Huaier extract also enhanced proteasome-mediated protein degradation of AR-FL and AR-V7 by downregulating proteasome-associated deubiquitinase ubiquitin-specific protease 14 (USP14). Furthermore, huaier extract inhibited AR-FL/AR-V7 transcriptional activity and their nuclear translocation. More importantly, our data demonstrated that huaier extract could re-sensitize enzalutamide-resistant prostate cancer cells to enzalutamide treatment in vitro and in vivo models. Our work revealed that huaier extract could be effective for treatment of prostate cancer either as monotherapy or in combination with enzalutamide.The androgen receptor (AR) plays a pivotal role in prostatic carcinogenesis, and it also affects the transition from hormone sensitive prostate cancer (HSPC) to castration-resistant prostate cancer (CRPC). Particularly, the persistent activation of the androgen receptor and the appearance of androgen receptor splicing variant 7 (AR-V7), could partly explain the failure of androgen deprivation therapy (ADT). In the present study, we reported that huaier extract, derived from officinal fungi, has potent antiproliferative effects in both HSPC and CRPC cells. Mechanistically, huaier extract downregulated both full length AR (AR-FL) and AR-V7 mRNA levels  With nearly 1,276,106 new cases expected worldwide in 2018, prostate cancer (PCa) represents the second-most frequent type of cancer in men following lung cancer, accounting for almost 7.1% of new cancer cases, and there were 358,989 prostate cancer–related deaths in 2018 around the world according to the GLOBOCAN 2018 (The androgen receptor (AR), consisting of four domains with 919 amino acids in full-length, is the core of cellularly physiological and pathophysiological process in prostate cell –7. The aRecently, enzalutamide, the second-generation of hormonal drug targeting full-length AR (AR-FL), were used to treat CRPCs . NeverthTrametes robiniophila murr (huaier), a type of medicinal fungus which is found on the trunks of Styphnolobium japonicum , has been used in Traditional Chinese Medicine (TCM) for approximately 1,600 years . Cell lines were routinely tested mycoplasma free and were authenticated by STR detection. The cell lines were cultured in RPMI1640 with 10% fetal bovine serum (FBS), and the culture media for 22Rv1 also contained 1× GlutaMAX™ , and 1mmol/L Sodium Pyruvate . Enzalutamide, MG132, and IU1 were purchased from MedChemExpress . Huaier extract was kindly provided by Gaitianli Medicine Co., Ltd. , and was prepared as previously described , 25.3 cells/well) in 100 μl of medium were seeded in 96-well plates. After 12-h culture in 5% CO2 at 37°C in a humidified incubator, the medium was replaced by different concentrated solutions of huaier extract, and then incubated for another 24–72 h. Afterward, 10 μl CCK-8 was added to each well and the cells were incubated for another 1–4 h at 37°C according to the instructions of the manufacture. Absorbance of each well was quantified at 450 nm by an enzyme-linked immunosorbent assay microplate reader .Cell proliferation was assessed by the cell counting kit 8 (CCK-8) assay according to the manufacturer’s Instruction . Briefly, PCa cells .The Transwell system  were used in invasion assay. In addition, 2 × 105 cells/well seeded, 36-h incubation.The migration assay was performed in the same way as the invasion assay except the following differences: no Matrigel using 1 × 10PCa cells were treated with huaier extract for 48 h, and the cells were collected in 100-ul binding buffer and incubated with PI and Annexin V-fluorescein isothiocyanate  in the dark for 15 min at room temperature. Then 400-ul binding buffer was added to each tube, and fluorescence activated cell sorting (FACS) was performed immediately.Cell-cycle analysis was performed using a Cell Cycle Assay Kit Plus  according to the manufacturer’s instructions. Briefly, PCa cells were treated with huaier extract for 48 h, and the cells were collected, washed with cold PBS twice and fixed with 75% ethanol at 4°C overnight. Then the cells were wash with PBS, and suspended in 1ml PBS, followed by the addition of 4-ul RedNucleus I  for 20 min in dark at room temperature. Fluorescence activated cell sorting (FACS) was performed at channel FL4 immediately.via jetPRIME.Cells were seeded in six-well plates and transfected with small-interfering RNA (siRNA)  using jetPRIME . To overexpress target genes in prostate cancer cells, pGL3-basic vector containing target genes cDNAs  transiently transfected the PCa cells For luciferase reporter assay, PC3 cells (2 × 105 cells per well of six-well plate) were transfected with pGL3-KLK3-Luc reporter plasmid or the control plasmid along with AR-FL or AR-V7. The luciferase activity was determined 24 to 48 h after transfection using a dual luciferase reporter assay system following the manufacturer’s instructions , the signal was normalized to Renilla luciferase control as relative luciferase units.via jetPRIME . After 24 h, cells were treated with either huaier extract (2 mg/ml) or enzalutamide (20 umol/L) for another 24 h and subsequently treated with dihydrotestosterone (DHT) (10 nmol/L) for 2 h. Cells were then fixed with Paraformaldehyde, and permeabilized with 0.4% Triton X-100. After blocking by goat serum for 1 h, cells were incubated with anti-AR-FL  and anti-AR-V7 antibodies  overnight at 4°C. The slides were then incubated in CoraLite488–conjugated affinipure goat anti-rabbit IgG  for 1 h. The images were obtained by the Olympus microscope equipped with PerkinElmer system.PC3 cells were transfected with vector containing AR-FL cDNAs , and AR-V7 cDNAs  For immunoprecipitation (IP), the cell lysates, prepared with agarose protein A+G  to eliminate nonspecific binding, were incubated with primary antibody overnight at 4°C, and mixed with agarose protein A+G for 6 h at 4°C. After washed three times by lysis buffer, the loading buffer was added and the agarose protein A+G were removed by centrifugation. Western blot was performed to analyze protein expression. For western blot, it was performed according to the standard method, briefly, 30 ug total protein was loaded on gel, after the progress of running the gel, transferring the protein, and incubating with antibodies, the protein image was acquired by the chemiluminescence detection system.via QuantStudio 3 Real-Time PCR system . Primers used for PCR were showed in RNAs were extracted using Trizol . One microgram of total RNA was used for cDNA synthesis by the SuperScript III Reverse Transcriptase Kit . For detection of the indicated genes, each cDNA sample was amplified using SYBR Green  The formalin fixed tissues were paraffin-embedded and sliced into 4-um slides. After deparaffinization, hydration, and antigen retrieval, blocking endogenous peroxidase and nonspecific antibody binding were carried out followed by incubation with primary antibody. DAB developing were carried out after the slides were incubated with secondary antibody labeled with HRP. Finally, the slides were counterstained with hematoxylin, dehydrated with ethanol, cleaned with xylene, and mounted with resin mounting medium. Images were taken with CIC XSP-C204 microscopy.6 22Rv1 cells were subcutaneously injected into the flanks of the mice. When tumor volume reached 50–100 mm3, mice were randomized into four groups and treated as follows: (i) vehicle control , (ii) enzalutamide , (iii) huaier extract , and (iv) enzalutamide  + huaier extract . Tumors were measured every five days and calculated using length × width2/2. Mice were also weighed one to two times per week to monitor for signs of drug toxicity. Tumor tissues were harvested after 25 days of treatment.The animal experiments were performed in strict accordance with the Guidelines for the Care and Use of Laboratory Animals of Shandong University. Five- to six-week-old male BALB/c-nu mice were purchased from Charles River Laboratories. 5 × 10The results were analyzed using SPSS software . Student’s t-test, one-way ANOVA, and two-way ANOVA were performed. Every experiment was performed in triplicate, and differences were considered significant when p-values < 0.05.In our study, we used two prostate cancer cell lines, LNCaP and 22Rv1, to verify the effect of huaier extract on prostate cancer. LNCaP, the hormone-sensitive prostate cancer (HSPC) cell line, expresses AR-FL, which is sensitive to AR-targeted therapy. 22Rv1, the CRPC cell line, expresses both AR-FL and AR-V7, which are resistant to AR-targeted therapy. First, to test the effect of huaier extract on PCa cells, we measured cell viability using a CCK-8 assay in which the cells were treated with different concentrations of huaier extract for different lengths of time. As shown in via targeting AR/AR-V7, we tested huaier extract inhibitory effect through CCK8 assays after overexpression of AR/AR-V7 in PCa cells  has been used to treat tumors for a long time in China. With more research and high-quality clinical trials on TCM, the treatment of cancer with TCM has attracted more and more international attention and cooperation. Many studies have demonstrated that huaier extract exerted antitumor properties in a range of tumors. According to a phase IV clinical trial evaluating the benefit of huaier extract on hepatocellular carcinoma (HCC), huaier extract can improve the recurrence-free survival (RFS) rate and reduce the tumor extrahepatic recurrence rate (ERR) in HCC . Li et ain vitro and in vivo models. Our data also provided the evidence that huaier extract would be used for CRPC.After a period of androgen deprivation treatment, HSPC is more commonly converted to CRPC. CRPC remains incurable, and all patients with CRPC eventually acquire resistance to the second generation of hormonal drug: enzalutamide . In our pt?>To further explore the underlying mechanisms that huaier extract inhibit the expression of AR-FL and AR-V7, we focused our attention on the modulators that could regulate AR transcription and post-translational modification (PTM). SMYD3, functioning as methyltransferase, plays a pivotal role in methylation of targeted proteins . Many stvia activating autophagy in PCa cells, which need to be explored in the future.PTM is responsible for the signal transduction of phosphate, ubiquitin, methyl, acetyl, and glycosyl groups from one protein to another , 47. Manvia targeting AR-FL and AR-V7 signaling pathways  and the National Science Foundation for Young Scientists of Shandong (ZR2020QH245).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.


