Rotavirus gastroenteritis (RVGE) remains a leading cause of hospitalization and death in children under five years of age in the Philippines. Rotavirus (RV) vaccination was introduced into the national immunization program (NIP) in 2012 but has since been limited to one region due to cost considerations and conflicting local cost-effectiveness estimates. Updated estimates of the cost-effectiveness of RV vaccination are required to inform prioritization of national immunization activities.We calculated the potential costs and benefits of rotavirus vaccination over a 10-year-period (2021–2031) from a government and societal perspective, comparing four alternative rotavirus vaccines: Rotavac, Rotasiil, Rotarix and Rotateq. For each vaccine, a proportionate outcomes model was used to calculate the expected number of disease events, DALYs, vaccination program costs, and healthcare costs, with and without vaccination. The primary outcome measure was the cost per DALY averted. Assuming each product would generate similar benefits, the dominant (lowest cost) product was identified. We then calculated the cost-effectiveness (US$ per Disability Adjusted Life Year [DALY] averted) of the least costly product and compared it to willingness-to-pay thresholds of 0.5 and 1 times the national GDP per capita , and ran deterministic and probabilistic sensitivity analyses.Introducing any of the four rotavirus vaccines would avert around 40% of RVGE visits, hospitalizations, and deaths over the period 2021–2031. Over the same ten-year period, the incremental cost of vaccination from a government perspective was estimated to be around $104, $105, $220, and $277 million for Rotavac, Rotasiil, Rotarix and Rotateq, respectively. The equivalent cost from a societal perspective was $58, $60, $178 and $231 million. The cost-effectiveness of the least costly product (Rotavac) was $1,148 ($830–$1682) from a government perspective and $646 ($233–1277) from a societal perspective. All other products offered similar benefits but at a higher cost. There is a >99% probability that Rotavac would be cost-effective at a willingness-to-pay threshold set at 0.5 times the national GDP per capita.Both Rotavac and Rotasiil are likely to be cost-effective options in the Philippines, but it is not possible to say definitively which product should be preferred. Rotarix and Rotateq are expected to offer similar benefits at more cost, so would need to be priced far more competitively to be considered for introduction. It has comprised approximately 44% of diarrheal hospitalizations and 28% of deaths globally A number of studies have shown RV vaccination to be cost-effective in low- and middle-income countries (LMIC), including non-Gavi countries Newer RV vaccines with different price points have subsequently been pre-qualified by the WHO 22.1A universal vaccine decision-support model (UNIVAC version 1.4.13) was used to calculate the potential cost-effectiveness and health impact of rotavirus vaccination over a 10-year period (2021–2031) in the Philippines. This static Microsoft Excel-based proportionate outcomes model was developed specifically for use by national multidisciplinary teams in LMICs To populate the model, we carried out a review of international and local literature, government records, and the PhilHealth claims database. Local data, especially primary data from local health agencies, were prioritized when available. A local expert panel comprised of 15 multidisciplinary specialists  were invited to two workshops held on December 9, 2020 and March 24, 2021 to review the accuracy, completeness, and appropriateness of the data inputs , prior to analysis. All demographic projections  were based on the UNPOP 2019 revision projections for the Philippines The potential benefits of RV vaccination were projected by estimating the number of RVGE disease cases, visits, hospitalizations, deaths, and Disability-Adjusted Life Years (DALYs), with and without vaccination, among children less than five years of age.‘Cases’ refer to children in the community with RVGE, regardless if brought to a healthcare facility. ‘Visits’ constitute the cases brought (but not admitted) to a healthcare facility . Meanwhile ‘hospitalizations’ are defined as cases admitted to an inpatient healthcare facility. RVGE was further classified as non-severe or severe. Non-severe RVGE was presumed to always lead to recovery. Thus, associated events include only cases (non-severe) and visits (outpatient clinic). In contrast, severe RVGE cases involve visits (emergency room), hospitalizations, and deaths.We used previously described methods to calculate the potential number of excess cases, hospitalizations, and deaths associated with intussusception, a possible serious adverse event following RV vaccination We estimated healthcare treatment costs with and without vaccination. We included costs associated with non-severe RVGE visits, severe RVGE visits and hospitalizations, and hospitalizations from intussusception. We calculated healthcare costs from two perspectives: 1.) the government perspective, representing all costs borne by the payor , and 2.) the societal perspective, pertaining to all incurred direct and indirect costs irrespective of payor.We conducted deterministic sensitivity analyses (DSA) using discount rates of 3%, 5%, and 10% (7% as the base case according to HTA guidelines) 2.22.2.1A summary of the model inputs can be found in 2.2.1.1Our literature search yielded several local incidence studies with a wide range of RV positivity rates (30–40%) To estimate DALYs, we obtained disability weights for moderate and severe diarrhea from the Global Burden of Disease study (GBD) 2.2.1.2We found no data on local incidence and mortality rates of intussusception. For hospitalization and deaths, we used region-specific estimates from a global systematic review and meta-analysis 2.2.2A summary of the model inputs can be found in To account for potential vaccine non-uptake and delay, we used local data on DTP vaccination coverage rates as a proxy for the expected RV vaccine coverage and timeliness. DTP vaccination rates of 72%, 71% and 69% for doses 1, 2 and 3, respectively, were the average annual coverage rates from 2014 to 2018 found in the Field Health Services Information System (FHSIS) In the Philippines, a case control study by Lopez et al. evaluated the effectiveness of Rotarix against RV positive hospitalizations in children aged <5 years Our estimates of the relative risk of intussusception in the 1-7- and 8-21-day periods after the first and second doses were based on a global meta-analysis of self-controlled case series (SCCS) studies 2.2.3A summary of the model inputs can be found in The government procurement price of Rotarix from 2018 was available from communication with the DOH and used as base case after adjusting for inflation to the 2020 price. The price estimates of the other RV vaccine types were accessed from the Market Information for Access to Vaccines (MI4A) database of the WHO 2.2.4A summary of the model inputs can be found in Detailed costing can be found in Supplementary Tables (Tables 1–4). Briefly, costs were classified into direct and indirect costs. Direct costs, whether medical or non-medical, included the costs of goods and services used in the course of treatment while indirect costs referred to the caregivers’ loss of productivity or income. Direct non-medical costs included transportation costs and meal costs for patients and caregivers while seeking treatment. The government perspective specifically involved only direct medical costs borne by the government as payor; the societal perspective entailed all direct and indirect costs, irrespective of payor.Costs of healthcare visits for RVGE were adapted from the costing scheme implemented by Lee et al. but substituted with contemporary (2020) prices 33.1A summary of healthcare costs and disease events with and without vaccination can be found in 3.1.1Over a 10-year period (2021–2031), total healthcare costs of RVGE without vaccination would be about $71 and $175 million USD from a government and a societal perspective, respectively. Concurrently, there would be an estimated 2 million cases of non-severe RVGE, 0.9 million cases of severe RVGE, and 65,000 cases of intussusception. In total, there would be about 1.3 million healthcare visits, 0.3 million hospitalizations, and 17,000 deaths due to RVGE.3.1.2With an RV vaccination program in place, healthcare costs of RVGE would reduce by 42–44% over 10 years compared to no vaccination, with an estimated $30–31 million USD averted from a government perspective and about $72–76 million USD averted from a societal perspective. This would result in an incremental net cost of approximately $104, $105, $220, and $277 million USD, from a government perspective, for the inclusion of Rotavac, Rotasiil, Rotarix and Rotateq, respectively, in the NIP for a 10-year-period. From a societal perspective, there would be an incremental net cost of about $58, $60, $178, and $231 million USD for the inclusion of Rotavac, Rotasiil, Rotarix and Rotateq, respectively.RV vaccination would also reduce cases of non-severe RVGE by 41–43% and severe RVGE by 48–51%, resulting in about 0.8 million non-severe RVGE and 0.4 million severe RVGE cases averted over 10 years. On the other hand, there would a potential increase of about 650 intussusception cases over 10 years, translating to an increase of 1% from baseline pre-vaccination cases. Overall, there would be a decrease in the number of hospital and clinic visits by approximately 0.5 to 0.6 million, hospitalizations by 0.15 million, and deaths by 8,000 over a 10-year period; these correspond to a reduction of 44–46%, 40–43%, and 35–37%, respectively.3.23.2.1In the base-case scenario, Rotavac was the least costly RV vaccine from both government and societal perspectives . The other three RV vaccines provided equivalent benefits but at higher costs. The cost-effectiveness of Rotavac (discounted US$ per DALY averted) was $1,148  from a government perspective and $646  from a societal perspective.3.2.2Supplemental Figs. 2 and 3 show the sensitivity of results to changes in key parameters. To illustrate this, each vaccine is compared to no vaccination, and we estimate sensitivity in the average cost-effectiveness ratio (each vaccine compared to no vaccination). Rotavac and Rotasiil had ACERs less than the 1 times GDP threshold from both government and societal perspectives at 3%, 5%, and 10% discount rates. Furthermore, Rotavac and Rotasiil remained cost-effective in all scenarios using high and low estimates for all parameters, from both government and societal perspectives. Rotarix had ACERs greater than 1 times GDP threshold in scenarios with low disease event rates and low vaccine efficacy from a government perspective. To achieve equivalent ACERs to Rotavac/Rotasiil, the price per dose of Rotarix and Rotateq would need to be lower, around $2.50 and $1.12, respectively, representing a 65% and 80% reduction in price from the base case values.3.2.3The least costly vaccine, Rotavac, has a 100% probability of being cost-effective, from both government and societal perspectives , Fig. 2,4This timely and country-specific analysis revealed that among the four WHO-prequalified RV vaccines, Rotavac would be the least costly. Choosing either Rotasiil, Rotarix, or Rotateq would result in higher vaccine program costs with no additional health benefits. However, we should note that the vaccine prices we used in the model for Rotavac, Rotasiil, and Rotateq were estimates and may not be reflective of the actual future tender prices of the vaccines. In particular, the prices for Rotavac and Rotasiil were from the MI4A data from Gavi-eligible countries We assumed the same incremental health system costs for all the four vaccines, using the incremental health system cost of Rotarix as reference While the four vaccines similarly averted costs and disease events, the two-dose Rotarix resulted in slightly fewer averted costs and fewer averted events compared to the three-dose RV vaccines . These differences are small, and the result of