Adherence to treatment guidelines and treatment success are low in Type 2 diabetes mellitus (T2DM). This study aims to capture the physician perspective on T2DM guideline adherence and identify levers for increasing adherence.A survey among German physicians captured the perceived value of 4 areas in the national treatment guideline (NVL), 13 possible barriers, and 9 possible enablers for guideline adherence. Perceived value was assessed by ranking 4 NVL areas by implementation difficulty and impact on treatment success. Barriers and enablers were assessed by rating their influence on guideline deviation and adherence. The consistency of results across subgroups was assessed using Fisher's exact test.Responses from 46 physicians showed a strong consensus about the value of each NVL area. Physicians perceived patient inability and demotivation to be the strongest adherence barriers . All queried enablers were approved by ≥ 50% of participants. Physicians considered cross-provider collaboration and electronic therapy decision support as strongest enablers . Consistency was high between subgroups.This study suggests that physicians consider patient-related factors to be stronger barriers for guideline adherence than physician-related factors. Finding opportunities to increase physician buy-in is important for better guideline adherence. In this study, physicians voiced appreciation for adherence enablers based on digital solutions to support the care process and to reduce the complexity of therapy decisions. Endocrinology; Evidence-based medicine; Health informatics; Health profession; Health promotion; Health technology; Diabetes mellitus, Type 2; Guideline adherence; Physician-patient relationship; Information systems. Glycemic control, i.e. achieving target levels of glycated hemoglobin HbA1c), is low c, is low. InternaTo address hyperglycemia and diabetes complications, medical guidelines have been issued. They aim to embed the latest scientific knowledge and best practices in daily medical practice, to improve treatment success. Guidelines are formally established in most countries but implementation success and monitoring of guideline adherence are lacking .Adherence rates to T2DM guidelines are low, typically at around 40–60%. Often, guideline adherence is measured by assessing which share of participants has received a lab test or examination . In some cases, adherence is measured longitudinally by assessing whether a treatment algorithm was followed or by evaluating whether certain structural requirements of the guideline were met , 8, 9.Achieving guideline-adherent disease management depends on patient factors, health care system factors, and physician factors. Patient factors include e.g. health literacy, lifestyle, or awareness of the disease. Health care system factors include e.g. availability of disease registry, collaboration among the interdisciplinary care team, or visit planning and follow-up. Physician factors include e.g. time for patient care, the ability to set clear treatment goals, and reactive vs. proactive approach to care , 11, 12.In Germany, 7.5 M adults (20–79 years) are supposed to have diabetes, 90% of them T2DM. An estimated 34% of these cases are undiagnosed. 11% of annual deaths are caused by diabetes, which is more than all infectious diseases combined .Since 2002, Germany has a national guideline program for T2DM which consists of 6 guidelines. The national T2DM treatment guideline (NVL) was last revised in 2014. A consolidated update of the guideline program is currently under review and expected to be published by the end of 2020.Diabetes care in Germany is provided by general practitioners (GP), diabetology specialists, and hospitals. 80–90% of patients with diabetes are monitored and treated by their GPs. The remaining patients are in the temporary or permanent care of one of 1,100 specialist practices. There, trained diabetologists manage uncontrolled episodes of diabetes or other more challenging cases. Physicians are supported by trained diabetes staff including diabetes consultants, diabetes assistants, and diabetes nurses. Emergencies, glycemic escalations, and severe complications require patients to be admitted to hospitals with specific qualifications to treat diabetes .Since 2003, Germany has a disease management program (DMP) for outpatient T2DM care, which aims to improve the health of T2DM patients. The DMP coordinates and evaluates evidence-based T2DM care across providers. In 2019, 4.3m patients participated in the DMP . A GP inAt the same time, optimal care becomes more complex. Physicians are faced with a large number of treatment options