Allergic rhinitis (AR), characterized by nasal itching, sneezing, and congestion, is a common disorder of nose. In the United States, AR affects 10% to 20% of adults. The negative impact of the high prevalence of AR has caused a great economic burdens worldwide. Modern Western Medicine mainly treats AR with antihistamine drugs, glucocorticoids, allergic immunotherapy (AIT), but it seriously affects patients compliance because of its long course of treatment, high medical costs and side effect. And now, as an important mean of treating AR, acupoint injection has been widely used in clinics, and has achieved significant efficacy.The following databases will be searched for relevant information before July 2020: PubMed, Embase, Cochrane Library, Web of Science, and CNKI. Major results: scores of Rhinitis Quality of Life (RQLQ), Rhinitis Total Symptom Scores (RTSS). Secondary results: levels of antigen-specific serum immunoglobulin E (IgE), total effective rate, adverse event. Data will be collected independently by 2 researchers, and the risk of bias in meta-analysis will be evaluated according to “Cochrane Handbook for Systematic Reviews of Interventions”. All data analysis will be conducted using Review Manager V.5.3. and Stata V.12.0.The curative effect and safety of acupoint injection treatment for AR patients will be evaluated systematically.The systematic review of this study will summarize the currently published evidence of acupoint injection treatment for AR to further guide its promotion and application.The private information from individuals will not be published. This systematic review also will not involve endangering participant rights. Ethical approval is not required. The results may be published in a peer-reviewed journal or disseminated in relevant conferences.https://osf.io/fa9dq.Open Science Framework (OSF) registration number:  AR affects 10% to 20% of adults in the United States. Due to the easy relapse of AR and the delay in the course of the disease, some AR patients are often complicated with asthma, nasal polyps, and rhinosinusitis. AR can cause adverse effects impacting patients quality of life .–6 The negative impact of the high prevalence of AR has caused a great economic burdens worldwide.Allergic rhinitis (AR) is a non-infectious inflammatory disease of nasal mucosa mediated by IgE after atopic individuals are exposed to allergens.Therefore, it is very necessary to actively treat AR and improve the quality of life of patients. Modern western medicine treatment of AR mainly antihistamine drugs, hormone therapy, allergy immunotherapy (AIT) and surgical treatment. Although drug treatment has a good short-term efficacy, it is easy to relapse after drug withdrawal. Hence, the aim of this study was to evaluate the efficacy and safety of acupoint injection in the treatment of AR by meta-analysis.AS thus, particularly, it is urgent and important to explore an alternative therapy. In recent years, acupoint injection has been widely used in the treatment of AR, and played a good role in improving symptoms and quality of life. Moreover, it is one of the methods of integrated traditional Chinese and western medicine in the treatment of allergic rhinitis, which as a comprehensive therapy, mainly based on the combination of acupoints, drugs and meridians. By injecting a small amount of liquid drugs into specific acupoints and producing stimulation, it can enhance and adjust the immune function of the body, and finally achieve the purpose of curing the disease.2In a randomized controlled trial (RCT), the efficacy and side effects of acupoint injection in treating AR have been evaluated systematically. We expect to provide reference for AR treatment in the field of acupoint injection.33.1The protocol of the systematic review has been registered. statement guidelines, and the important protocol amendments will be documented in the full review.Registration: OSF Preregisration.2020, Sep.8. osf.io/fa9dq. This systematic review protocol will be conducted and reported strictly according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA)3.23.2.1Inclusion criteria are all randomized controlled trials (RCTs), which main treatment of AR is acupoint injection. The language of the trials to be included only Chinese or English.3.2.2Following studies will be excluded:1.Repeated publications2.Review of literature and cases3.Animal studies4.Incomplete literature5.Non-randomized controlled trials3.3We will include RCTs of participants of 18 years or older, of any sex, race/ethnicity, and the patients are in accordance with diagnostic criteria of AR established by the American Academy of Otolaryngology or Chinese Medical Association of Otorhinolaryngology. We will exclude patients who are also participating in clinical trials for other drugs or treatments; patients with mental or legal disabilities should not be treated with this therapy; pregnant or lactating women, mental patients, etc.; patients with severe diseases of the heart, brain, liver, kidney or hematopoietic system.3.4Interventions included treatment with acupoint injection. The control group only received conventional western medicine treatment. The routine treatment of each RCT may not be identical, but the use of acupoint injection is the only difference between intervention and control.3.53.5.11.Rhinitis Quality of Life (RQLQ);2.Rhinitis Total Symptom Scores (RTSS);3.5.21.antigen-specific serum IgE;2.total effective rate;3.adverse events.3.63.6.1This review will include the following electronic databases from their inception to Aug 2020: Electronic database includes PubMed, Embase, Cochrane Library, Web of Science, CNKI Fig. . The res3.6.2The following MeSH terms and their combinations will be searched:AR;RCT OR RCTs;acupoint injection OR point injection OR acupuncture point injection.The search strategy for PubMed is shown in Table . Other e3.73.7.1Two researchers (ZC and SX) will independently assess abstracts and titles of studies identified by literature search. Duplicates will be omitted using EndNote software (V.X9.0). Secondly, we will download full text of the relevant studies for further selection according to the inclusion criteria. If there is any different opinion, 2 researchers will discuss and reach an agreement. If a consensus could not be reached, there will be a third researcher (RL) who make the final decision. The details of selection process will be displayed in the PRISMA flow chart.3.7.2The assessment of risk of bias will be carried out by 2 independent reviewers (ZC and YH), using the Cochrane Collaboration's “Risk of bias” tool. Study bias will be conducted as either: “unclear,” “low,” or “high” risk for the following criteria: random sequence generation, allocation concealment, blinding, incomplete data, selective outcome reporting, and other bias. The assessment of the bias has caused controversy, there is a need for discussion with a third reviewer (RL). The graphic representations of potential bias within and across studies using Rev Man V.5.3.5.3.7.3Statistical analyses will be conducted using the risk ratio with 95% confidence intervals (CIs). Odds ratio (OR) and relative risk (RR) are commonly used for dichotomous outcomes data. For continuous outcomes, the weighted mean difference (WMD) or the standard mean difference (SMD) will be analyzed.3.7.4The unit of analysis will be the individual participant.3.7.5Among the results of several studies with insufficient data or missing data, the corresponding author will be contacted to complement the contents. If the corresponding author cannot be contacted, the data alone will be conducted.3.7.6I2value of the forest, plot will be calculated to assess heterogeneity, according to the Cochrane Handbook. The I2value is classified into 4 levels: little or no heterogeneity (0%–40%), moderate heterogeneity (30%–60%), substantial heterogeneity (50%–90%), and considerable heterogeneity (75%–100%).The assessment of heterogeneity will be conducted by Review Manager (V.5.3.5). Chi-squared test and 3.7.7If the numbers of available studies are sufficient, funnel plots will be assessed reporting biases.3.7.8I2 ≥ 50%). If there is considerable variation in results (I2 ≥ 75%), the meta-analysis will not be performed. The narrative and qualitative summary will be available.Review Manager (V.5.3.5) will be used to analyze. The test indicated little or no heterogeneity; a fixed effect model will be used for data. The random effect model will be adopted when there is considerable heterogeneity  may be affected heterogeneity.3.7.10I2 value will be used to quantify statistical heterogeneity.Sensitivity analysis will be used to assess the robustness of the results. It is possible to determine according to methodological quality, sample size, and analysis-related issues. The studies that follow a sequence will be removed from all the inclusion reviews. The Chi-Squared test and 3.7.11The assessment of evidence for all outcomes will be summarized using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach. The quality of evidence will be rated as high, moderate, low, and very low quality.4–10 Western medicine prefer to relieve symptoms of AR, and most patients are treated with antihistamine drugs, glucocorticoids or AIT. Although its fast effect in improving symptoms, there are side effects, immune therapy for a long time and high medical bills, seriously affect the patients compliance, also prone to adverse reactions.Obviously, allergic rhinitis has become a public health problem that needs to be paid attention to. AR belongs to type I hypersensitivity that is mainly mediated by IgE, during which a variety of immunoreactive cells , inflammatory mediators  and inflammatory cells  are activated.–12 The effects of drugs include not only differentiation of traditional Chinese medicine, but also modern pharmacology. It has the advantages of short course of treatment, excellent therapeutic effect, low cost, and good security, which has been widely used in the clinical treatment of various diseases. The report show that acupoint injection can significantly improve the clinical symptoms and quality of life of AR patients. In addition, acupoint injection can relieve the nasal mucosa inflammation by reduce the levels of H1R and H4R proteins and decreasing the gathering of EOS in the nasal mucosa. Through many clinical samples, it is fully realized that acupoint injection has its advantages in treating AR.–15Study proves that acupuncture can generate a curative effect on this disease by restoring Th1/Th balance, decreasing IgE levels, reducing nasal mucosal inflammatory cell infiltration. Acupoint injection therapy is based on the basic theory of acupuncture, and under the guidance of meridian theory, it combines meridians, acupoints and drug effects. Through the action of meridians and acupoints, the drug effect can be magnified, so only a few drugs can produce a good curative effect. The mechanism of acupoint injection therapy is to ameliorate the pathological conditions and regulate the function of the body through the synergistic effect of acupuncture and drugs.In short, this systematic review and meta-analysis can help identify the potential value of acupoint injection in the treatment of AR and improving patients QOL. Furthermore, this study will provide a basis for patient selection treatment options in future.Conceptualization: Zhenfeng Chen, Shasha Xing.Data curation: Shasha Xing, Yingzhuan Huang, Renfeng Li.Formal analysis: Zhenfeng Chen, Donghan Xu, Qiumei He.Funding acquisition: Renfeng Li.Investigation: Shasha Xing, Yingzhuan Huang.Project administration: Renfeng Li.Quality assessment: Renfeng Li, Zhenfeng Chen.Software: Yingzhuan Huang, Shasha Xing, Xiaofan Luo.Supervision: Zhenfeng Chen.Validation: Zhenfeng Chen, Lizhen Wang.Writing – original draft: Zhenfeng Chen, Shasha Xing, Yingzhuan Huang.Writing – review & editing: Shasha Xing, Renfeng Li.