a third dose delivered a few weeks later and providing more durable overall protection for the three-dose vaccine products. However, as noted earlier, there are no known head-to-head studies demonstrating differential impact by product. If we assume all four have the same health impact, a Rotarix vaccination program over 10 years would still cost $116,580,596 USD more than a Rotavac vaccination program.In our analysis, vaccine price is a crucial determinant of differential cost-effectiveness by vaccine type. To demonstrate, a Rotarix program with only two vaccine doses would incur less incremental health system costs than the three-dose vaccines; yet, due to a higher vaccine price per dose, the overall vaccination program cost for Rotarix was among the highest. The importance of vaccine price has been previously illustrated in a local CEA by Lam et al., who concluded that the 2015 prices of Rotarix and Rotateq warranted a reduction of about 70% in price for the vaccines to be cost-effective The cost-effectiveness ratios in our study can only be interpreted vis-à-vis our pre-defined willingness-to-pay threshold. While our set threshold has been used traditionally, there has been no explicit threshold in the Philippines from the payor’s side In the Philippines, inclusion of vaccines in the NIP is also influenced by the ability to address health inequities Notably, in our analysis, there could be a potential increase of intussusception cases over a 10-year period for the expansion to a national RV vaccination program. Should this potential risk be observed, we estimate that around 275 rotavirus hospitalizations could be prevented per intussusception hospitalization (or about 140 rotavirus deaths prevented per intussusception death). However, our estimates of the number of excess cases of intussusception were pessimistic for a number of reasons. First, we used pre-vaccine hospitalization rates from the WHO Western Pacific Region, which are far higher than other the rates in other WHO regions While the WHO position strongly emphasizes that the benefits of RV vaccination on severe diarrhea far outweigh the risks for intussusception, it is recommended that active intussusception surveillance accompany the introduction of RV vaccination to monitor and assess this risk Our analysis included some limitations. First, we used international data for some model parameters, such as health system costs, thus restricting local applicability of our results. Although local disease rates for RVGE were available, the data originated from urbanized areas, which under-represented rural communities which may have a different disease burden. Furthermore, the UNIVAC model did not account for herd immunity, effects of breastfeeding, and possible long-term sequelae of RVGE such as malnutrition and adverse neurodevelopmental outcomes. Altogether these areas constitute current gaps in local research.5A rotavirus vaccination program over a 10-year period using any of the four WHO-prequalified RV vaccines  compared to no vaccination would avert 30–50% of visits, hospitalizations, and deaths from RVGE. Rotavac vaccination had the best value-for-money with the lowest ICER compared to no vaccination. However, given the uncertainty in actual vaccine prices, both Rotavac and Rotasiil are likely to be cost-effective options in the Philippines. Rotarix and Rotateq are expected to offer similar benefits but at a higher cost, so would need to be priced far more competitively for introduction consideration.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Conclusions: The cross-reactivity and sequence homology between α-synuclein and frequently consumed foods, reinforces the autoimmune aspect of Parkinson’s disease. It is hypothesized that luminal food peptides that share cross-reactive epitopes with human α-synuclein and have molecular similarity with brain antigens are involved in the synucleinopathy. The findings deserve further confirmation by extensive research.Introduction: Parkinson’s disease is characterized by non-motor/motor dysfunction midbrain neuronal death and α-synuclein deposits. The accepted hypothesis is that unknown environmental factors induce α-synuclein accumulation in the brain via the enteric nervous system. Material and Methods: Monoclonal antibodies made against recombinant α-synuclein protein or α-synuclein epitope 118–123 were applied to the antigens of 180 frequently consumed food products. The specificity of those antibody-antigen reactions was confirmed by serial dilution and inhibition studies. The Basic Local Alignment Search Tool sequence matching program was used for sequence homologies. Results: While the antibody made against recombinant α-synuclein reacted significantly with 86/180 specific food antigens, the antibody made against α-synuclein epitope 118–123 reacted with only 32/180 tested food antigens. The food proteins with the greatest number of peptides that matched with α-synuclein were yeast, soybean, latex hevein, wheat germ agglutinin, potato, peanut, bean agglutinin, pea lectin, shrimp, bromelain, and lentil lectin.  Environmental factors play a major role in human chronic diseases . In addiHowever, this complex formation between antibodies and food proteins can result in the activation of a complement cascade and binding to C1q. These anti-nutrient antibodies can cross-react with human self-molecules. This autoimmune cascade can also be evoked when sequence homology or shared immunogenic epitopes exist between those food-originated foreign proteins and the host’s self-determinants . This phNo less interesting is the relationship between neuroinflammatory, neurodegenerative or neuropsychiatric conditions and food consumption. In this regard, bovine milk and dairy products , gluten A very logical suggestion was recently suggested by Riccio and Rossano . They coMonoclonal mouse IgG1 antibody made against recombinant aSN was purchased from R&D Systems, Minneapolis, MN, USA. Monoclonal rabbit IgG antibody made against aSN epitope 118–123, was purchased from Abcam, Cambridge, MA, USA.For the preparation of the food antigens, food products in either raw or cooked form were purchased from the supermarket. Food proteins undergo structural epitope transformation when the food is cooked, and dietary preparation was conducted to reflect dietary proteins in either raw or cooked form as represented in typical human diets. A total of 180 different food extracts, representatives of various meats, seafoods, grains, fruits, vegetables, seeds, nuts, beans, spices, gums and more were prepared, in a process similar to the one used in our earlier study . The difFood antigens at a concentration of one mg/mL were prepared. For coating the ELISA plate, the optimal concentration of each food antigen was determined by examining the concentration of antigens that gave the most reproducible results in quadruplicate. Accordingly, the stock solution was diluted from 1:50–1:200 in 0.1 M carbonate-bicarbonate buffer (pH 9.5). One hundred microliters were added to each well of the polystyrene flat bottom ELISA plate. The plates were then incubated overnight at 4 °C, and then washed 5 times with 200 microliters PBS containing 0.05 % Tween 20 at a pH of 7.4. 200 microliters of 2.5 % bovine serum albumin (BSA) were then added to prevent the nonspecific binding of immunoglobulins, after which the plates were incubated at 4 °C overnight. Plates were subsequently washed as described above, and then unimmunized mouse serum, rabbit serum, and mouse or rabbit monoclonal antibodies made against aSN at optimal dilutions of 1:100–1:300 were added to different groups of wells and were incubated at room temperature for 1 h. They were then washed 5 times with PBS-Tween buffer. We then added one hundred microliters of monoclonal antibody made against aSN, followed by incubation, washing, and the addition of secondary antibody or alkaline phosphatase labeled anti-rabbit or anti-mouse IgG to different wells, after which the plates were incubated again. 100 microliters of para-nitrophenylphosphate (PNPP) in 0.1 mL diethanolamine buffer 1 mg/mL containing 1 mM MgCl2 and sodium azide at a pH of 9.8 were added to start the enzymatic reaction, which was stopped 45 min later with 50 microliters of 1 N NaOH, so that the samples were ready for quantitative analysis by an ELISA reader. The microtiter reader recorded the optic density (OD) at 405 nm to provide quantitative antibody reactivity levels in comparison with control wells coated either with human serum albumin (HSA) as negative control or aSN as positive control. Each analysis was performed in quadruplicate.To demonstrate the specific binding of aSN antibodies not only to synuclein, but to different food antigens, serial dilutions and inhibition studies in the presence of different food antigens in the liquid phase of the antigen-antibody reaction was performed. Different sets of different ELISA wells were coated with the food antigens. In each set four or five foods were chosen as being representative of the antigens that showed from moderate to strong reactions with aSN antibodies. For the plates using mouse monoclonal antibody, these foods were honey, shrimp, yeast, tofu and quinoa. For the plates using rabbit monoclonal antibody, the foods were wheat germ agglutinin (WGA), lentil lectin, latex hevein, thyme and potato. The aSN antibodies were added serially in dilutions ranging from 1:100 to 1:12,800 to the appropriate set of microtiter strips. After incubation, washing, the addition of a second antibody, and the completion of other ELISA steps, the ODs were recorded at 405 nm.The inhibition study was done by the addition of various concentrations of a specific antigen to an antibody in liquid phase first, followed by the addition of the antigen-antibody mixture to ELISA microwells coated with the same antigen. For example, to prove that the binding of aSN antibody to yeast, quinoa, tofu, shrimp and honey is specific, the following steps were taken:1. Eight different ELISA plate wells were coated with optimal concentrations of yeast or another antigen used in this inhibition study.2. 100 microliters of monoclonal anti-aSN were then added to each of 8 different test tubes.3. The first tube, tube #1, containing anti-aSN antibody was to serve as the baseline reaction of antibody to each tested antigen, so no antigen  was added to this tube. To the other seven tubes containing anti-aSN antibody, we added increasing concentrations of yeast protein. Tube #2 received 2 micrograms, tube #3 received 4 micrograms, and tubes #4–8 received 8, 16, 32, 64, and 128 micrograms of yeast antigen respectively.4. The eight tubes now contained 100 microliters of aSN-antibody and from 0 to 128 micrograms of yeast protein. The tubes were individually mixed, and then the contents of each tube were added to the appropriate ELISA wells coated with yeast protein described in Step #1.After incubation, washing, the addition of anti-mouse or anti-rabbit IgG labeled with enzyme, and the completion of additional ELISA steps, the ODs were recorded. The inhibition of anti-aSN antibody binding to specific food antigen-coated ELISA wells by the same antigen in liquid phase was demonstrated graphically to be in proportion to the increased amount of protein used for inhibition. All of these same steps were also used for quinoa, tofu, shrimp and honey, which were the other food antigens used in the inhibition study using mouse monoclonal antibody.The inhibition of anti-aSN antibody made against the short epitope AA 118–123 of aSN was done in a similar manner using food proteins that had moderate to strong reactions with aSN antibody.We used the NIH/US National Library of Medicine’s Basic Local Alignment Search Tool (BLAST) sequence matching program to study degrees of possible mimicry between the amino acid sequences of aSN and food proteins such as soy bean, yeast, pea lectin, lentil lectin, bean agglutinin , wheat germ agglutinin, peanut protein, pineapple bromelain and shrimp. These are foods that had moderate to strong reactions with the aSN antibody. We also ran a sequence match search with proteins for coconut, a