good.

Tree sapling vitality and recovery following the unprecedented 2018 drought in central Europe

Background:Ongoing climate change is anticipated to increase the frequency and intensity of drought events,thereby affecting forest recovery dynamics and elevating tree mortality.The drought of 2018,with its exceptional intensity and duration,had a significant adverse impact on tree species throughout Central Europe.However,our understanding of the resistance to and recovery of young trees from drought stress remains limited.Here,we examined the recovery patterns of native deciduous tree sapling species following the 2018 drought,and explored the impact of soil depth,understory vegetation,and litter cover on this recovery.Methods:A total of 1,149 saplings of seven deciduous tree species were monitored in the understory of old-growth forests in Northern Bavaria,Central Germany.The vitality of the saplings was recorded from 2018 to 2021 on 170 plots.Results:Fagus sylvatica was the most drought-resistant species,followed by Betula pendula,Acer pseudoplatanus,Quercus spp.,Corylus avellana,Carpinus betulus,and Sorbus aucuparia.Although the drought conditions persisted one year later,all species recovered significantly from the 2018 drought,albeit with a slight decrease in vitality by 2021.In 2018,the drought exhibited a more pronounced adverse effect on saplings in deciduous forests compared to mixed and coniferous forests.Conversely,sapling recovery in coniferous and mixed forests exceeded that observed in deciduous forests in 2019.The pivotal factors influencing sapling resilience to drought were forest types,soil depth,and understory vegetation,whereas litter and forest canopy cover had a negative impact.Conclusion:Long-term responses of tree species to drought can be best discerned through continuous health monitoring.These findings demonstrate the natural regeneration potential of deciduous species in the context of climate change.Selective tree species planting,soil management practices,and promoting understory diversity should be considered when implementing adaptive management strategies to enhance forest resilience to drought events.