Sitona discoideus , using the single sensillum recording technique. Our study using five pheromone-related compounds and 42 plant volatile compounds indicates that S. discoideus have highly specialized ORNs for pheromone and plant volatile compounds. Different groups of ORNs present in both males and females of S. discoideus were highly sensitive to 4-methylheptane-3,5-dione (diketone) and four isomers  of 5-hydroxy-4-methylheptan-3-one, respectively. Our results also indicate that male S. discoideus, using the sensory input from antennal ORNs, can distinguish both diketone and the RR-isomer from others, and RS- and SS-isomers from others, although it was unclear if they can distinguish between RS-isomer and SS-isomer, or between diketone and the SR-isomer. It also appeared that female S. discoideus could distinguish between RS-isomer and SS-isomers. The antennae of S. discoideus thus contain sex-specific sets of ORNs for host- and non-host plant volatile compounds. Both sexes of S. discoideus have highly sensitive and selective ORNs for some green-leaf volatiles, such as (Z)-3-hexenol and (E)-2-hexenal. In contrast, male antennae of S. discoideus house three distinct groups of ORNs specialized for myrcene and (E)-β-ocimene, 2-phenylethanol, and phenylacetaldehyde, respectively, whereas female antennae contain three groups of ORNs specialized for (±)-linalool and (±)-α-terpineol, myrcene and (E)-β-ocimene, (±)-1-octen-3-ol, and 3-octanone. Our results suggest that S. discoideus use a multi-component pheromone communication system, and a sex-specific set of ORNs with a narrow range of response spectra for host-plant location.Antennal olfactory receptor neurons (ORNs) for pheromone-related and plant volatile compounds were identified and characterized in the lucerne weevil,  Sitona discoideus (Gyllenhål) (Coleoptera: Curculionidae), commonly called the lucerne weevil, was first reported in New Zealand in 1974 when larvae were found feeding on burr medic (Medicago polymorpha L.) near Napier in the North Island (Esson M. sativa L.) hay used for horse fodder  considered to be synonymous with S. lepidus and S. flavescens (Coleoptera: Curculionidae)    in New Zealand which continues through winter into early spring (November). Adults of both species feed on the foliage and the larvae feed on the root nodules and roots. Feeding by larvae of both S. discoideus and S. obsoletus can cause significant yield losses in lucerne  and -5-hydroxy-4-methyl-3-heptanone (minor), during the autumnal post-aestivatory flight period -isomer is the major aggregation pheromone component for two beetles of the Sitophilus genus, the rice weevil and the maize weevil  provide clues to potential species-specific sets of ORNs and their potential use for host and mate location in insects. In a recent study, we have identified ORNs for pheromone-related compounds and plant volatiles for S. discoideus used in the experiments were collected in the Canterbury region of New Zealand. Males and females were distinguished based on the shape of the ventrite  and the four stereoisomers  of 5-hydroxy-4-methyl-3-heptanone Fig. , and 42 Presentation of test chemicals to the insect antennae was similar to previous studies  glass electrode  filled with 0.1 M KCl was inserted into a membranous part of the abdomen to serve as the reference electrode. An electrochemically sharpened tungsten electrode (tip diameter < 0.1 μm) was used as a recording electrode and the position of the electrodes was controlled with micromanipulators . An Ag-AgCl junction was used to maintain electrical continuity between the reference electrode and the ground input of a high-input impedance headstage preamplifier . The AC signals through the preamplifier were further amplified, digitized at 12,000/s sampling rate, and processed with a PC-based signal processing system  and software .n = 2~27 depending on sensillum types) of the subtracted values were classified into seven different categories of ORN response strength. The ORNs were then classified into different types using an algorithm with step-by-step analysis according to their response profiles across the test compounds  and 13.24 ± 1.59 spikes/s  in pheromone-related ORNs of males and females, respectively, and at 3.44 ± 1.21 spikes/s  and 2.95 ± 1.12 spikes/s  in plant volatile-related ORNs of males and females, respectively. Mineral oil used as a solvent for green leaf volatiles elicited no activity in any of the ORNs tested. In contrast, hexane used as a solvent for all other test compounds elicited significant responses from some ORNs. In the S. discoideus antennae examined, 58 sensilla  were found to contain ORNs responsive to pheromone-related compounds  of sitophilure, 5-hydroxy-4-methylheptan-3-one  and the four stereoisomers  of sensilla in the females -2-hexenal (E2–6:Ald), whereas two six-carbon acetates (hexyl acetate and (Z)-3-hexenyl acetate (Z3–6:OAc)) and a seven-carbon ketone (2-heptanone) elicited very weak responses only in some of these sensilla (Table E)-2-hexenol (E2–6:OH), (Z)-2-hexenol (Z2–6:OH), and (Z)-3-hexenol (Z3–6:OH), elicited the strongest responses from the GLV-specific sensilla in the males.Approximately 58% (M-PL-A and M-PL-B) of plant-volatile responsive sensilla in males and 45% (F-PL-A and F-PL-B) in females contained ORNs that were highly sensitive to green leaf volatiles (GLVs), exhibiting strong responses only to some of the nine green leaf volatiles tested (Table E)-β-ocimene and 2-phenylethanol, elicited strong responses from the ORNs in two types of male-specific sensilla (M-PL-D and M-PL-E), whereas two compounds, (±)-citronellal and (±)-linalool, elicited strong responses in two types of female-specific sensilla (F-PLC and F-PL-D)  and 55% in females  contained ORNs responsive to some of 18 plant volatile compounds that are not GLVs, in which each type of sensillum exhibited a distinct response spectrum to a narrow range of volatile compounds .The presence of sex-specific sets of specialized ORNs for different pheromonal compounds implies that male S. discoideus. All the ORNs responsive to plant volatile compounds exhibited narrow response spectra, suggesting that these compounds can be discriminated from each other with high precision in S. discoideus. A large proportion  of plant volatile-responsive ORNs in S. discoideus are specialized for green leaf volatiles (GLVs) -β-ocimene, 2-phenylethanol and phenylacetaldehyde -β-ocimene, 2-phenylethanol, (±)-linalool and 3-octanone) among these seven compounds are produced by lucerne, a main host plant of S. discoideus -β-ocimene, whereas the females of both S. discoideus and S. obsoletus can detect the oxygenated monoterpenoids linalool, α-terpineol and citronellal  -β-ocimene, 2-phenylethanol and phenylacetaldehyde could be of help when weevils migrate to, or from, their estivation sites. Much stronger responses of males to these compounds could indicate that the males, which migrate first, are directed by these compounds, and that the females are later attracted by pheromonal compounds and/or GLV volatiles released by the first-arriving males. Although Goldson et al. -β-Ocimene was the most abundant compound in the emission, which also contained induced amounts of linalool and 1-octen-3-ol. Typically in Lepidoptera, fatty acid amino acid conjugates from saliva are involved in this response to herbivory by volatile upregulation (Yoshinaga et al. S. discoideus, have been found in the emission from clovers (Kicel et al. S. obsoletus also have specialized ORN for these compounds, since these compounds have not been tested in this species. It would be interesting to see if these compounds are involved with any sex-specific behavior in S. discoideus.Our study indicated that n et al.  indicateS. discoideus contain more than one ORN in the same sensillum. Each of eight different types of olfactory sensilla among ten different types of olfactory sensilla responsive to plant volatile compounds in S. discoideus contained an ORN that did not show any responses to 42 plant volatile compounds tested, suggesting that they are highly specialized for a narrow range of volatile compounds, if they are olfactory. It has been suggested that co-compartmentalization of ORNs facilitates the discrimination of closely moving odor filaments, by providing high temporal and spatial resolution (Baker et al. S. discoideus. In this context, it would be interesting to investigate why two ORNs for 1-octen-3-ol and 3-octanone, specialized for each compound respectively, are co-compartmentalized in the same sensillum, and to understand if they are involved in any female-specific behavior, as they are present only in female antennae.Our study shows that the majority of antennal olfactory sensilla in S. discoideus possess a characteristic set of highly specialized ORNs for pheromone-related compounds and specific plant volatiles. Field observations suggested that olfactory cues are involved in the aestivation flights of S. discoideus in summer towards hedge rows (Goldson et al. S. discoideus. Therefore, examining behavioral activities of the volatile compounds would be worthwhile in developing semiochemicals for this species. There is obviously a need for behavioural studies to validate the role of antennally active compounds which would link the observed electrophysiological patterns with beetle behaviour, preferably including field trials with synthetic blends and effective trap types.Our study showed that