food that did not react with this antibody.t-tests were performed to evaluate mean differences of optical densities between controls and antigens. A Bonferroni adjustment was conducted to account for type 1 errors with multiple comparisons and alpha was set to <0.001.Statistical analysis was performed using STATA 14.2 software. Independent Based on earlier studies about the cross reactivity of food components and human tissue antigens ,47,48,49Anti-aSN antibodies had significant reactivity with 86/180 foods, much higher in comparison to the ODs of reagents, controls and the mean ODs of the other 94 foods +3SD (0.50).The resulting means +SD of each antigen’s quadruplicate ODs were calculated and are presented in p < 0.0001).Comparing the reaction of aSN antibody with alpha synuclein (3.82), which was close to the maximum OD of 4.0, the reaction of anti-aSN antibody was strongest with honey (OD 3.0—very high), followed by soy sauce (OD 2.5—high), potato (OD 2.3—high), tofu, radish and soybean oleosin (OD 2.2—high), yeast and soy (OD 2.1—high). Overall, not counting the highest reaction of aSN antibody with honey, 17 foods were graded as high (OD 1.81–2.5), 56 were graded as moderate, 12 were graded as low, and 94 foods had individual ODs that were equal to or less than the mean OD of all these 94 foods plus 3SD, or 0.50 or less, representing the insignificant or negative results A–C. InteThe aSN epitope 118–123 (VDPDNE) is localized at the polar, negatively charged and acidic C-terminal tail of aSN, representing a cleavage site, playing an important role in fibrillar synucleinopathy ,51,52,53In order to semi-quantify the levels of the reactivity of this epitope’s antibody with the studied food antigens, the following key for the ODs were used: 0–0.51 = insignificant or negative; 0.52–1.0 = low; 1.10–1.80 = moderate; 1.81–2.5 = high; and >2.5 = very high. Whereas the complete sequence aSN protein antibody reacted significantly with 86 food antigens, the monoclonal antibody made against this specific epitope resulted in reactivity with a total of 32 food antigens, ranging from low to high reactivity.p < 0.0001).The reaction of anti-aSN antibody was strongest with WGA (OD 3.5—very high), followed by pea lectin (OD 3.4—very high), peanut butter and lentil lectin (OD 2.9—very high). There were no food antigens in the high zone range, 10 food products in the moderate range, and 19 in the low range. All the rest of the tested food antigens were below the cut-off level of 0.52 and were considered negative A,B. The Specificity of anti-aSN antibody binding to various food antigens is demonstrated by serial dilutions for the complete aSN sequence in The binding of anti-aSN antibody to aSN as the control in the presence of HSA, or anti-aSN antibody binding to various cross-reactive food antigens was examined using different concentrations of cross-reactive food components, in a liquid phase. The binding of rabbit monoclonal antibody binding to aSN and various cross-reactive food antigens was examined in the presence of different concentrations of HSA or the same food antigens in the liquid phase of the assay. Inhibition in the binding of rabbit monoclonal anti-aSN Epitope 118–123 antibody to 5 different food antigens and aSN are shown in Amino acid sequences for proteins from peanut, yeast, potato, shrimp, pineapple bromelain, bean agglutinin , pea lectin, lentil lectin, soybean agglutinin, wheat germ agglutinin, and latex hevein, which had moderate to very strong reactions with aSN antibody A–C, wereSaccharomyces cerevisiae), soybean agglutinin, latex hevein, wheat germ agglutinin, peanut, pea lectin, potato, bean agglutinin, and shrimp. Bromelain had two matches, while lentil lectin only had one, but all the other proteins had from four to more than twenty matches with aSN. Even when the cutoff was set for percentage of identity at 50%, the resulting number of matches for many of those proteins was so overwhelming that not all of the instances of similarity could practically be shown in a table , but it only had a 46% match with the aSN sequence (KE-GVVAA---AE).Parkinson’s disease is characterized by the abnormal folding of aSN, a protein localized in the substantia nigra in the form of Lewy bodies. Regarding the source of misfolded aSN in the brain, the current hypothesis is that chemicals, infections or other unknown environmental factors penetrate the peripheral nervous system through the gastrointestinal system or through the olfactory bulb in the nasal cavity, subsequently moving on to the adjacent enteric nerve, from there finally reaching the brain, then culminating in widespread aSN pathology at the later stages ,54,55,56In the present study, the hypothesis was that if aSN shares molecular similarity with so many foods, then monoclonal antibodies made against aSN should react with some of those food proteins. Looking at it from the other direction, if anti-aSN reacts with different food antigens, then amino acid sequence similarities should exist between those food proteins and aSN.Following the above-mentioned hypothesis, it can be envisioned that if foods that cross-react with aSN are partially or undigested in the gut due to local eco events, the aSN in those food particles may become misfolded, and its handling by the enteric nerve may help in the transportation of the misfolded aSN into the brain. Based on the increased barriers permeability in PD, the leaky gut and the permeable BBB may also allow aSN aggregates to migrate cephalically .E. coli protein fibril, and two other protein amyloids, silk and Sup35, produced by certain microbiota, was found to accelerate the aggregation of amyloid protein A and aSN in animal models of amyloidosis and PD [Molecular mimicry between food antigens and self-tissue proteins is one of the possible mechanisms in the initiation of autoimmune diseases ,49,58,59s and PD ,63. Besis and PD ,51,52,53s and PD ,55 and ts and PD ,64,65, nWhile antibody made against the short 118–123 epitope of aSN had low to strong reactions with 32 different food antigens, the monoclonal antibody that was prepared against the full-sequence recombinant aSN reacted with 86 out of the 180 tested food antigens. These reactive antigens were prepared from seeds, nuts, beans, fruits, vegetables, seafood, fish, chicken and mammalian meats A–C. The The reactivity of aSN antibodies with many food antigens was confirmed by the high degree of peptide sequence homologies between aSN and yeast, soybean agglutinin, wheat germ agglutinin, latex hevein, potato, peanut, bean agglutinin , pea lectin, shrimp, pineapple bromelain, and lentil lectin . Using tThe plethora of these matches between aSN sequences and food antigens may explain why monoclonal antibodies made against aSN proteins reacted with so many food antigens out of the 180 in the present study. It should be noted that the study was limited to the identification of general cross-reactive antibody responses, while the BLAST search was just limited to 12 food antigens. The results may indicate that the aSN antibodies reacted against shared conformational epitopes in the food proteins. The current study design did not specifically include analyses that would capture conformational or non-linear epitopes, but screened any of the food sequences that matched with the aSN sequences, especially the highly recurring ones, hence possibly be conformational epitopes. Conformational epitopes could be major targets of autoantibody production in autoimmune diseases ,67, resuThe present results open a new horizon for food specific antigens that might drive PD. It is a new insight that reflects a connection between the disease and commonly consumed foods that enter the gut lumen, where they are digested, absorbed, sensed and sampled by the pluripotent epithelial and sub-epithelial innate immune cells ,39. The In support of the dual-hit hypothesis of PD by Braak et al. ,55,56, CLike many other proteins in the gut, aSN may undergo PTMP by acetylation, phosphorylation, oxidation, ubiquitination, glycation, nitration, deamidation, transamidation and sumoylation. This enzymatic PTMP reaction can result in the alteration of protein structures, affecting their functional and aggregatory capacity ,77. FurtSimultaneously, aggregated aSN, microbial generated toxins, antigens, amyloids, antibodies, inflammatory cytokines, and undigested food antigens can affect the functional integrity of the tight junctions ,6,39,43.Although the presence of some of these factors  in the blood may not be directly pathogenic for the brain, it is possible that in the context of BBB permeability, these factors may contribute to the severity of diseases in the brain . Thus, tIt is not known if the cross-reactive food specific antibodies against aSN epitopes will induce gain or loss of aSN functions. Most recently, strategies to decrease synucleinopathy by tackling the aSN molecule have been described, including targeting the spread, production, aggregation, and degradation of aSN . One wonThe presence of antibodies in the blood that cross-react with aSN may interfere with the accurate measurement of aSN antibodies used in support of PD diagnosis and progression assessment of the disease.Compromised BBB, which is often observed in PD patients, may result in the entry of food cross-reactive antibodies into the central nervous system, where their binding to aSN can further contribute to Lewy body pathology.The presence in the blood of food antigens that cross-react to aSN may interfere with treatment modalities in which anti-aSN antibodies are used to target misfolded proteins in PD.High cross-reactivity of a food-specific antibody with aSN may affect some physiological functions of aSN, thus putting individuals at risk for synucleinopathy.Glutamate neurotransmission and glutamate receptors play a pivotal role in brain physiology and are altered in PD ,97. On tSeveral consequences of aSN antibodies cross-reacting with a variety of food proteins or specific peptides can be postulated:It should be stressed that those possibilities need further investigation. There are some limitations for the present study. It is an in vitro study, but there would definitely be advantages in investigating in vivo, by feeding or injecting the aSN-cross-reactive food epitopes into animal models and observing if they develop PD-like symptoms or pathology. Ingested nutrients are subjected to enzymatic digestion in the stomach and small intestine. The post-digestion antigenicity of the presently studied food products were not explored. As it is, the clinical and pathophysiological impacts of the cross-reactive antibodies were not explored, and thus any conclusions are purely hypothetical, and it is not truly known whether aSN-cross-reactive food epitopes are pathogenic or protective. The strengths of the study are the plethora of food products, which span a wide range of common nutrients. The specificity of the antibodies’ level determination was double checked by dilution, inhibition and epitope similarity. Furthermore, the putative cross-reactivity between aSN and food proteins was supported by ELISA testing and BLAST matching with many lectins, agglutinins and different forms of the foods, such as soybean, soy sauce and tofu, peanut, peanut agglutinin and peanut butter.Interestingly, in addition to the luminal aSN ,36,37,38Multiple commonly consumed specific food products induce antibodies that cross-react with aSN and its 118–123 sequence. Our findings raise the possibility that antibody’s cross-reactivity supported by specific sequence’s molecular mimicries between nutrients and aSN is operative thus might represent an additional autoimmune feature to PD neurodegeneration. If substantiated in vitro or on PD animal models or in human subjects, this study’s findings might open a new avenue of research in PD pathophysiology. While the biological and pathophysiological cross-reactive and sequence homology effects are far from being unraveled, it is hoped that the current findings will encourage the scientific and clinical communities to explore the food cross-reactive and molecular mimicry phenomena in the PD enigma.