to choose from: In the past 10 years, the European Medicines Agency alone has approved 35 new medicines for diabetes treatment . New resIn daily practice, most physicians work with administrative IT systems. These systems serve billing purposes, can document the care process, and have some reporting capability. Solutions that integrate relevant T2DM patient data from different sources and which assist the physician in therapeutic decision making, possibly with embedded guideline information, are emerging but not broadly disseminated , 22.In summary, many T2DM patients do not achieve glycemic control and are treated by physicians that are busy handling a breadth of conditions with high administrative burden – leaving little time per patient. At the same time, refined guidelines and a growing number of treatment options add complexity to the treatment choice. Physicians do not have the electronic tools at hand to manage this complexity without additional workload. These factors may all contribute to low guideline adherence.Therefore, this paper aims to capture the physician perception of guideline adherence and identify leverage points for increasing adherence.This paper draws on previous research where perspective on guideline adherence was collected from 9 outpatient physicians in Southern Germany and correlated with data from their patients . The pre2This study is based on a survey among physicians in primary and specialist practice as well as hospital-based physicians with experience in the treatment of T2DM patients in Germany. Survey participants were recruited in 2 ways: First, customers of Roche Diabetes Care in the Baden-Württemberg region (“BW physicians”) who were asked to complete the questionnaire during routine visits of the sales team. Second, participants of the 2019 Congress of the German Diabetes Association  who were asked to complete the questionnaire during breaks between scientific presentations.–4 demographic questions about physicians' role, experience with T2DM, type of practice, and participation status in the German national T2DM program.–1 question about awareness of German national diabetes guidelines–2 questions about the perceived value of 4 aspects of the NVL. Physicians were asked to rate each aspect by the difficulty of implementation in daily clinical practice and by the impact on treatment success. The 4 aspects were monitoring adherence, screening adherence, targeting adherence, and therapy adherence –2 questions about 13 potential barriers and 9 potential enablers for guideline adherence  by difficulty of implementation and impact on treatment success. For the difficulty of implementation, rank 1 was “easiest to implement” and rank 4 was “most difficult to implement”. For impact on treatment success, rank 1 was “highest influence on treatment success” and rank 4 was “lowest influence on treatment success”. Results were analyzed by combining the 4 ranks to 2 groups : A response was interpreted as “higher difficulty of implementation” if it was ranked 3 or 4 and as “lower difficulty of implementation” if it was ranked 1 or 2. A response was interpreted as a “higher impact on treatment success” if it was ranked 1 or 2 and interpreted as a “lower impact on treatment success” if it was ranked 3 or 4. These grouped results were analyzed for each NVL area separately and combined (difficulty-impact matrix) by calculating absolute and relative frequencies.Barriers and enablers for guideline adherence were assessed with a 4-point Likert scale. For barriers, the scale ranged from “1 = never cause for a deviation from the guideline (lower influence)” to “4 = very frequent cause for a deviation from the guideline (high influence)”. For enablers, the scale ranged from “1 = does not increase guideline adherence (low influence)” to “4 = strongly increases guideline adherence (high influence)”. For analysis, the 4 points of the scale were assigned either to the group of “higher” or “lower” influence: Higher influence was assigned to a response when the physician rated a barrier or enabler 3 or 4. Conversely, a lower influence was assigned when the physician rated a barrier or enabler 1 or 2. The grouped results were analyzed for each barrier and enabler by calculating absolute and relative frequencies.The consistency of results between subgroups was evaluated using Fisher's exact test . 5 different groupings were considered: Physician type , physician role , physicians' self-reported knowledge of the T2DM treatment guideline (good or very good vs. lower), physician experience with T2DM (>10 years vs. less), and recruitment .3118 physicians were invited to participate in the study. 