In Germany, an ageing population is affected by societal and political changes due to demographic transition, e.g. by a prolonged working life for older employees. Demographic change also influences persons of higher working age with a migrant background. In 2018, 25% of all employees in Germany had a migrant background. Those affected by poor health at a higher working age can benefit from medical rehabilitation services, which aim to prevent early retirement and disabilities. So far, the utilisation of medical rehabilitation has been lower among persons of foreign nationality (often the only available proxy for migrant background), compared to that of Germans. The aim of this scoping review is to assess the utilisation of medical rehabilitation services by those with migrant background (PMB) and those without (non-PMB) and to identify the differences between these groups. We included 25 studies in our analysis, which were mainly secondary analyses of routine data and also a small number of primary studies. The results were inconsistent: studies published before 2018 showed a lower use of rehabilitation services for persons of foreign nationality compared to Germans. However, no differences were found between PMB and non-PMB in studies published in 2018 or later. PMB, as well as foreign nationals, showed poorer health before medical rehabilitation utilisation and had a higher chance of occupational disease and a lower education level. We identified a lower work-related performance, as well as barriers (e.g. information deficits) in the utilisation of rehabilitation services for groups of PMB. Our review is limited in that we cannot generalise our results to all PMB living in Germany. This is because of the heterogeneity, the limited number of studies and lack of representativeness in some studies. In many cases, studies only analyse the nationality, but they lack information about the second generation PMB. Future studies should survey the utilisation of medical rehabilitation services by migrant background rather than by nationality and focus on changes in the provision of rehabilitation measures following diversity-centred strategies. Demographic transition is accompanied by societal and political changes, such as an ageing labour force, the increase of the statutory retirement age  and a prolonged working life for older employees. In 2019, the average age on reaching retirement was 64.1 years [The number of older employees with a migrant background (PMB) in the German workforce is increasing. PMB are born outside or in Germany, with at least one parent born abroad, or of foreign nationality . Apart fStudies have shown that older employees with a migrant background, especially those of foreign nationality, have a higher risk of poorer occupational health than non-PMB in manual and low-skilled jobs. Incapacity to work and occupational diseases, or accidents are more common for foreign nationals. As are disability pensions, due to years of physically and mentally demanding occupations –16. LessThis scoping review assesses the utilisation of medical rehabilitation services by PMB  comparing groups of different origins, as well as PMB and non-PMB.Population-Intervention-Comparison-Outcome criteria, which is used to formulate a research question when searching for relevant studies and evidence [We conducted a scoping review, which is a type of review that aims to identify the state of research, examines research gaps and gives a broad overview of a topic –25. Our evidence : “Are thPopulation), who have utilised medical rehabilitation services in Germany (Intervention). We included studies which examined PMB compared to non-PMB, German nationals compared to foreign nationals and also studies comparing migrant subgroups (Comparison). We chose the following broad concept of utilisation as we expected few studies on the subject, specifically for the outcomes we have focussed on (Outcome): utilisation, medical indications, treatment outcomes and rehabilitation (treatment) success, work-related performance and returning to work after rehabilitation, satisfaction with rehabilitation, perceptions/expectations, as well as needs/intentions and barriers.We searched for all study types—cohort studies, surveys, randomised clinical trials and qualitative studies—with a sample of persons of working age, 18 to 60+ years , until the end of January 2020. Our search was conducted in 2018 and updated in 2020.We carried out the search  journals. We included the abstracts in our data extraction sheet and extracted the full text when available.We summarised the most relevant characteristics answering our research question in Tables . In both tables, the category “linked source” is integrated to show that some studies, or parts of studies are published and described in shorter or more detailed form, e.g. in other journals or books. We explained and included more detailed results for interested readers in the full text and in our additional files. We showed the most frequently used outcomes of the studies in the category “research focus” . Five studies were published in conference abstract books and four as final project reports. Lastly, we identified three studies through other sources, such as documentation, presentation at a conference or book chapters , e.g. of the German statutory pension insurance. Fewer studies analysed “place of birth” of the persons and/or of their parents (8 of 25) and “(native/spoken) language” (6 of 25), using a combination between “place of birth” and “(native/spoken) language” in three cases. In addition, two studies applied a name-based algorithm to identify Turkish rehabilitants in the data from the German statutory pension insurance  and the “work-related performance” (12 of 25). Ten of 25 studied the aspects “rehabilitation (treatment) success/treatment outcome” after utilisation of rehabilitation Table . Also, 5About half of the studies examined differences between PMB and non-PMB with a focus on the indications muscle/skeletal/connective tissue 12) and mental illnesses (12). This was followed by neoplasms (11) and diseases of the cardiovascular system (11). Indications relating to metabolism and digestion were investigated less frequently (6). Four studies analysed all of the aforementioned indications  and a lower effectiveness of rehabilitation due to a lack of culture- or gender-sensitive treatment concepts of health care providers , 51, 42.The included studies showed that effectiveness of rehabilitation and rehabilitation success is lower for PMB than for non-PMB. This included a lower (work-related) performance and less capability to return to work , 60, 42.Additionally, Erbstößer and Zollmann (2015)  reported.Important differences between German and non-German nationals, as well as between different PMB groups were seen in secondary data analyses in terms of indications. PMB used more rehabilitation services because of indications relative to “muscle/skeletal/connective tissue”, “mental illness (incl. addiction)” and “neoplasms” , 60. HowWe identified 25 studies and found that non-German nationals used medical rehabilitation services less frequently than persons with German nationality. Moreover, we discovered differences in several aspects of the utilisation of rehabilitation services between PMB and non-PMB and between different migrant groups. These differences were statistically non-significant or did not have much of an effect on the use of rehabilitation services , 31. PMBThe use of services was mainly investigated for the group of Turkish nationals and PMB in general, followed by nationals from former Yugoslavia, the former Soviet Union and Mediterranean countries. Fewer studies and sub-studies examined ethnic-German resettlers or focused on other migrant groups, e.g. Polish PMB, or PMB of Arabic origin living in Germany. Among the databases, we found five qualitative studies examining the use of rehabilitation and other aspects. In recent studies (published since 2018), no clear evidence for differences in utilisation of PMB in comparison to non-PMB was found. This could indicate a change in rehabilitation utilisation over time. Possible reasons could be the changes in service provision towards a more diversity-sensitive approach. Besides, young PMB may be more knowledgeable and willing to use health services such as medical rehabilitation , 62. AnoIn comparison to our results, other countries showed a lower use of medical rehabilitation for ethnic minorities. In the USA, non-Whites (for example Blacks and Hispanics) had a lower chance of discharge to rehabilitation in post-hospitalisation care after trauma than Whites, thereby not having the chance to use rehabilitation . In a CaThere are limitations comparing the utilisation of specific health care services, such as rehabilitation, between Germany and other countries. The rehabilitation systems vary widely due to their historical development. While in Germany medical rehabilitation takes place in hospitals away from the place of residence (mainly as inpatient care), the opposite is the case in other European countries , 65, 66.Further possible causes for our results could be that there are other influencing factors for the utilisation of health services such as medical rehabilitation, e.g. social inequalities. Socioeconomic-related inequalities can influence the utilisation of health services worldwide –69. In rWe identified barriers such as lack of information, knowledge of the rehabilitation system or cultural barriers. Also, unaddressed needs, as well as dissatisfaction can hinder utilisation and success of rehabilitation , 27, 70.Nationality is the common characteristic for migrant background registered in German routine data . InsuffiWe included studies indexed in databases and grey literature that we could identify online. The literature search was not as comprehensive as in a systematic review. Our aim was to examine the state of research, research gaps and to give an overview on the current state of knowledge on the research focus. Our search resulted in the inclusion of 25 studies which focussed on the use of medical rehabilitation services. A possible reason for the low number of included studies is the difficulty of recruiting PMB for studies . BesidesWe did not find differences in the utilisation of rehabilitation services in recent studies between PMB and non-PMB. However, we found out that in previous studies, non-German nationals used medical rehabilitation services less frequently than persons with German nationality. There is a need for studies examining the use of rehabilitation and assessing migrant background by more than just nationality to allow more precise comparisons between PMB and non-PMB. Comparative studies, which survey more than one aspect of utilisation, are also important in order to detect possible differences between groups. Furthermore, there is a need for research focusing on factors enabling utilisation of rehabilitation, improving health outcomes after rehabilitation and evaluating diversity-sensible strategies in order to understand possible changes in the provision of rehabilitation services.Additional file 1: Table S1. Studies, technical information to answer the review question. Table S2. Studies, content-related information to answer the review question