To our knowledge, there are no published studies that describe the physical activity (PA) levels and objectively measure them through accelerometry in toddlers (2–3 years old) attending early childhood education and care (ECEC) institutions during the COVID-19 pandemic. The aims of this study were two-fold: (a) to analyse toddlers’ PA levels and sedentary behaviour (SB) during school hours in ECEC institutions, as well as the rate of adherence to specific recommendations on total PA (TPA) and moderate–vigorous PA (MVPA); and (b) to evaluate the characteristics correlates  of young children and the school environment on toddlers’ TPA, light PA (LPA), MVPA, and SB during school hours in ECEC institutions. PA was evaluated with ActiGraph accelerometers. The main findings were that: (a) toddlers engaged in very high amounts of TPA and MVPA during ECEC hours; (b) girls and boys displayed similar levels of LPA, TPA, and SB, while girls had lower levels of MVPA, compared to boys, and younger toddlers were less active than older ones; (c) BMI was not associated with PA of any intensity or SB; (d) playground and classroom density were not associated with higher levels of PA of any intensity, though classroom density was associated with SB. These ECEC institutions provide and challenge the new COVID-19 scenario, as well as supportive environments for toddlers’ PA. Between January 2020, when the World Health Organization (WHO) declared a new coronavirus disease 2019 (COVID-19) outbreak to be a public health emergency of international concern , travel Regarding PA in toddlers, international PA guidelines recommend 180 min/day of total PA  and no more than one hour sedentary screen time , progresMultiple correlates of PA, explained by ecological models of health behaviours , have poAt the environmental level, Hunter et al.  suggesteOn the other hand, the pandemic has driven schools to make adaptations to their internal operations, creating measures for prevention, hygiene, and health promotion in the face of COVID-19 . This neGiven the major challenges to leisure-time PA that young children and adults have been facing in the COVID-19 era ,36, ECECThe study used a cross-sectional, correlational design. A total of 180 young children, aged 24 to 36 months from seven different public ECEC institutions in metropolitan Valencia, Spain, were recruited by way of convenience sampling after a formal invitation to participate in the study. Inclusion criteria were: children aged 2–3 years; being able to walk without assistance; and attending the ECEC institution at least three days per week. Young children whose parents signed consent, who attended an ECEC, and who were developmentally typical were eligible to participate in the study. Young children (n = 60) who did not attend the school for the five consecutive days of data collection or went home early  were excluded from the final analyses.After applying these criteria, the final sample was made up of 120 toddlers  and offered full-time instruction and care. In all the ECEC institutions, there was a recess time, lunchtime, and nap time, lasting a mean of 45 min, 30 min, and 90 min, respectively. In all the ECEC institutions, lunch was served in the same building, while nap time was in the toddlers’ classroom. Two educators were present in each toddler classroom. In addition, during the week of the measurements, the teachers responsible for each classroom were asked about the number of children attending regularly. Of the seven participating ECEC institutions, three had specific structured PA sessions within their curricular practices , while the other four worked PA curricular contents in an integrated, interdisciplinary way. All ECECs had fixed equipment in their outdoor playgrounds.The rules of social distancing, hygiene, and cleanliness, as well as other logistical and institutional rules for both teachers and children remained in place from the beginning of the school year (September 2020) to the end (June 2021). These policies were common for all the ECEC institutions. Following governmental regulations for maintaining a safe distance and avoiding contact between different groups, one of the restrictions imposed to prevent the spread of COVID was the creation of stable classroom groups that do not socialise with children outside their group. Furthermore, school principals were asked how the different groups were organised during recess time to comply with the new COVID-19 regulations, including what modifications had been made to the play area and how many children per space participated during playtime.PA levels were measured with ActiGraph accelerometers , for which, the use has been validated to assess PA in toddlers ,42. For 2) in each ECEC institution were measured manually with a meter. Classroom sizes ranged from 35 m2 to 42 m2. For the outdoor playground area, Google Earth Pro (GEP) software was used to provide an estimate of the playground spatial area (m2) at each of the ECEC institutions using aerial pictures of the playgrounds and the polygon measurement tool. Playground sizes ranged from 67 m2 to 240 m2). Classroom and playground densities (toddlers/m2) were calculated as described elsewhere [The indoor classroom and indoor playground area  minutes, comprising a mean 111.85 ± 21.78 min of LPA and 42.50 ± 17.50 min of MVPA. The mean duration of SB was 164.47 ± 33.85 min. 2 (range 35 to 42), and the playground area, 157.58 m2 (range 67 to 240). The classroom and playground densities, measured as the average number of young children per square meter of classroom and playground space, were 2.78 children/m2 (range 2.30 to 3.5) and 11.36 children/m2 (range 4.61 to 16), respectively.Regarding the size of the ECEC, the average classroom was 38.06 mp < 0.001) and between LPA and age (p < 0.05). No other associations with objectively measured PA levels were observed for gender or age, nor for BMI, playground density, classroom density, or the presence of structured PA sessions.International recommendations for PA indicate that toddlers should have at least 180 min per day of PA at any intensity level ; howeverThe main findings indicate that throughout the school day, young children had a mean 154.35 ± 30.60 min of TPA, 111.85 ± 21.78 min of LPA, 42.50 ± 17.50 min of MVPA, and 164.47 ± 33.85 min of SB, with boys and girls exhibiting similar levels of TPA, LPA, and SB. In adherence to the Institute of Medicine recommendations, included toddlers exceeded the minimum one-quarter of time spent in the school facilities in TPA . These rThe restrictions implemented in response to COVID-19  had a negative impact on preschoolers’ PA levels ,37,45. TThe COVID-19 pandemic could exacerbate the health effects of the obesogenic environment ,48, so sChildren’s age was associated with LPA. Specifically, older children had approximately 16 min more LPA than younger ones. In the same line, using an observational system, Gubbels et al.  found thRegarding toddlers’ gender and PA levels, boys exhibited higher levels of MVPA than girls . This is in keeping with systematic review, showing a consistent association between gender and PA in young children . The litHowever, further studies are necessary to analyse specifically how MVPA is modulated by the different curricular practices  and by the location where these practices are performed (indoors/outdoors). In any case, following Lahuerta-Contell et al. , reachinOne of the novelties of this study relates to the exploration of the concordance between toddlers’ PA levels and ECEC classroom density. In line with its findings , indoor environmental characteristics, such as the ‘modified open-plan space’, are positively associated with PA in toddlers, so providing enough spaces for playing likely keeps children engaged and active . Indeed,2, are associated with a 22% greater likelihood of meeting PA recommendations than schools with smaller playgrounds [Regarding playground density, no association with PA levels was observed, which contradicts pre-pandemic studies in Dutch toddlers, wherein the size of the outdoor playground was positively correlated with children’s activity level during the outdoor time . Accordiygrounds ; howeverygrounds . Furtherygrounds . In factygrounds  recentlyygrounds . Other vGiven that most young children in this sample met recommendations (one-quarter of the time with active behaviours) and, in fact, almost completed the full 3 h of recommended PA per day during school hours, it is clear that these ECEC institutions provide, each with its own identity, and challenge the new COVID-19 scenario, with supportive environments for toddlers’ PA. However, taking into account that it is essential to examine curricular practices and environmental characteristics related to the school curriculum of ECEC centres relevant to children’s PA and SB while they are in the ECEC institutions, especially during the COVID-19 era, future studies analysing teachers’ perceptions of the barriers to PA that COVID-19 has created in schools are also needed.To our knowledge, this is the first study in the pandemic era (October 2020–May 2021) to examine toddlers’ PA levels and SB during ECEC time using objective measures with accelerometers. This is a novelty in COVID-19 studies, as previous studies have only described self-reported PA. A large sample of children across multiple ECEC institutions and classrooms was recruited, ensuring heterogeneity of centre characteristics.On the other hand, this study is not without some limitations, including those inherent to its cross-sectional design and the lack of collecting socio-economic information from families. In addition, while accelerometry is an objective measure of PA and sedentary time, there is no consensus on the optimal cutoff in toddlers . As otheThe main findings of this study were that: (a) toddlers engaged in very high amounts of TPA and MVPA during ECEC hours, meeting 85% of the recommended levels of daily PA; (b) girls and boys displayed similar levels of LPA, TPA, and SB during school hours; (c) girls had lower levels of MVPA, compared to boys; (d) younger toddlers were less active than older ones; (e) BMI was not associated with PA of any intensity or SB; (f) playground and classroom density were not associated with higher levels of PA of any intensity, though classroom density was associated with SB. Taken together, 85% of the PA recommendations were met during school hours in the pandemic time. The present study shows that, in this age group (2–3 years), it is essential to focus on the analysis of total PA time; in other words, ECEC institutions and practitioners should encourage active behaviours, regardless of intensity. However, intervention studies aiming to promote PA at these ages should pay attention to the correlates of age and gender for each PA intensity.