52 physicians filled out the questionnaire (response rate: 44%). 6 questionnaires were excluded from the analysis because 2 participants did not complete the full survey and 4 participants delegated filling out the questionnaires to non-physicians. Most participants were outpatient diabetology specialists and practice owners and had more than 10 years of experience in treating T2DM. >90% of physicians reported good or very good knowledge of the NVL and participated in the DMP. All physicians who did not participate in the DMP where hospital-based physicians where the DMP does not apply.Subgroup analysis showed no significant differences with regards to adherence barriers, except for item 10: None of the physicians with ≤10 years of experience vs. 50% of physicians with >10 years of experience in treating T2DM patients viewed disapproval with the guideline as a reason for guideline non-adherence (p = 0.014).Subgroup analysis for adherence enablers showed significant differences in 2 areas: All assistant and salaried physicians but only 61% owners and department heads believed that alignment of guideline and reimbursement system would increase adherence (p = 0.020). 88% of physicians with ≤10 years of T2DM experience but only 42% of physicians with >10 years of T2DM experience rated financial incentives for patients as an adherence enabler (p = 0.047). As a trend, the largest differences between subgroups (16–36%) were observed in physicians' education as a possible enabler for adherence.4This study shows that T2DM-experienced physicians in Germany have a consistent perspective on the value of the NVL. Participants rated patient-related factors as the strongest barriers for guideline adherence, followed by deficiencies in the patient-physician relationship and high physician workload. Physicians considered electronic physician support systems and improved cross-sectoral collaboration as the strongest enablers for improved guideline adherence.In many areas, this study aligns with previous research: Studies have shown that physicians often do not intensify T2DM therapy despite knowing that it is indicated  , 27. ThiBarriers and enablers for adequate T2DM care have been studied previously. A review article identified patients' and physicians' beliefs and attitudes as major factors for adherence. Beyond that, physician adherence was also influenced by their diabetes knowledge. And, patient adherence was also influenced by their health literacy, financial situation, comorbidities, and social support system . A more DAWN2 also researched enablers for T2DM guideline adherence. Health providers in 13 countries rated patient education, provider education, and availability of additional specialized resources, and better collaboration within the care team as key areas for improvement in diabetes care . Lower sElectronic therapy decision support for physicians was the enabler which scored second highest in this study. When guideline information is embedded in these systems, when they can track whether monitoring and screening tasks are completed in adequate intervals, highlight contraindications, and alert about thresholds for therapy intensification, these systems can support physicians in clinical decision making. In recent years, information technology interventions, including clinical decision support systems (CDSS), have shown to improve glycemic control in people with T2DM. Some studies also report improvements in the care process and patient satisfaction . PreviouThis study had strengths and limitations. The forced ranking of NVL areas for the difficulty to implement and the impact on treatment success methodologically only allows analysis of relative differences, not the absolute perception of “high” or “low”. This may have led to over- or understating physicians' responses. This study is based on self-reporting. Results reflect the subjective perception of physicians. Selection bias may have occurred in the group of physicians who were invited to participate by Roche Diabetes Care. However, there was very little difference in how these physicians rated compared to the randomly selected participant group recruited at the DDG congress. The sample size in some of the subgroups is very small and p-values must be interpreted with caution. A larger sample size in these subgroups would be required to confirm consistency between groups. As a strength, this study was able to capture feedback on a broad set of adherence barriers and enablers from the same group of respondents, which allowed an interesting perspective on physician-related factors influencing guideline adherence. For the