This retrospective study evaluated the association of hyperreflective foci (HRF) with treatment response in diabetic macular oedema (DME) after anti-vascular endothelial growth factor (VEGF) therapy. The medical records, including of ophthalmologic examinations and optical coherence tomography (OCT) images, of 106 patients with DME treated with either intravitreal ranibizumab or aflibercept were reviewed. The correlations between best-corrected visual acuity (BCVA) changes and HRF along with other OCT biomarkers were analysed. The mean logMAR BCVA improved from 0.696 to 0.461 after an average of 6.2 injections in 1 year under real-world conditions. Greater visual-acuity gain was noted in patients with a greater number of HRF in the outer retina at baseline (p = 0.037), along with other factors such as poor baseline vision (p < 0.001), absence of epiretinal membrane (p = 0.048), and presence of subretinal fluid at baseline (p = 0.001). The number of HRF after treatment was correlated with the presence of hard exudate (p < 0.001) and baseline haemoglobin A1C (p = 0.001). Patients with proliferative diabetic retinopathy had greater HRF reduction after treatment (p = 0.018). The number of HRF in the outer retina, in addition to other baseline OCT biomarkers, could be used to predict the treatment response in DME after anti-VEGF treatment. Among the different treatments for DME, anti-vascular endothelial growth factor (anti-VEGF) therapy, compared with laser photocoagulation and steroid treatment, has been shown to lead to superior visual outcomes5. Furthermore, intravitreal injection (IVI) of anti-VEGF agents such as ranibizumab and aflibercept has been shown to significantly improve the vision and macular anatomy of patients with DME in clinical trials, especially when injections were administered under strict loading and re-treatment protocols8. However, the frequency of re-treatment in the real world was often lower than that recommended, therefore resulting in relatively inferior outcomes compared with those of the clinical trials10.Diabetic macular oedema (DME), occurring in 3–9% of patients with diabetes, is a sight-threatening condition arising in cases of diabetic retinopathy13. Bolz et al. firstly reported the presence of HRF and their characteristics on optical coherence tomography (OCT) in patients with DME14, but their nature remained unclear despite several possible origins having been proposed, including lipid extravasation from a compromised vasculature, microglia proliferation, or retinal pigmented epithelium (RPE) migration15. In addition, previous studies have also reported controversial results regarding the correlation between HRF and visual acuity, especially that the presence of HRF in different retinal layers at baseline might impact visual improvement after anti-VEGF therapy differently20. Therefore, unlike other OCT biomarkers such as SRF, which have been determined to predict the treatment response of DME21, the role of HRF as a predicting factor for DME has not been determined.The treatment response of DME after anti-VEGF injection differs between clinical trial and real-world studies and varies among patients. To better understand the prognosis of DME after anti-VEGF therapy, several clinical biomarkers have been correlated with treatment outcomes, including central foveal thickness (CFT), external limiting membrane disruption, ellipsoid zone disruption, subretinal fluid (SRF), and presence of hyperreflective foci (HRF)The purpose of this study was to investigate the treatment response of DME and its correlation with OCT biomarkers, especially the presence of HRF in different retinal layers.The demographic data of the 106 patients with diabetic macular oedema who were enrolled are summarised in Table A greater number of HRF in the outer retina at baseline was correlated with better BCVA improvement at the 12-month follow-up (p = 0.037). However, the number of HRF in the inner retina was not associated with BCVA improvement. Other factors correlated with BCVA improvement are listed in Table 30. The differences in study designs and their results are summarised in Table We identified 13 studies that reported on the association between HRF and final visual acuities or visual improvementsThe number of HRF at different anatomical locations and different time points Fig.  was corrAmong all 106 cases enrolled, 65 eyes received IVI treatment with ranibizumab and 41 eyes with aflibercept. There was no difference between the groups at baseline except for more ERM in the aflibercept group  and a higher incidence of SRF in the ranibizumab group . The two groups had comparable visual improvements at every time point. There were slightly fewer total injections in the aflibercept group than in the ranibizumab group (6.5 ± 2.1 vs. 5.7 ± 1.9) with borderline significance (p = 0.080). There was no interaction between the amounts of HRF in the outer retina and the drug type used in the regression model in predicting VA improvement (p = 0.207).In this present study, we demonstrated the efficacy of anti-VEGF therapy in treating DME, both anatomically and functionally, under real-world conditions. The treatment effect was sustained during the 1-year follow-up with an average of six injections. In addition, a greater number of HRF in the outer retina at baseline predicted better visual improvement, as did other OCT biomarkers such as absence of ERM and presence of SRF.14. These HRF were commonly distributed through the retinal layers, including the subretinal space. In addition to their presence in DME and advanced DR, HRF were also found in the early stage of DR with inadequate glucose and blood pressure control31. The different origins of HRF, including extravasated lipids from compromised microvessels in diabetic retinopathy14, microglia activated by inflammation32, and migrating RPE cells33, might contribute to the wide distribution of HRF observed in our study.Bolz et al. first introduced the characteristics and localisation of HRF found on OCT among patients with DME20. In the present study, we found that patients with a higher number of HRF in the outer retina had enhanced visual improvement at the 1-year follow-up, which was in accordance with the findings of two recent reports29. Other studies have reported inferior visual outcome or limited improvement after anti-VEGF therapy with the presence of HRF at baseline30. The inconsistency might have resulted from the wide range of HRF present at baseline across studies. Furthermore, the HRF in different studies may have had different origins, thus showing different predictive values. In addition, not every study separately evaluated HRF in different retinal layers. In our study, we evaluated HRF according to their presence in different retinal layers; hence, the exact role of HRF in outcome prediction could be better appreciated.Despite their undetermined nature, HRF have been linked to visual outcome, yet the results varied among different studies Table . Uji et 34. However, in a real-world retrospective study, better HbA1c was correlated with superior visual and anatomical outcomes35. Framme et al. also showed that a greater number of HRF was found in patients with higher HbA1c16. In this study, higher HbA1C at the time of diagnosing DME, possibly representing a more ischaemic retinal environment, was related with a greater number of HRF in the inner and outer retina after anti-VEGF therapy. This effect lasted for 12 months after first injection.To date, there is limited literature focusing on the relationship between other baseline biomarkers and the presence of HRF before and after treatment. In the current study, we noted a strong correlation between the presence of hard exudate at baseline and the presence of HRF in every layer before and after treatment. This finding supported the hypothesis that HRF represents lipid extravasation as subclinical hard exudate. Furthermore, patients with PDR at baseline exhibited fewer residual HRF in the outer layer after treatment. The high concentration of VEGF in patients with PDR possibly caused increased vascular permeability and leakage, which further formed HRF. As a result, the HRF in these eyes had a better response under adequate anti-VEGF therapy, and thus fewer HRF after treatment. The level of HbA1c, representing the average status of blood glucose control, was reported to be independent of the prognosis of DME treatment in a large clinical trial32. Nevertheless, Chatziralli et al. demonstrated that both intravitreal anti-VEGF and steroid therapy could result in a similar degree of HRF reduction in retinal vascular disease, along with comparable visual outcomes24. Our study also found significantly decreased HRF after anti-VEGF therapy, and the reduction of HRF was similar among eyes treated with different anti-VEGF agents. The efficacy of different anti-VEGF agents in treating DME has been evaluated in previous studies and showed conflicting results. While one study with a small number of cases reported similar efficacy between aflibercept and ranibizumab36, other studies showed superior efficacy of aflibercept38. Moreover, in DRCR.net protocol T, they reported a better visual gain with aflibercept compared