Patient-controlled analgesia (PCA) is an effective method of postoperative pain, there have been many studies performed that have compared the efficacy of hydromorphone with continuous sufentanil. The purpose of this systematic review is to compare the efficacy and safety of hydromorphone and sufentanil.Seven databases were searched for controlled trials to compare the efficacy and safety of hydromorphone and sufentanil. After selecting the studies, extracting the data, and assessing study quality, the meta-analysis was performed on several of the studies with RevMan 5.3.12 = −1.52, 95% CI , P <.05). The sedation intensity of the hydromorphone group at 12, 24, and 48 hours were lower than those of the sufentanil group, with no statistical difference ], P > .05). The PCA requests in the hydromorphone group were less than that in the sufentanil group, and there was no significant difference . The incidence of adverse events in the hydromorphone group was less than that in the sufentanil group, and there was a statistical difference: .Thirteen studies comprised of 812 patients were found. The pain intensity of the hydromorphone group was significantly lower than that of the sufentanil group at 12 hours. With no statistical difference at 24 to 48 hours (MDCompared with sufentanil, PCA with hydromorphone was more effective in relieving pain and PCA requests 12, 24, and 48 hours after operation, and significantly reduced the incidence of adverse events, but it did not have an advantage in sedation intensity. Patients can adjust the time and dose of injection of analgesics as needed to meet the analgesic requirements.Postoperative pain is an acute pain that occurs immediately after surgery, usually lasting no more than 7 days. Effective postoperative analgesia not only alleviates the pain of the patient, but also helps to accelerate the recovery of the disease.–7 Sufentanil is a potent opioid analgesic with high selectivity of mu agonists. It has a definite analgesic effect and has the characteristics of stable cardiovascular function, and it is a cheap synthetic opioid with a high therapeutic index and a quick response, is an attractive drug for postoperative pain.–12 In recent years, many clinical studies on the efficacy and safety of these 2 drugs for PCA have been made, however, these results were controversial. Therefore, we made the systematic review and meta-analysis comparison of the effects between the 2 drugs.Hydromorphone is a new kind of opioid analgesic. It has the characteristics of strong analgesic effect, long duration, and less adverse events than fentanyl. It is suitable for the treatment of postoperative acute pain, and it would provide long-acting pain relief due to its hydrophilicity and induce fewer adverse events due to its lipophilicity. It is well suited for Enhanced Recovery After Surgery protocols.2 and Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines for systematic reviews. The ethical approval and informed consent were unnecessary since the meta-analysis was aimed to summarize the previous studies.Data collection and analysis were performed by the best practice Cochrane Association guidelinesWe sought randomized controlled trials (RCTs) that the clinical effects of hydromorphone and sufentanil for PCA after an operation. Reports were identified by Pub Med, EMBASE TM, Cochrane Central Register of Controlled Trials, and 3 Chinese databases, including the Chinese Biomedical Literature Database, China National Knowledge Infrastructure, and Wan fang Data using the following search terms as keywords and text words: “hydromorphone”, “sufentanil”, “PCA” “patient-controlled analgesia,” “self-administered,” “pain,” “analgesia,” “postoperative,” and “surgery.” Alternative spellings of the search terms were also used. Without restriction to regions, publication types, or languages confining the search to studies published between inception and July 2, 2018.2.1Inclusion criteria were:1.RCTs;2.adult surgical patients receiving postoperative PCA;3.the use of opioid for a PCA strategy; and4.postoperative pain-related outcomes and PCA-related adverse events.Exclusion criteria were:1.hydromorphone was not compared with sufentanil;2.animal trials, reviews, and other genres, repeated publication;3.only abstract and lack of full text, or full text does not provide sufficient raw data;4.abstracts of scientific meetings, unpublished observations, and correspondence.Two reviewers identified all studies that appeared to fit the inclusion criteria for the full review. Two reviewers independently selected studies for inclusion in the review. Any disagreements were referred to a third reviewer. If data were reported in a format that did not allow inclusion in the meta-analysis, we contacted the authors and asked them to release data. We identified a total of 13 RCTs. The study characteristics for each included trial are shown in Table 2.2All studies comparing hydromorphone to sufentanil for PCA were included. The outcome were: pain intensity, as measured by the Visual Analogue Scale (VAS) score, VAS is the most common measurement to assess pain intensity. It is scored on a range of either 0 to 10. Sedation intensity, as measured by the Ramsay score at the 12/24/48 hours after operation; PCA requests for analgesia, adverse events of patients. We also conducted subgroup analyses to explore the various types of PCAs on the incidence of postoperative pain management. We separated PCIA and epidural PCA for analysis.2.3P < .05 was considered significant. Heterogeneity was analyzed using a Chi-Squared test on N-1 degrees of freedom, with an alpha of 0.05 used for statistical significance and with the I2 test. I2 values of 25%, 50%, and 75% correspond to low, medium, and high levels of heterogeneity. A fixed-effect model was used unless statistically significant high heterogeneity (I2 > 75% was considered as significantly high heterogeneity) existed between studies. A random-effects model was employed if heterogeneity existed. An assessment of the methodological quality of the included studies into the meta-analysis was conducted in line with the Cochrane handbook.,29 Review Manager (Rev Man 5.3) was used to plot the quality assessment.Results that were pooled from the included studies were meta-analyzed. For continuous data, a Mantel–Haenszel Chi-Squared test was used and expressed as the mean difference with 95% CI, and for dichotomous data an inverse variance was used and expressed as risk ratio with 95% CI. In both cases 33.1A total of 64 citations were identified for eligibility through the systematic literature search. After exclusion of duplicate publications and full-text review of the relevant studies. A total of 13 cohort studies encompassing 812(411 patients were hydromorphone group and 401 patients were sufentanil group) individuals were included in the quantitative synthesis . A random-effect model was used to analyze the data. Meta-analysis showed that pain score of the hydromorphone group was significantly lower than that of the sufentanil group at 12 hours. With no statistical difference at 24 to 48 hours , see Figures Eleven studies% CI −2.1, −1.97, 3.3.2,23,25–27 provided Ramsay score data for patients at different time points 12 to 24 hours after surgery. Statistical heterogeneity was found among the studies . Random effect model was used to analyze the data. Meta-analysis showed that the sedation scores of the hydromorphone group at 12 to 24 hours were lower than those of the sufentanil group, with no statistical difference. , see the Figures Eight studies3.3.3–21,27 reported the number of PCA requests. Statistical heterogeneity  was found among the studies. Random effect model was used to analyze the results. Meta-analysis showed that the number of PCA requests in the hydromorphone group was less than that in the sufentanil group, and there was no significant difference , see the Figure Eight studies3.3.4–27 reported the incidence of postoperative adverse events. The main adverse events include postoperative nausea vomiting (PONV), somnolence, pruritus. Therefore, we set up 3 subgroups for analysis. Statistical heterogeneity was found among the studies . Random effect model was used to analyze the incidence of postoperative adverse events. Meta-analysis showed that the incidence of adverse events in the hydromorphone group was less than that in the sufentanil group, and there was a statistical difference , see Figure Thirteen studies3.3.5The funnel plot was used to analyze the incidence of adverse events. As a result, the distribution of the inverted funnel plot is asymmetrical, suggesting that there may be large publication bias and clinical heterogeneity in the included literature, see Figure 4,30 characterized by obvious analgesic effect and long duration of action. However, it always causes vertigo, pruritus, nausea, and vomiting. Hydromorphone, as a novel powerful opioid with a clear analgesic effect, has been reported both at home and abroad. A meta-analysis of Felden  has shown that the clinical efficacy of hydromorphone in acute pain is slightly superior to morphine. There are many studies on the clinical efficacy of hydromorphone and sufentanil in PCA, but evidence-based medicine is lacking for their efficacy and safety.Patient-controlled analgesia pump, which is characterized by high efficiency, no blind zone for analgesia, and stable blood concentration, has been widely used in postoperative rapid analgesia. At present, sufentanil is the representative used in intravenous analgesia in a clinic,,32Our meta-analysis showed that compared with sufentanil, hydromorphone can significantly reduce postoperative pain for 12 hours, there was no significant difference in sedation, and PCA requests, but the incidence of adverse events in the hydromorphone group was significantly better than that in the sufentanil group, especially in PONV and somnolence. It may be due to the hydrophilicity of hydromorphone, which can provide long-lasting analgesic effects and cause fewer adverse events. American Pain Society recommends against routine basal infusion of opioids with i.v. PCA in opioid-naive adults.  Therefore, adverse events such as PONV in the included studies may be related to the basal infusion.Although some studies have found that basal infusion of sufentanil PCIA can effectively relieve pain with few adverse events,  in which the postoperative VAS score was higher than 10 (the highest score is 10). Therefore, we contacted the author but did not receive any reply, so we excluded this article.Our study revealed an article by Hua,,17,26,27 We analyzed separate experiments with different doses.There are multiple-dose comparative studies in some articles.There are shortcomings and limitations in this study:1.This systematic review included 13 studies, all of which were Chinese articles. Although we searched the Cochrane Central Register of Controlled Trials, MEDLINE, EMBASE, and WHO International Clinical Trials Registry Platform, we found few English language studies.2.There were some differences in the dosage of each study, and the loading dose/basal infusion/locking time of PCA drugs included in the study were also different.The literature reveals that, between 1996 and 1999, 25 sedation assessment tools were published, of which 3 have been rigorously tested for validity and reliability in adults: the motor activity assessment scale, the Ramsay sedation scale and the sedation agitation scale (SAS) Their study can be criticized, however, only the Ramsay scale had been validated adequately for use in the critical care environment, so more studies have chosen Ramsay scale.5The results demonstrate that compared with sufentanil, PCA with hydromorphone is more effective in relieving postoperative pain at 12, 24, and 48 hours and reducing PCA request, and significantly decreases the incidence of postoperative adverse events. However, its effect on analgesia is not obvious. The quality of clinical studies is relatively low and the dosage in most studies is different; therefore, high-quality multicenter, randomized, parallel-controlled, and blind trials are needed for further study. The studies are of low quality and are all of Chinese origin, so this meta-analysis conclusion is only suitable for Chinese.Conceptualization: Zhongbiao Nie, Bin Lu, Yao-yao Guo.Data curation: Zhongbiao Nie, Zhi-Hong Li, Bin Lu, Yao-yao Guo, Ran Zhang.Formal analysis: Zhongbiao Nie, Bin Lu.Methodology: Zhongbiao Nie, Zhi-Hong Li, Bin Lu, Ran Zhang.Software: Zhongbiao Nie, Zhi-Hong Li, Ran Zhang.Writing – original draft: Zhongbiao Nie, Zhi-Hong Li, Ran Zhang.Writing – review & editing: Zhongbiao Nie, Zhi-Hong Li, Ran Zhang.