first time, the role of data-driven, digital aspects are researched in this context. Our study showed the value of different, typical aspects of T2DM treatment guidelines to physicians. It is, to our knowledge, the first assessment of physician perspective on T2DM guideline adherence in the German-speaking area.5This study suggests that physicians consider patient factors to be stronger adherence barriers than physician factors. Opportunities to increase physician adherence must be leveraged in addition to increasing patient competence and engagement and building a health care system suitable for chronic care. Physicians voiced appreciation for adherence enablers based on digital solutions. With the high physician workload and often challenging patient-physician relationship, such enablers must be designed so that they do not increase workload and complexity for physicians. Existing practice management systems could be enhanced with automated appointment reminders and follow-up functionalities, with patient dashboards integrating key data from different sources, or with the ability to immediately see deviations from most current guidelines. This can save valuable physician time, increase evidence-based decision making, and allow physicians to refocus on the conversation with their patients.S. Brenner: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.W. Oberaigner: Conceived and designed the experiments; Analyzed and interpreted the data; Wrote the paper.H. Stummer: Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.The authors declare no conflict of interest.No additional information is available for this paper.


Background: The role of microbiota in Lynch syndrome (LS) is still under debate. We compared oral and fecal microbiota of LS saliva and stool samples with normal healthy controls (NHC). Methods: Total DNA was purified from feces and saliva to amplify the V3–V4 region of the 16s rRNA gene. Sequences with a high-quality score and length >250 bp were used for taxonomic analysis with QIIME software. Results: Compared to NHC, LS fecal samples demonstrated a statistically significant increase of Bacteroidetes and Proteobacteria and a significant decrease of Firmicutes at the phylum level and of Ruminococcaceae at the family level. Moreover, LS oral samples exhibited a statistically significant increase of Veillonellaceae and Leptotrichiaceae and a statistically significant decrease of Pasteurellaceae. A beta-diversity index allowed differentiation of the two groups. Conclusions: A peculiar microbial signature is associated with LS, similar to that of sporadic colorectal cancer and Crohn’s disease. These data suggest a possible role of proinflammatory bacteria in tumor development in a condition of genetic predisposition, such as LS. MLH1, MSH2, MSH6, and PMS2 genes, which guarantee DNA mismatch repair (MMR). Failure of the MMR system provokes generalized genome instability, especially in the short repetitive coding and noncoding sequences [Lynch syndrome (LS) is an autosomal dominant hereditary cancer syndrome with variable penetrance and expressivity . LS accoequences  with abuequences ,6.Tumors from patients with LS express numerous neoepitopes, which could elicit an antigen-specific cytotoxic T-cell response ,8,9. TumChanges to the microbiota contribute to sporadic colorectal carcinogenesis . An incrThe contribution of the microbiota to colorectal carcinogenesis in patients with LS is relatively unknown. Yan et al. recently profiled the microbiota from fecal and histological specimens of 100 LS patients at baseline and a one-year follow-up, and they observed that dysbiosis developed with colonic preneoplastic lesions . A colectomy can change the overall microbiome structure. Nonetheless, baseline differences in mucosal and fecal community function were concordant with previously observed changes in later-stage CRC and weakly predictive of interval adenoma development . MoreoveWe aim to characterize the salivary and fecal microbial population of LS patients compared to healthy controls. We are specifically interested in investigating the overall microbiota composition, diversity, and taxonomic pattern abundance in LS patients.EPCAM gene: class IV and class V variants were collectively considered to be pathogenic, according to the InSiGHT criteria by Thomson et al. [Patients were consecutively enrolled from 2017 to 2019 at IRCCS San Raffaele Scientific Institute, a tertiary referral hospital in Milan, Italy. Patients with LS had their DNA sequenced with a Next Generation Sequencing (NGS) panel that included the 4 MMR genes and the n et al. .MLH1 pathogenetic variant, 14 subjects