to ranibizumab at 1 year, especially in patient with worse baseline vision (worse than 20/50)41. However, the difference was not significant at the 2-year follow-up40. In our study, which only included patients with baseline vision worse than 20/50, the visual improvements were comparable in both groups in a clinical practice setting, with a 12.5-letter gain in the ranibizumab group and a 10-letter gain in the aflibercept group, which were both inferior to the results of the clinical trial. In addition, different anti-VEGF agents did not affect the predictive value of HRF in our study.A previous study linked the presence of HRF to a higher grade of inflammation, which might contribute to poor response to anti-VEGF therapy43, especially when the patients had been treated aggressively21. Conversely, the presence of ERM contributed to inferior functional and anatomical outcomes44. In addition to those baseline OCT biomarkers, the BCVA response at 3 months also independently predicted the visual outcome at 12 months  were included in this study. All patients were treated with two or three monthly loading injections, followed by as needed injections according to the BCVA change and OCT findings at each follow-up visit. Each patient visited the clinic every 4–8 weeks for at least 1 year. The choice between ranibizumab and aflibercept was based on the physician’s preference. The exclusion criteria included a serum haemoglobin A1c (HbA1c) level greater than 10% at baseline, a history of receiving vitrectomy, glaucoma, previous treatments for DME  within 6 months prior to the first IVI in our hospital, presence of macular scar, less than two loading injections of anti-VEGF, and presence of coexisting retinal vascular disease.30, including inner , outer (from the outer plexiform layer to the ellipsoid zone), and SRF (within the SRF) layers. The amounts of HRF within the central 3 mm of the macula were manually counted by two retinal specialists (TTL and CHH), and the central 3 mm of the macula was defined as 1500 μm on both sides from the foveal centre using the built-in software calliper.We collected baseline data including of age, sex, HbA1c at the time of first anti-VEGF treatment, hypertension, lens status, previous treatment , BCVA in logMAR, fundus photography, fluorescein angiography, and OCT images. The OCT was performed using the RTVue XR Avanti with AngioVue OCTA system , and fluorescein angiography using Heidelberg retina angiography HRA 2 . The presence of hard exudate on fundus photography, the presence of PDR on fluorescein angiography, CFT, the presence of ERM, intraretinal cysts (IRCs), SRF, and the number of HRF on OCT were recorded. The following data were also collected at 3, 6, and 12 months after first injection: BCVA in logMAR, CFT, number of injections, presence of ERM, IRCs, and SRF, and the number of HRF. HRF were defined as discrete, circumscribed, hyperreflective dots on OCT, with size ranging from 20 to 40 μm  AND (diabetic macular edema). In addition, we reviewed the reference lists of all selected articles to identify other potentially relevant studies. The eligibility criteria were: (1) recruited patients with DME who received any type of treatment; (2) evaluated the presence of HRF, either qualitatively or quantitatively, using OCT; (3) reported on the association of HRF with treatment response of DME, as either final BCVA or BCVA improvement. Two retinal specialists (TTL and CHH) independently reviewed the titles and abstracts of all identified studies and extracted the data from all eligible studies.For the analysis of BCVA, CFT, and the amount of HRF, the comparisons of measurements between baseline and follow-up visits were performed with paired Student’s t-tests. Linear regression analyses were performed to evaluate the predictive factors for visual outcome and CFT at the 12-month follow-up. The candidate predictive factors included demographic data, OCT biomarkers, drug type, interaction between drug type and HRF, and injection numbers. Age, sex, and pre-treatment logMAR BCVA  or CFT (for CFT reduction) were adjusted in all models. Linear regression analyses were also performed to evaluate the factors associated with the number of HRF. The candidate predictive factors included age, sex, HbA1c, presence of HTN, treatment history, and presence of PDR or hard exudate. The statistical analysis was performed using SPSS . A P value lower than 0.05 was considered statistically significant.Supplementary Information


The aim of this study was to determine the secondary sex ratio (SSR) of offspring in assisted reproduction technology (ART) in Jilin Province, China, and to analyse the influencing factors associated with SSR.A cross-sectional study of 3833 babies including singletons and twins born to 2990 couples treated by ART between May 2011 and December 2018 was performed.(p < 0.05). Comparing to in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI)  decreased the percentage of male babies.The main outcomes of this study were that the SSR of ART babies in Jilin Province was 50.64% and the SSR was associated with fertilization methods This study suggests that the SSR of ART births in Jilin Province was lower than the normal level and ICSI had a significant effect on SSR. Though we need more samples to study in the future, we still need to think about the impact of ICSI on SSR in ART. Human sex ratio is an important indicator of the gender balance of men and women. It is influenced by many factors. The ratio after conception is the primary sex ratio (PSR). And the ratio at birth is the secondary sex ratio (SSR). SSR is significantly lower than PSR due to abortion and many other factors. The normal value of SSR is approximately 100 females to 105 males [With the rapid development of assisted reproduction technology (ART ), more and more test-tube babies were born by ART . It may There had been lots of studies showing the SSRs brought by ART and exploring the influencing factors of the SSR. Jilin Province as a large province in northern China, there were no research on this type had been reported. We used the data one of the large reproductive centre in the province to enumerate the SSR and try to analyse the influencing factors of it. This study will put forward referable suggestions for maintaining the gender balance in Jilin Province.The data was collected from Centre for Reproductive Medicine, First Hospital of Jilin University, Changchun, China. The study subjects included babies born to 2990 couple patients who performed ART from May 2011 to December 2018. Oocyte donation cycles, pre-implantation genetic diagnosis cycles and pre-implantation genetic screening were not included. Our subjects included singletons and twins. One male baby and one female baby twins were excluded when we performed logistic regression analyses. And for multi-cycle couples, we only included the first delivery cycle. The study had been approved by the ethics committee of the First Hospital of Jilin University. And written informed consent was obtained from each participant.SSR in this study was the ratio of male births to total births. The variables in the analyses included number of cycles, maternal age, paternal age, infertility duration, maternal BMI, infertility type, infertility factor, fertilization methods, embryo transfer morphology, embryo transfer stage and birth type. The number of cycles referred to the current cycle number of the patient’s delivery. Infertility duration referred to the number of years of infertility without contraception. Above two variables were all divided into groups by class interval. And the class interval of maternal age group and paternal age group were 10 years old. The maternal BMI was divided into three groups according to the World Health Organization criteria. And birth type included singletons and twins two groups.p < 0.10. The significant variables were put into multivariable logistic regression to estimate the influencing factors for SSR. P < 0.05 was considered to be significant.SPSS software  was used to analyse the data. Univariate logistic regression analyses were performed to examine the correlations of various variables with SSR. Statistical significance was set to A total of 3833 babies were included from 2990 mothers in this study. There were 1941 male babies and 1892 female babies. The total SSR of the sample was 50.64%. The SSR of singletons and twins were 50.12% and 51.30%, respectively. The SSRs of different infertility types, factors, methods and embryo transfer morphologies were different (see Table p < 0.10) in all variables .In terms of paternal age, the babies from highest paternal age group had a higher SSR than that from youngest age group (p < 0.10). And for fertilization methods, the SSR was significantly lower in ICSI group than IVF group (p < 0.05) . ICSI was associated with SSR  and decreased the SSR comparing to IVF.Table The main findings of this study were as follows: the SSR of ART babies in Jilin Province was 50.64%; SSR was associated with fertilization methods. And comparing to IVF, ICSI might decrease the percentage of male babies.SSR is often used as an important indicator of population health and fertility. As far we know, the normal value of SSR is 51.22% . It was Although our results only showed an association between fertilization methods and SSR, this result was consistent with the results of other similar studies , 13, 14.There are some limitations in our study. Firstly, our sample size is relatively small. As a newly developed technology in Jilin Province in recent years, the sample size was limited when the gender of the offspring was excluded as one male and one female. What’s more, the information we have collected is not comprehensive. We should collect information such as the test results of some reports from the parents, etc. This is the incomplete information caused by the incomplete electronic medical record system in the early stage. We think that in the future, with the gradual improvement of our system, we will analyse the impact on SSR from a more comprehensive perspective.In conclusion, we found that the SSR of assisted reproductive technology births in Jilin Province was lower than the normal level through this study. And ICSI had a significant effect on SSR. The use of ICSI decreased SSR comparing to IVF. Although we need more samples to study in the future, we still need to think about the impact of ICSI on SSR in the assisted reproduction technology.