Accumulating evidence has demonstrated that gene alterations play a crucial role in LUAD development, progression, and prognosis. The present study aimed to identify the hub genes associated with LUAD. In the present study, we used TCGA database to screen the hub genes. Then, we validated the results by GEO datasets. Finally, we used cBioPortal, UALCAN, qRT-PCR, HPA database, TCGA database, and Kaplan–Meier plotter database to estimate the gene mutation, gene transcription, protein expression, clinical features of hub genes in patients with LUAD. A total of 5930 DEGs were screened out in TCGA database. Enrichment analysis revealed that DEGs were involved in the transcriptional misregulation in cancer, viral carcinogenesis, cAMP signaling pathway, calcium signaling pathway, and ECM–receptor interaction. The combining results of MCODE and CytoHubba showed that ADCY8, ADRB2, CALCA, GCG, GNGT1, and NPSR1 were hub genes. Then, we verified the above results by GSE118370, GSE136043, and GSE140797 datasets. Compared with normal lung tissues, the expression levels of ADCY8 and ADRB2 were lower in LUAD tissues, but the expression levels of CALCA, GCG, GNGT1, and NPSR1 were higher. In the prognosis analyses, the low expression of ADCY8 and ADRB2 and the high expression of CALCA, GCG, GNGT1, and NPSR1 were correlated with poor OS and poor PFS. The significant differences in the relationship of the expression of 6 hub genes and clinical features were observed. In conclusion, 6 hub genes will not only contribute to elucidating the pathogenesis of LUAD and may be potential therapeutic targets for LUAD. Lung cancer is a common and severe disease which ranks the top among cancers worldwide in terms of mortality for both men and women . In JanuLung adenocarcinoma (LUAD) is one of the subtypes of non-small cell lung cancer (NSCLC) with high morbidity and mortality . As we aPrevious studies have shown that several genetic abnormalities were associated with the initiation and development of LUAD ,10, but https://tcga-data.nci.nih.gov/tcga). These RNA-seq data (HTSeq-count) from Illumina HiSeq RNASeq platform consisted of 502 LUAD samples and 49 adjacent non-cancerous samples, and were achieved from the publicly available Genomic Data Commons (GDC) data portal .High-throughput gene expression data of LUAD tissues and normal lung tissues were extracted from the TCGA Data Portal (2(fold change [FC])| > 2, P value < 0.01, and false discovery rate (FDR) < 0.01 were considered as the threshold values for DEG identification. We selected the top 300 up-regulated and 300 down-regulated genes for our study. The heatmap and volcanoplot were performed with ‘gplots' and ’ggplot2’ packages of R software.In order to identify the DEGs, R software (version 4.0.5) were applied to compare the expression profiles of LUAD tissues with those of normal tissues. Differential expression analysis of individual genes was carried out by edgeR Bioconductor package . The |loP<0.05 was considered to indicate a statistically significant difference in the functional enrichment analysis.The Database for Annotation Visualization and Integrated Discovery (DAVID)  is a onlIdentified DEGs were mapped into the online search tool for the retrieval of interacting genes  database  to evaluThe Jvenn, an interactive Venn diagram viewer , was uset-test statistical score was conducted in consistent pathways and the mean of the DEGs was calculated. A permutation test with 1000 times was utilized to detect the signifcantly involved hallmark pathways. The adjusted P-value using Benjamini and Hochberg (BH) and FDR method by default was used to correct for the occurrence of false positive results. Significant involved genes were defined with an adjusted P-value < 0.01 and FDR < 0.25.Gene set enrichment analysis  was applied to predict associated up-regulated and down-regulated genes and the signifcantly changed pathways based on the expression profle from TCGA database . In eachhttp://www.ncbi.nlm.nih.gov/geo/). The search strategy was as follows: (‘lung cancer’ OR ‘lung adenocarcinoma’). Gene expression profiles of datasets GSE118370, GSE136043, and GSE140797 were obtained from GEO. The dataset GSE118370 consists of 6 LUAD samples and 6 normal lung samples, GSE136043 consists of 5 LUAD samples and 5 normal lung samples, while GSE140797 consists of 7 LUAD samples and 7 normal lung samples. The data were analyzed on the GPL570 platform Affymetrix Human Genome U133 Plus 2.0 Array and the GPL13497 platform Agilent-026652 Whole Human Genome Microarray 4×44K v2. To identify DEGs between LUAD tissues and normal lung tissues, we employed the Limma package in R software (version 4.0.5) for processing  and ADRB2 , and high expression of CALCA , GCG , GNGT1 , and NPSR1  were correlated with significantly poor OS in LUAD patients (P=0.0029] and ADRB2 , and high expression of CALCA , GCG , GNGT1 , and NPSR1  were correlated with significantly poor PFS in LUAD patients  and progression-free survival (PFS) were analyzed for patients with LUAD using the Kaplan–Meier survival plot. Briefly, the 6 genes were uploaded to the database and Kaplan–Meier curves were plotted. The results indicated that low expression of ADCY8 , and TNM stage . The expression level of ADRB2 in female was significantly higher than in male , and the expression level of ADRB2 was negatively correlated with T stage , nodal metastasis , distant metastasis , and TNM stage . The expression level of CALCA in male was significantly lower than in female , and the expression level of CALCA was positively correlated with nodal metastasis , distant metastasis , and TNM stage . The expression level of GCG was negatively correlated with age , and the expression level of GCG was positively correlated with nodal metastasis , distant metastasis , and TNM stage . The expression level of GNGT1 was positively correlated with T stage  and nodal metastasis . The expression level of NPSR1 was positively correlated with T stage , nodal metastasis , and TNM stage  (Pearson’s chi-squared test showed that the expression level of ADCY8 was negatively correlated with T stage  , Table 8LUAD is one of the important subtype of NSCLC with high morbidity and mortality . LUAD is2+ overload triggers cell death. ECM–receptor interaction pathways are the most up-regulated gene-enriched signaling pathways, which play an important role in the process of tumor shedding, adhesion, degradation, movement, and hyperplasia. The roles of ECM in other cancers have been proved. Previous studies reported that the ECM was up-regulated in prostate cancer tissue [The GO analysis results indicated that the up-regulated DEGs were primarily associated with nucleosome assembly, telomere organization, cellular protein metabolic process, extracellular region, nucleosome, nuclear chromosome, sequence-specific DNA binding, protein heterodimerization activity, and hormone activity, while the down-regulated DEGs were primarily enriched in angiogenesis, receptor internalization, cell surface receptor signaling pathway, plasma membrane, extracellular region, integral component of plasma membrane, heparin binding, ion channel binding, and receptor activity. Summarily, most of the functional enrichment was related to the structure and function of chromosomes. As we all know, chromosomal instability is a major form of genomic volatility and contributes to abnormal chromosomal structure and numbers. Micro-satellite instability and increased frequency of base-pair mutations are other described forms of genomic instability, and the genome instability that is a hallmark of cancer almost certainly contributes to further alter sequences in regulatory regions that can promote tumor progression . Furtherr tissue  and partr tissue . In addir tissue .By constructing a PPI network with DEGs, our study identified the top 10 degree hub genes, including ADCY8, ADRB2, ALB, CALCA, F2, GCG, GNGT1, INS, NPSR1, and SST. Then, a significant module was subsequently constructed with 14 nodes, which gained the highest MCODE score. After combining the results of MCODE and CytoHubba, 6 hub genes were chosen, including ADCY8, ADRB2, CALCA, GCG, GNGT1, and NPSR1. ADCY8 is a member of the adenylyl cyclase family of genes and produces the enzyme adenylyl cyclase (AC8). Orchel et al.  found thConsidering the enrichment results of the 6 hub genes in the present study, it was demonstrated that LUAD was associated with E2F targets, G2M checkpoint, glycolysis, mitotic spindle, and PI3K-Akt-mTOR signaling. E2F family members play a major role in cell cycle regulation and DNA synthesis in mammalian cells . PreviouIn addition, we also use GEO database to verify the above results. We found 77 DEGs by screening common differentially expressed genes from GSE118370, GSE136043, and GSE140797 datasets. The function of 77 DEGs was mainly enriched in cell adhesion. Furthermore, the enriched KEGG pathways of 77 DEGs included ECM-receptor interaction, dilated cardiomyopathy, focal adhesion, and PI3K-Akt signaling pathway. Subsequently, we screened out 8 genes through the establishment of PPI network and the application of Cytoscape plugin MCODE, which includes the 6 genes screened from TCGA database. So the 6 genes are used as hub genes for further research.in vitro and in vivo verification.Subsequently, cBioPortal was used to summarize the possible genetic alterations for 6 hub genes in LUAD. We found that the mutation is the most common mutation in 6 hub genes. Mutations in transcription factors have long been known to contribute to tumorigenesis, and previous studies indicated that overexpressed oncogenic transcription factors could alter the core autoregulatory circuitry of the cell . MutatioIn this paper, we used UALCAN database, RT-PCR, and HPA database to study the expression differences of 6 hub genes between LUAD tissues and normal tissues. The results showed that the expression levels of GCG, GNGT1, NPSR1, and CALCA were higher in LUAD tissues than in normal lung tissues, and the expression levels of ADCY8 and ADRB2 were lower in LUAD tissues than in normal lung tissues. Previous studies have shown that altered expression levels of the 6 hub genes. For example, ADCY8 hypermethylation and altered expression have been observed in endometrial cancer ,73. FeigDuring the survival analysis of the present study, KMplot and TCGA database were used to assess the effect of expression levels of the 6 hub genes in patients with LUAD. Notably, high expression of ADCY8 and ADRB2 and low expression of CALCA, GCG, GNGT1, and NPSR1 were correlated with significantly better OS and PFS in LUAD patients. Previous studies have reported the relationship between some of these genes and prognosis of cancers. For example, Wu et al.  found thFinally, we analyzed the clinical data of TCGA database to clarify the relationship between the expression level of 6 hub genes and the clinical manifestations of patients with LUAD. The results showed that the expression level of ADCY8 was negatively correlated with T stage, distant metastasis, and TNM stage. The expression level of ADRB2 in female was significantly higher than in male, and the expression level of ADRB2 was negatively correlated with T stage, nodal metastasis, distant metastasis, and TNM stage. The expression level of CALCA in male was significantly lower than in female, and the expression level of CALCA was positively correlated with nodal metastasis, distant metastasis, and TNM stage. The expression level of GCG was negatively correlated with age, and the expression level of GCG was positively correlated with nodal metastasis, distant metastasis, and TNM stage. The expression level of GNGT1 was positively correlated with T stage and nodal metastasis. The expression level of NPSR1 was positively correlated with T stage, nodal metastasis, and TNM stage.In summary, it can be seen that the expression levels of ADCY8, ADRB2, CALCA, GCG, GNGT1, and NPSR1 are significantly related to the OS and PFS of LUAD, which are also important indicators for the evaluation of the prognosis of LUAD and the evaluation of further treatment. The analysis of the 6 genes indicated that they could effectively distinguish between LUAD tissues and normal tissues, which may increase the accuracy of predicting LUAD.In conclusion, ADCY8, ADRB2, CALCA, GCG, GNGT1, and NPSR1 may be potential biomarkers and therapeutic targets for LUAD.