with an MSH2 pathogenetic variant , and one female subject with an MSH6 pathogenetic variant. Thirty-seven patients provided salivary samples : nine subjects with an MLH1 pathogenetic variant , 23 subjects with an MSH2 pathogenetic variant , two female subjects with an MSH6 pathogenetic variant, and three female subjects with a PMS2 pathogenetic variant. Four MLH1 patients, 10 MSH2 patients, one MSH6 patient, and one PMS2 patient had undergone hemicolectomy for CRC: nine had received  a right hemicolectomy and seven  a left hemicolectomy. Two patients  received a gastrectomy for gastric cancer. Two patients had a pancreatic resection: one MSH6 patient for pancreatic cancer, and one PMS2 patient for a neuroendocrine tumor.Seventeen patients with LS provided fecal samples : two female subjects with an Healthy normal control subjects were tested for their likelihood of harboring a mutation in the MMR genes, employing PREMM5 predictive Model  . The stuHealthy, age-matched controls provided 11 salivary samples  and 21 fecal samples .Salivary samples were retrieved with an oral wash with sterile physiological solution and immediately frozen at −80.0 °C. Patients received instructions on how to collect their fecal samples: within seven days of their upcoming visit, they sampled a small volume of feces in a 0.5 mL Eppendorf tube that contained RNAprotect Tissue Reagent . They kept fecal samples in their freezers at home and then brought them to the clinic using iceboxes. LS patients and normal controls had to be completely healthy in the three months before sampling, without using antibiotics or anti-inflammatory drugs. All LS patients were cancer-free at the time of sample collection and analysis. Patients on chemotherapy could not participate in the study. On average, 9.2 years had elapsed from previous surgery and chemotherapy to the day of sample collection. Based on findings from Feng et al. , this guThis study conformed to the ethical guidelines of the 1975 Declaration of Helsinki , and it was reviewed and approved by the Institutional Review Board of IRCCS Ospedale San Raffaele on 12 July 2010 . Patients and controls both provided written informed consent for study participation.The microbiota analysis was performed by 16S amplicon sequencing. Total DNA was purified from fecal and oral samples using the QIAamp PowerFecal DNA Kit and the Dneasy Blood & Tissue Kits , following the manufacturer’s instructions. The V3–V4 region of the 16S rRNA gene was amplified starting from 200 ng of extracted DNA using the AccuPrime Taq DNA Polymerase , the following primers: V3-16S-Fw: CCT ACG GGN GGC WGC AG and V4-16S-Rev: GAC TAC HVG GGT ATC TAA TCC, and the following amplification protocol: 94 °C for 2 min, 35 cycles of 94 °C for 30 s, 56 °C for 30 s, 68 °C for 1 min, and finally stored at 4 °C. Amplicons were purified using AMPure XP beads . A second PCR step was performed for indexing and adding Illumina sequencing adapters to each sample. The Nextera XT Index Kit  and the KAPA HiFi HotStart PCR Kit  were used with the following amplification protocol: 95 °C for 3 min, 8 cycles of 95 °C for 30 s, 55 °C for 30 s, 72 °C for 30 s, 72 °C for 4 min, and then stored at 4 °C. A second purification step with AMPure XP beads was performed to clean up the final library. The purified DNA was quantified using a Qubit Fluorometer  and a 2100 Bioanalyzer System . Libraries were diluted and mixed for pooling with unique molecular tags. The pool was loaded on a MiSeq reagent cartridge. Sequences with a high-quality score and length >250 bp were used for the taxonomic analysis with Quantitative Insights Into Microbial Ecology v1.9.1 software (QIIME ).t-test with Benjamini–Hochberg FDR multiple test correction. To determine if factors like sex and age had an impact on the statistical outcome, we performed a binomial logistic regression using the healthy/pathological status as the dependent variable and the relative abundance of bacteria, age, and sex as the independent variables. Weighted UniFrac distance metric and principal component analysis were used to perform the beta-diversity analysis: the Adonis statistical method was used to calculate the differences in beta diversity between the groups, describing the strength and significance that a variable has in determining the variation of distances in a beta-diversity graph. Microsoft Office Excel 2010™  and Graphpad Prism 5™  were used to perform statistical tests. All tests were two-sided, with a significance level set at 0.05.The analysis excluded less abundant bacterial taxa, <1% in all samples, at any taxonomic level. Bacterial relative abundance was reported as an