Erasmoneura vulnerata (Fitch) (Hemiptera: Cicadellidae) is native to North America, and was found in Europe for the first time  in 2004 where it remained a minor pest of grapevine for more than ten years. Recently, its importance increased in commercial vineyards located in north-eastern Italy, where outbreaks of E. vulnerata populations with severe leaf symptoms were observed despite insecticide applications. Information on its biology and ecology is needed for the development of management strategies. Here, we investigated the phenology and seasonal abundance of E. vulnerata in commercial vineyards. We found that E. vulnerata can complete three generations per growing season. Vineyard colonization by overwintered adults showed a clear edge effect, suggesting an effect of overwintering sites  adjacent to vineyards. The impact of natural enemies on pest populations appeared to be limited and mostly related to egg parasitoids. Organic vineyards were more heavily infested by E. vulnerata compared to conventional vineyards, likely due to the low effectiveness of natural insecticides typically used in the former farms. The results generated by this study provide implications for the management of this pest in European vineyards. The leafhopper Erasmoneura vulnerata (Fitch), detected in Europe for the first time  in 2004, has remained a minor pest of grapevine for more than 10 years. The first outbreaks of E. vulnerata were reported in 2016 in commercial vineyards located in north-eastern Italy. High population densities and severe leaf symptoms  were observed in late summer despite the application of insecticides. Investigations were carried out from 2017 to 2019 in 10 vineyards located in Veneto region (Vicenza and Verona provinces) to shed light on the seasonal abundance of E. vulnerata on different Vitis vinifera cultivars. Pest phenology was studied in six vineyards where the impact of insecticides was minimal. Erasmoneura vulnerata completed three generations in each of the growing seasons. Vineyard colonization by overwintered adults showed a clear edge effect, suggesting the influence of overwintering sites  in vineyard margins. The impact of natural enemies on pest populations appeared to be limited and mostly related to egg parasitoids. Organic vineyards were more heavily infested by E. vulnerata compared to conventional vineyards, likely due to the minimal efficacy of natural insecticides typically used in the former farms.The Nearctic leafhopper  Erasmoneura vulnerata (Fitch) (Hemiptera: Cicadellidae) is native to Northern America, and has belonged to the genus Erythroneura for an extended period of time [Erasmoneura, that comprises 11 taxa including Erasmoneura variabilis (Beamer), a key pest in California vineyards [E. vulnerata was collected on American and European grapevine species, as well as on alternative hosts, primarily Parthenoccissus quinquefolia (L.) Planch, Ilex decidua Walter and Cercis canadensis L. [E. vulnerata has been reported as being a severe pest of grapevine in the USA [The leafhopper  of time ,2,3. In  of time  placed tineyards ,5,6,7,8. the USA ,10, but  the USA ,13).Erasmoneura vulnerata was recorded in Europe for the first time in 2004  [E. vulnerata behavior in Italy were described by Girolami et al. [E. vulnerata could develop two or three generations per year [E. vulnerata into newly invaded areas and its abundance in vineyards. On untreated vines, E. vulnerata dominated over the native leafhoppers Empoasca vitis (Göethe) and Zygina rhamni Ferrari. In contrast, the opposite situation was reported in commercial vineyards where insecticides were applied to control Scaphoideus titanus Ball and other pests [n Italy) . Some asi et al. . Nymphs i et al. ,14. Infoper year ,17. Pester pests .E. vulnerata has spread to new areas both in Italy and Slovenia [Vitis vinifera) vineyards located in the Veneto region (Treviso and Vicenza provinces). In late summer, symptoms caused by this leafhopper were spread on more than 90% of the canopy, and population densities exceeded 10 nymphs per leaf in some vineyards [E. vulnerata has been recorded in southern Switzerland [Since 2005, Slovenia ,18, but ineyards . Surpristzerland .E. vulnerata phenology were carried out on untreated Vitis labrusca vineyards, and V. vinifera cultivars were used for semi-field experiments [E. vulnerata like for other leafhoppers [E. vulnerata was studied in some V. vinifera vineyards, and the effect of field margins on adult E. vulnerata colonization patterns in vineyards was also investigated. Vineyard management  was also considered as a factor influencing the seasonal abundance of E. vulnerata.This new scenario prompted the need for in-depth investigations on colonization patterns, phenology, and abundance in areas where the insect has become a significant pest in vineyards. Previous observations of eriments . These ofhoppers  ThereforE. vulnerata colonization patterns  were carried out in four vineyards per year, from 2017 to 2019 . In particular, we observed E. vulnerata adults on Parthenocyssus tricuspidata, Rosa spp. and Prunus spp. The presence of hedgerows contiguous to vineyards was also considered to be a factor potentially affecting E. vulnerata overwintering [E. vulnerata, E. vitis and Z. rhamni; they were identified using keys published by Dmitriev [Observations carried out before bud break revealed the occurrence of many intering , and thuDmitriev  and VidaDmitriev . Traps wS. titanus. In AC vineyard, a total of 30 leaves were inspected every 7–10 days in the laboratory using a dissecting microscope. Individual leafhoppers were counted considering their identity and age, and age was subdivided into three categories . The occurrence of potential predators or parasites of leafhoppers on leaf samples was also evaluated, as well as egg parasitoid emergence holes. Data from leaf samplings were coupled with data from adults captured on four yellow sticky traps placed within the grapevine canopy. Traps were analyzed in the laboratory under a dissecting microscope and were renewed every sampling date. The same procedure was applied in AO vineyard, where a total of 60 leaves (15 leaves per cultivar) were analyzed. In 2018, 30 leaves were collected in LO2 and MO vineyards approximately every 10–15 days and were then analyzed in the laboratory following the previously described procedures. Four yellow sticky traps were also placed in each vineyard. Mineral oils were applied in MO vineyard. The same approach was applied in 2019 in LO1 and MO vineyards. Pyrethrins were applied in LO1 vineyard, and kaolin in MO vineyard.In 2017, leaf sampling was regularly carried out on insecticide-free plots of AC vineyard (cv. Cabernet Sauvignon), while in AO vineyard  pyrethrins were only applied in late July to control E. vulnerata abundance was tested comparing four organic and four conventional vineyards located in the Vicenza and Verona provinces . We selected AO, LO2, MO, and MO2 among organic vineyards (MO2 vineyard was located at Monteforte D’Alpone and comprised Garganega cultivar), while AC, LC, MC, and RC were selected among conventional vineyards (RC vineyard was located at Roncà and comprised Garganega cultivar). In each vineyard, 30 leaves and four yellow sticky traps were analyzed in the laboratory approximately every two weeks, from May to September. Leafhoppers were identified at species level focusing on E. vulnerata, E. vitis, and Z. rhamni. The number of egg parasitoid (Hymenoptera: Mymaridae) emergence holes was also taken into account to calculate parasitism rates. These values were calculated by dividing the number of parasitoid emergence holes by the sum of the nymph hatching holes and the parasitoid emergence holes, expressed as a percentage. A number of emerging cages [Anagrus spp., which were reared in the laboratory from grapevine leaves infested by E. vulnerata. A fragment of the cytochrome c oxidase subunit 1 (COI) was amplified and sequenced using the procedure described in Martinez-Sañudo et al. [In 2018, the effect of vineyard management  on ng cages , mitochoo et al. .