The purpose of our study was to evaluate the effectiveness of thermal ablation (TA) for Bethesda IV thyroid nodules, and to compare TA and surgery in terms of treatment outcomes, complications, and costs.This study was approved by the local ethics committee. From January 2017 to December 2019, 30 patients elected TA and 31 patients elected surgery for treatment of Bethesda IV thyroid nodules. Demographics information and conventional ultrasound before treatment for each patient was obtained. For the TA group, the ablation extent was 3 mm beyond the edge of the tumor to prevent marginal residual and recurrence. Patients were followed up at 1, 3, and 6 months after intervention, and every 6 months thereafter. Postoperative complications, operation time, hospitalization time, blood loss, and incision length were recorded.P<0.001).In the TA group, the volume reduction ratio (VRR) was 94.63 ± 8.99% (range:76%-100%) at the final follow-up. The mean follow-up time was 16.4 ± 5.2months (range:12–24 months). No recurrences, no metastatic lymph node, and no distant metastases were detected during follow-up. The TA group had fewer complications, shorter operation time, smaller incision length, less blood loss, shorter hospitalization time, and lower treatment costs compared to the surgery group (all TA is technically feasible for the complete destruction of Bethesda IV thyroid nodules, and also safe and effective during the follow-up period, with high VRR and low complication rates, especially in patients who were ineligible for or refused surgery. Thyroid nodule has become increasingly common in clinical practice. This trend possibly stems from the improved detection of asymptomatic and incidental thyroid nodules due to the application of high-resolution ultrasound or other imaging. It has been reported that the incidence of thyroid nodules in adults can be up to 60% .Ultrasound-guided fine-needle aspiration (FNA) is the preferred procedure to evaluate suspicious thyroid nodules, and the Bethesda System for Reporting Thyroid Cytopathology stratifies the malignancy risk based on FNA result. However, the diagnosis of Bethesda IV accounts for 2%-25% . The indTA, such as radiofrequency ablation (RFA) or microwave ablation (MWA), has been validated for its efficacy and safety in treating thyroid benign nodules and autonomously functioning thyroid nodules –7. RecenIn this study, we analyzed the therapeutic outcomes of TA for 30 Bethesda IV thyroid nodules patients after a mean follow-up of 16.4 ± 5.2months (range 12–24 months). Moreover, we also analyzed 31 Bethesda IV thyroid nodules patients who had undergone surgery. Two methods in terms of technical effectiveness, treatment outcomes, complications, and costs were evaluated.This study was approved by the ethics committee of the Chinese People’s Liberation Army General Hospital (S2019-211-01). Written informed consent was obtained from all patients prior to ultrasound-guided FNA, TA, and surgery. We reviewed the medical records of 61 patients who were diagnosed as Bethesda IV thyroid nodules by FNA in our department from January 2017 to December 2019, including 30 patients of ultrasound-guided TA treatment and 31 patients of surgical treatment  a solitary suspicious thyroid nodule was detected by ultrasound; (2) located in the thyroid gland and with no thyroid capsule contact; (3) no imaging evidence of extrathyroidal invasion, lymph node metastasis, or distant metastasis; (4) ultrasound-guided FNA confirming Bethesda IV cytology; (5) refusal of or ineligibility for surgery due to medical contraindications or other reasons; and (6) complete records and more than 12 months of follow-up.For the surgery group, the inclusion criteria were as follows: (1) a solitary suspicious thyroid nodule was detected by ultrasound; (2) no imaging evidence of extrathyroidal invasion, lymph node metastasis, or distant metastasis; (3) ultrasound-guided FNA confirming Bethesda IV cytology; and (4) complete clinical data.For the TA group and the surgery group, the exclusion criteria were: (1) suspicious cervical lymph node metastasis, (2) a history of head or neck irradiation, (3) severe diseases that cannot tolerate interventional and surgical treatment, and (4) a history of severe bleeding and coagulopathy.Demographics information for each patient was obtained, and all patients underwent conventional ultrasound before treatment to evaluate nodule characteristics, including the diameters in three dimensions , volume, position, composition, echogenicity, shape, margin, and echogenic foci. The volume of each nodule was calculated as V =πabc/6 . For all patients, Bethesda IV thyroid nodules was diagnosed based on cytology findings by ultrasound-guided FNA.The ablation techniques include RFA and MWA, which are selected according to nodule characteristics, the relationship with adjacent organs, the patient’s own condition, and the physician’s experience. All TA procedures were performed by one physician with rich experience in thyroid ultrasound and interventional ultrasound, including initial diagnosis and preoperative evaluation. TA was performed in an outpatient ultrasound interventional room. Patients were placed in the supine position. Ultrasound was used to guide the positioning, and then conventional sterilization and sterile drapes were carried out. 1% lidocaine was used for local anesthesia. In order to protect normal structures, using the Hydrodissection technique, the tumor was separated from the critical structures by infusing a mixture of normal saline , 12. BefThe Siemens Acouson Sequoia 512  scanner with a 6.0 MHz linear array transducer was used for guidance. The bipolar RFA generator  and a 18-gauge bipolar RF applicator with a 1.5cm active tip  were used. Under the guidance of ultrasound, the electrode was inserted into the nodule. Ablation started at 3W of power, and the moving-shot technique was applied , 14. If An MTI-5DT machine  and XR-B1610W superficial organ ablation needle  were used. When the ablation position was determined, the skin was incised 3mm subcutaneously, and the 17G-100 microwave needle was applied by moving-shot technique. After MWA was started the initial power was set to 25W. If there was no hyperechoic zone in the ablative area after 5-10s ablation with this power, the power was gradually increased (up to 35 W). Multifaceted and multipoint mobile ablation was performed until the lesion nodules were completely covered by hyperechogenic vaporization area. The ablation extent was 3 mm beyond the edge of the tumor to prevent marginal residual and recurrence. Contrast-enhanced ultrasound was performed immediately after ablation to evaluate the ablation area. If the ablation was incomplete, the ablation was supplemented; otherwise, the ablation was completed. After observation for 2 hours, patients were discharged with stable vital signs.Surgery was performed by surgeons with rich experience in thyroid surgery. The decision to perform different surgical methods was made by individual surgeons and patients based on patient preferences in consultation with the surgeon.After anesthesia was satisfied, the routine neck and shoulder surgery area was disinfected. An arc incision was taken to cut skin, subcutaneous tissue, and latissimus muscle. The thyroid gland was explored, the character of the nodule and the adjacent tissue was evaluated, and the resection was performed. The blood vessels were carefully separated and ligated. During the operation, the recurrent laryngeal nerve and parathyroid gland was protected. The tissue was cut and sent to frozen examination. Neurological function and bleeding were evaluated after surgery.For the TA group, patients were followed up at 1, 3, 6, and 12 months after intervention, and every 6 months thereafter. The therapeutic effect was evaluated by ultrasound and volume of ablation was detected. The percentage reduction of the ablation area in volume was calculated as follows: Volume reduction ratio (VRR) = [ × 100]/initial volume. Postoperative complications  , operati2 test or Fisher exact test was used for comparison between groups. Wilcoxon’s signed rank test was used to compare changes in the maximum diameter and volume of ablation before TA and at each follow-up point. P < 0.05 was considered statistically significant.Using SPSS 23.0 software, continuous variables were expressed as mean ± SD, and t-test was used for comparison between groups; categorical data are presented as frequencies and percentages, and Pearson xP > 0.05). Ultrasound characteristics of all the nodules are shown in P > 0.05). The ultrasound characteristics of all thyroid nodules including position, composition, echogenicity, shape, margin, and echogenic foci also showed no significant differences between the two groups (P > 0.05).61 patients with Bethesda IV thyroid nodules were evaluated, including 30 patients of ultrasound-guided TA treatment and 31 patients of surgical treatment. The general data are provided in P <0.001), except for immediately and 3 months after ablation (P > 0.05) (P <0.001) (P <0.001) , Hürthle cell adenomas , nodular goiters , follicular thyroid carcinoma (FTC)   includiP <0.001, Of the 30 patients in the TA group, only one patient had postoperative pain after ablation, which resolved spontaneously in 2 hours. For the surgery group, 31 patients had postoperative pain. Thyroid function was assessed 1 month after surgery. Four patients were diagnosed with hypothyroidism and received prescriptions for levothyroxine sodium, and one patient had subclinical hypothyroidism. Parathyroid function was assessed 1 day and 1 month after surgery. Four patients had transient hypoparathyroidism, three of which were asymptomatic; the remaining patient had mild facial paresthesia. The serum parathyroid hormone and calcium level of all the four patients were restored within 3 months after calcium supplement treatment. Therefore, a significant difference was noted  at 24 months. According to this standard, effective treatment was defined as a VRR > 50% of the initial nodule volume . The theIn terms of complications, our study shows that TA was less invasive than surgery. In the TA group, only one patient had postoperative pain which resolved spontaneously. In the surgery group, in addition to postoperative pain, there were also complications of hypothyroidism and hypocalcemia, which needed drug supplement treatment. TA offers better protection of thyroid function. The TA group had a shorter intervention time, smaller incision length, less blood loss, shorter hospitalization time, and lower treatment costs compared to the surgery group. All patients of the TA group were treated in one session. If repeated TA for recurrence of nodule is required, the cost would be higher than surgery. However, this risk is low and requires longer follow-up to verify . TherefoThere were many limitations in our study. The number of patients was small, and follow-up time was not long enough. Validation of the results requires a large population, a long follow-up period, and prospective and multicenter studies. For the TA group, the final pathology of Bethesda IV thyroid nodules has not been diagnosed. In the future, development in the field of molecular testing and emergence of new technologies are needed to assist in the differential diagnosis between follicular adenomas and FTC.In conclusion, this study demonstrated ultrasound-guided TA is technically feasible for the complete destruction of Bethesda IV thyroid nodules, and is also safe and effective during mean 16.4 ± 5.2months follow-up period, with high tumor VRR and low complication rates. Surgical treatment is the first choice, but for patients who are ineligible for or refuse surgery, TA may be an available option. The persistence and repeatability of these findings in our study need to be demonstrated by larger populations and longer follow-up periods.