® . Distance from the margin, time of sampling, and their interaction were considered as sources of variation in the model and were tested using an F test (α = 0.05). The transect was considered as a random effect in the model. Pairwise comparisons of catches placed at different distances were performed using Tukey’s test (α = 0.05) on the least-square means. The degrees of freedom were estimated with Kenward–Roger method. Prior to the analysis, data were checked for model assumptions. The model was run on data transformed to log (n + 1), while untransformed data are shown in figures.Data from these experiments aimed at investigating the effects of distance from vineyard margins on leafhopper colonization and were analyzed using repeated measures linear mixed model with the MIXED procedure of SASE. vulnerata, E. vitis and Z. rhamni adults on traps, nymphs on leaves, as well as on the parasitism rate by the Mymaridae were analyzed separately with a repeated measures linear mixed model with the MIXED procedure of SAS® . In this analysis, vineyard management , time of sampling, and their interaction were considered as sources of variation and were tested with an F test (α = 0.05). Comparisons between vineyard management on each date were performed using Tukey’s test (α = 0.05) on the least-square means. The degrees of freedom were estimated using the Kenward–Roger method. Prior to the analysis, data were checked for model assumptions, and leafhoppers captures were transformed to log (n + 1), while arcsin of the square root was applied to the parasitism rate data. Untransformed data are shown in the figures.Effects of vineyard management on the abundance of E. vulnerata catches were abundant in late April when adults moved inside the vineyards, while catch numbers declined in May and slightly increased in June  and the most abundant catch numbers were detected at the vineyard margin . There were no differences between catches on traps located at 0 m and 20 m . The remaining comparisons were not significant . Empoasca vitis and Zygina rhamni catch numbers were much lower, and reached the highest levels in June .Adult  in June . The effE. vulnerata catches were higher than those of E. vitis and Z. rhamni in early spring of 2018 . Differences among treatments (increasing distances from the margin) were also significant  and the number of catches was higher at the vineyard margin . The remaining comparisons were not significant . The number of Z. rhamni and E. vitis catches reached the highest levels in June .Numbers of adult  of 2018 . Adult E in June , and theE. vulnerata catches increased in April when adults moved inside the vineyards, while later catches declined in number , and the highest number of catches were detected at the vineyard margin . The remaining comparisons were not significant . The number of E. vitis and Z. rhamni catches were much lower .The number of ch lower , and theE. vulnerata adults were caught on yellow sticky traps of the selected vineyards (E. vitis and Z. rhamni catches was much lower (data not shown). In subsequent weeks, the number of E. vulnerata catches declined. In AC vineyard, early instar nymphs of E. vulnerata were first detected in the second half of May, and their densities peaked in late May (At bud break in 2017 (April 10th), high numbers of ineyards . The numlate May . Older nE. vulnerata reported in S. titanus. A slight increase in early instar nymphs detected in late August and early September could indicate the onset of the third generation. The occurrence of other leafhoppers in this vineyard was negligible (data not shown).In AO vineyard, observations were carried out on four different varieties, and the phenology of E. vulnerata adults were abundant; however, when regular sampling started in early May, the number of adult catches in the selected vineyards (MO and LO2) were relatively low , as well as Neuroptera , were seldom observed preying on leafhopper nymphs in leaf samples. Nymphs of Allothrombium sp. (Acari Prostigmata) were also detected feeding on leafhopper nymphs. Parasitism by Hymenoptera Mymaridae was ascertained in all vineyards through the observation of emergence holes left by parasitoid adults on leaf veins. Among the Mymaridae that emerged from grapevine leaves in the laboratory, 14 individuals belonged to Anagrus parvus Soyka sensu Viggiani [Anagrus atomus (Linnaeus) [A number of Heteroptera (Viggiani  and onlyinnaeus) .E. vulnerata catches was higher in organic vineyards  and differed by the time of sampling and by the interaction vineyard management * time . The number of adult E. vulnerata catches was higher in organic vineyards on multiple sampling dates  as well as the effect of time and the interaction vineyard management * time . In particular, the number of E. vitis catches was higher in conventional than in organic vineyards on multiple dates . On some dates, there were more Z. rhamni adults in conventional vineyards . Vineyard management did not affect E. vitis or Z. rhamni nymphs  or Z. rhamni .p = 0.015). In organic vineyards, the highest parasitism rates were observed in late spring (55.5%) and late summer (54%). The effect of time was significant , while the interaction vineyard management * time was not . This is likely due to the decline in parasitism rates during mid-summer in both situations.Higher parasitism rates from Mymarids were found in organic vineyards (mean 42%) compared to conventional vineyards (mean 23%)  abundance implied the need for insecticide applications to reduce possible damage, likely leading to an effect on population densities. Based on observations made in two subsequent years, authors suggested that, in Colorado, E. vulnerata was able to complete two generations per year. To explain why E. vulnerata was less abundant than E. ziczac in these vineyards, Zimmerman et al. [E. vulnerata nymphs that colonize upper leaf surfaces which, in turn, leads to increased exposure to natural enemies. Years later, Triapitsyn et al. [E. vulnerata was found to be the dominant species among leafhoppers, while E. ziczac was not detected. These contradictory findings suggest that the pest status of E. vulnerata in native areas is not defined.In their review, Olivier et al.  considern et al. , had alrn et al.  suggesten et al.  surveyedn et al.  to shed E. vulnerata in northern Italy. These findings can have serious implications for other Italian regions and European countries  where this pest has been detected more recently. Prior to this study, the damage potential of E. vulnerata was a matter of discussion in North America [E. vulnerata outbreaks in commercial V. vinifera vineyards in southern Europe suggest a need for investigating factors influencing its potential as a new pest. Previous observations on the pest’s phenology carried out on Vitis labrusca in Italy suggested that this species could complete two or three generations per year [E. vulnerata completes three generations per year in V. vinifera vineyards. In the present study, several V. vinifera cultivars  were found to be equally infested, providing no evidence for variable susceptibility among cultivars located on the same farm. Overwintering is a crucial phase for the success of vineyard colonization by E. vulnerata. Zimmerman et al. [E. vulnerata during the spring. In this phase, vineyards were also colonized by the native leafhoppers E. vitis and Z. rhamni, but their incidence in investigated vineyards was very low. Data suggest the possible role of interspecific competition and the need to investigate this phenomenon in controlled conditions. In a vineyard near those investigated in the present study, high numbers of adult E. vulnerata at bud break seriously impacted shoot growth, resulting in the need for control measures.Our results highlight the most recent pest status of  America ,29 and w America . Recent per year . The pren et al.  detectedn et al. . In the E. vulnerata in Northern Italy, tolerance to some pesticides has been suggested [E. vulnerata and found higher pest population densities in organic vineyards. In Italy and in other European countries, organic vineyards can be managed with copper-based fungicides, sulfur, and a few natural insecticides, mostly pyrethrins. Results strongly suggest that these insecticides are less effective than conventional insecticides in keeping leafhoppers at acceptable density levels. Interestingly, predation and parasitism did not increase significantly in organic vineyards to compensate for the low efficacy of pyrethrins or other natural insecticides. Natural control of E. vulnerata and E. ziczac by egg parasitoids in Colorado appeared to be negligible (parasitism rate < 2%) [A. parvus, is frequently associated with Z. rhamni in north-eastern Italy [Among other factors promoting the pest status of uggested . In thiste < 2%) , while trn Italy ,30.E. vulnerata control. Knowledge of the “edge effect” in adult colonization could help growers in early detection of this pest and lead to the adoption of adequate control measures. This is particularly important when high leafhopper numbers infest plants at bud break, imposing a threat on shoot growth. In this regard, based on the information provided in the present study, yellow sticky traps should be placed at the vineyard margins in March. This will provide useful information on adult colonization patterns. Data obtained on natural control by Mymarids suggest properly managing areas surrounding vineyards to preserve alternative hosts that promote Mymarid overwintering [E. vulnerata and their negative side effects on beneficials suggest the need for alternative control measures. Trials have been carried out with kaolin based on the results obtained from other leafhopper species [Our results have implications for intering . Finally species . These tE. vulnerata completes three generations per year in V. vinifera vineyards in Northern Italy. Also, there is no apparent difference in susceptibility among the most common cultivars tested in this study. Our study strongly supports an edge effect on E. vulnerata vineyard colonization during the spring. This aspect should be considered during the application of modern IPM strategies. Also, future investigations should aim to identify control solutions in organic farms where natural insecticides are not very effective. Moreover, the economic impact of E. vulnerata on grapevine yield and quality should be investigated to define economic threshold levels and reduce pesticide use.In conclusion,