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by institutional review board of General Hospital of Chinese PLA(S2019-211-01). The patients/participants provided their written informed consent to participate in this study.XL and YKL: integrity of the whole study, analysis of data, and review of the final manuscript. MZ and YL: management of data and manuscript writing. LY, JX, and NL: statistical analysis of data. All authors contributed to the article and approved the submitted version.This work was supported by the National Natural Science Foundation of China under Grant [No. 81771834].The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The threshold inflection point of AFC was 8n. Female age, BMI, AFC and COH protocol were independent predictive factors associated with live birth in POR patients classified by the POSEIDON criteria. The smooth curve fit and threshold effect analyses provide clinical management strategies for these patients. In addition, the early-follicular-phase long-acting GnRH-agonist long protocol seems to have a higher live birth rates than other protocols. It is worth highlighting that BMI should be considered as well in the POSEIDON criteria.The mechanisms underlying poor ovarian response (POR) in assisted reproductive technology remain unclear, there is no consensus on the management of poor responders, the POSEIDON stratification classifies infertility patients into “expected” or “unexpected” groups to provide a more nuanced picture of POR, but few researchers have discussed the independent predictive factors (smoothed plots and the threshold effect) for live birth in POR patients classified by the new criteria. We conducted a retrospective cohort study using clinical data from 6,580 POR patients classified by the POSEIDON criteria in the First Affiliated Hospital of Zhengzhou University, and explored the live birth based on the results before and after the threshold inflection point of each independent influencing factor. Among 6,580 poor ovarian reserve patients classified by the POSEIDON criteria, 1,549 (23.54%) had live births, and 5,031 (76.46%) did not have live births. Multivariate logistic regression analysis showed that female age , body mass index , antral follicle counting  and controlled ovarian hyperstimulation protocol were independent factors predicting live birth in patients with POR. The threshold effect analysis found that the inflection point of female age was 34 years old, and when age was > 34 years old, the probability of live birth in POR patients dropped sharply . The inflection point of BMI was 23.4 kg/m The Patient Oriented Strategies Encompassing Individualized Oocyte Number (POSEIDON) group proposed a new stratification method for poor ovarian response (POR) patients in 2016 . DiffereAccurate predictive factors of ovarian reserve and pregnancy outcome in infertile women with poor ovarian response (POR) remain unknown and are one of the main puzzles of assisted reproductive technology treatments. Although many studies have been performed to identify predictors of IVF/ICSI outcome, there is still no real consensus , 10. ThiToday, large data and evidence-based medicine have progressively developed as the standard approach for clinical management strategies in the field of reproductive medicine , 14. At This retrospective cohort study evaluated clinical data from 6,580 POR patients classified by the POSEIDON criteria who underwent IVF/ICSI in the reproductive medical center of the First Affiliated Hospital of Zhengzhou University from June 2013 to December 2018. All the included case data were recorded and sorted by dedicated personnel. The quality of data entry is strictly controlled to ensure the completeness and authenticity of the data. This study strictly followed the relevant requirements of the Declaration of Helsinki of the World Medical Association. The study was a retrospective study approved by the Medical Ethics Committee and did not require the informed consent of patients.We compared the experimental data of patients in the “live birth group” and “non-live birth group” and determined the statistically significant influencing factors for the two groups of patients. Based on these results, further single-factor and multifactor logistic regression analysis was performed to screen for independent influencing factors for live birth in POR patients; then, a smooth fitting curve model was constructed based on independent influencing factors, and the linearity between these influencing factors and the occurrence of live birth was observed. The relationship was further analyzed based on the model observation results, and finally, the probability of live birth was explored in detail based on the results before and after the threshold inflection point of each independent influencing factor.There patients were put on either the early-follicular-phase long-acting GnRH-agonist long protocol, Mid-luteal phase short-acting GnRH-a long protocol, GnRH antagonist protocol, GnRH agonist short protocol or natural cycle for IVF/ICSI followed by embryo transfer in the same cycle. The starting dose of gonadotropin  were administrated on the basis of patient`s antral follicle count, age, BMI and previous ovarian response to stimulation. The dosage was also adjusted constantly according to patient`s response. When dominant follicles measuring > 16mm account for 60% or a follicle reached 20mm in mean diameter, trigger was normally performed using 250ug r-hCG  in combination with 2000IU u-HCG . Oocyte retrieval under the guidance of transvaginal ultrasound was performed 37 hours after trigger. The diagnosis of spontaneous abortion is after the pregnancy is confirmed, the clinical history, physical examination, female endocrine and vaginal ultrasound confirm that the embryo stops developing before 20 weeks of pregnancy , 16. LivPOSEIDON “unexpected” group: age < 35 years, AFC ≥ 5, AMH ≥ 1.2 ng/mL and ≤ 9 oocytes retrieved in the first stimulation cycle; age ≥ 35 years, AFC ≥ 5, AMH ≥ 1.2 ng/mL and ≤ 9 oocytes retrieved in the first stimulation cycle; POSEIDON “expected” group: age < 35 years, AFC < 5, AMH < 1.2 ng/mL; age ≥ 35 years, AFC < 5, AMH < 1.2 ng/mL , 17, 18.http://www.r-project.org; version 3.6.1), EmpowerStats (http://www.empowerstats.com) and SPSS 19.0 . The continuous variable data are expressed as the mean ± standard deviation (Mean ± SD) and were compared using Student’s t test or the Wilcoxon rank sum test; categorical variables are expressed as frequencies (percentages) and were compared using the chi-square test. We then applied multiple regression analysis to estimate the independent relationship between live birth and female age, BMI, AFC and COH protocol with an adjustment for potential confounders. P < 0.05 indicated that a difference was statistically significant, to avoid inflating the probability of a Type I error, P-values were adjusted with the Holm-Bonferroni method.All analyses were performed using the statistical packages R  and 95% confidence intervals (Cis) for the threshold turning points of the independent influencing factors. P < 0.05 indicated that a difference was statistically significant.We collected effective experimental data from 6,580 POR patients classified by the POSEIDON criteria, the “unexpected” group (n=3639) and the “expected” group (n=2951), and 1,549 (23.54%) had live births, 5,031 (76.46%) did not have live births, and 1309 of these women (35.97%) succeeded had live births in “unexpected” group, 240 of these women (8.13%) succeeded had live births in “expected” group. There were statistically significant differences in female age, primary infertility, infertility years, BMI, pasal P, AMH, PRL, AFC, number of treatment cycles, initiation dosage of Gn used, duration of Gn used, oocyte number, and use of a controlled ovarian hyperstimulation protocol between the two groups , primary infertility , infertility years , BMI , AMH , PRL , AFC , No. of treatment cycles , initiation dosage of Gn used , duration of Gn used , oocyte number  and COH protocol were factors for predicting live birth in patients with POR. Multivariate logistic regression analysis showed that female age , BMI , AFC  and COH protocol were independent factors for predicting live birth in patients with POR Multivariate logistic regression analysis showed that female age, BMI, and AFC were independent predictive factors associated with live birth in POR patients classified by the POSEIDON criteria. The adjusted smooth curve fit showed a nonlinear correlation between them. Age and BMI had a negative correlation with live birth, and AFC had a positive correlation with live birth. However, these influencing factors did not have a simple linear relationship with live birth . When age ≤ 32 years of age, the probability of spontaneous miscarriage and female age were not significantly related . The inflection point of BMI was 23.4 kg/m2; when BMI > 23.4 kg/m2, it had a negative correlation with live birth , but when BMI > 23.4 kg/m2, there was no significant relationship between them . The threshold inflection point of AFC was 8n. However, there was still a roughly linear positive relationship between AFC and live birth before and after the inflection point Our study first analyzed the independent factors predicting the possibility of live birth in POR patients using smooth curve fit and threshold effect analyses. The results confirm that the POSEIDON classification criteria are indeed very accurate in predicting the live birth rate for POR patients, which is very similar to our results, however, it seems that the results indicate that BMI should be considered as well in the POSEIDON criteria, and AFC threshold of 8 instead of 5 may be more relevant. AMH was not an independent factor, as this is also included in the POSEIDON criteria. We all know that various advances in assisted reproductive technologies have led to a significant improvement in the efficacy of treatment for couples who with infertility in recent decades, and various embryo manipulation techniques and controlled ovarian stimulation specifications continue to be improved , 20. HowOur study found that the older the female was, the lower the probability of live birth in POR patients. In fact, some studies have suggested that the blastocyst euploidy rate drops from 60% before 35 years to 30% after 40 years and that the embryo euploidy rate decreases by 2.4% per year with increasing female age. These studies suggest a negative correlation between age and the live birth rate , 22. DifThis study found that AFC and BMI are other important factors for predicting live birth in patients with POR. AFC is a good indicator of reactive ovarian reserve function in infertile patients , 28. BunThe early-follicular-phase long-acting GnRH-agonist long protocol seems to have a higher live birth rates than other protocols for POR patients. This protocol is designed to suppress the pituitary gland for 28 days, and a standard full dose of GnRH-a before ovarian stimulation in IVF-ET might improve the pregnancy and live birth rates per fresh ET , 32. SomIn summary, our study can more accurately predict the probability of a live birth and provide clinical management strategies for these patients. However, the main limitation of our study is that it is a retrospective study that could not exclude all potential biases, and pregnancy is a process of continuous change, during which there are many confounding factors. For example, maternal infection factors, environmental factors, emotional factors, etc., were not taken into account in this study, and prospective, large-scale and multicenter clinical trials are still needed to confirm our findings in the future.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Ethics Committee of The First Affiliated Hospital of Zhengzhou University. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.GL and FL conceived of and designed the experiments. GL, TY and LH selected and supervised suitable patients. GL, JL, HJ, and LF obtained basic clinical data including age, body mass index, FSH, LH, estradiol, progesterone and AMH levels, total dosage of gonadotropin used, duration of gonadotropin use, oocyte number, and live birth rate per transfer. GL provided overall supervision. GL, GH and FL drafted the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the National Natural Science Foundation of China (81771534), the Key Science and Technology Foundation of Henan Province (SBGL202002048), the Key Research Projects of Henan Higher Education Institutions (18A320057) and the Medical Science and Technology Project of Henan Province (212102310049).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.