average ± standard error percentage. The statistical significance of differences in means and proportions among LS patients and controls was tested with Welch’s We analyzed fecal samples from 17 LS patients and 21 normal controls and oral samples from 37 LS patients and 11 normal controls. The analysis excluded groups at any taxonomic level representing <1%.In fecal samples, alpha diversity—the variation of microbes in a single sample—had no statistically significant difference between the two cohorts. Operational taxonomic units (OTUs) and the Shannon index demonstrated lower values among LS patients, but without reaching statistical significance . Beta diAnalysis of the microbiota composition revealed significant differences between patients and controls according to the relative abundance of bacteria. We identified 21 bacterial phyla, and five had an average relative abundance >1%: Bacteroidetes, Firmicutes, Proteobacteria, Actinobacteria, and Verrucomicrobia. Patients with LS had a statistically significant increase of Bacteroidetes  and Proteobacteria  and a decrease of Firmicutes  compared to control subjects . At the In salivary samples, alpha diversity did not detect statistically significant differences between LS patients and control subjects in either observed OTUs or Shannon index analyses. However, patients with LS had lower values of alpha diversity . Beta diAs for fecal samples, analysis of the relative abundance of bacteria revealed statistically significant differences. There were 23 different phyla, and five had an average relative abundance >1%: Actinobacteria, Bacteroidetes, Firmicutes, Fusobacteria, and Proteobacteria. In LS patients, compared to control subjects, we observed a statistically significant increase of Actinobacteria  and Firmicutes  and a decrease of Proteobacteria  .At the family level, we identified 156 families, 17 of which showed an average bacterial relative abundance >1%: Actinomycetaceae, Micrococcaceae, Porphyromonadaceae, Prevotellaceae, Paraprevotellaceae, Gemellaceae, Carnobacteriaceae, Streptococcaceae, Lachnospiraceae, Ruminococcaceae, Veillonellaceae, Fusobacteriaceae, Leptotrichiaceae, Burkholderiaceae, Neisseriaceae, Campylobacteraceae, and Pasteurellaceae. In particular, we observed in LS patient samples an increase of Veillonellaceae  and Leptotrichiaceae  and a decrease of Pasteurellaceae  compared to control samples .t-test with Benjamini–Hochberg FDR multiple test correction. Conversely, salivary samples demonstrated statistically significant differences between groups at the genus level for the Veillonella (p < 0.01) and Haemophilus (p = 0.026) genera (p < 0.01)  genera , and at t-test with Benjamini–Hochberg FDR multiple test correction analysis . No statistically significant differences were observed for age and sex variables. In saliva samples, there were statistically significant differences in the same bacterial taxa that were statistically different with Welch’s t-test with Benjamini–Hochberg FDR multiple test correction analysis . No statistically significant differences were observed for age and sex variables.Binomial logistic regression confirmed the statistical significance of these findings, and it showed that age and sex did not influence the statistical outcome. Indeed, in fecal samples, there were statistically significant differences in the same bacterial taxa that were statistically different with Welch’s Lynch syndrome is an autosomal dominant cancer syndrome with variable penetrance and expressivity. LS results from pathogenetic variants in genes that guarantee DNA mismatch repair. This cross-sectional study details the microbiome composition of fecal and oral specimens from a cohort of patients with LS and compares it to that of age-matched healthy controls. Fecal samples from subjects with LS had a statistically significant increase of Bacteroidetes and Proteobacteria, a significant decrease of Firmicutes at the phylum level and of Ruminococcaceae at the family level, and a notable reduction of Lachnospiraceae . A significant decrease of Firmicutes was recently demonstrated in both stool samples and intestinal lavage fluid of CRC patients compared to controls . MoreoveWNT inactivation and biallelic APC loss), and then MMR deficiency occurs, prompting the transition to CRC. As evidence suggests, MMR deficiency in LS patients develops in adenomas that are larger than 8 mm [APC mutations, which implies that MMR deficiency can occur during adenoma formation. Kloor and colleagues [The statistically significant differences in the oral and fecal microbiota of LS individuals, compared to normal healthy controls, would add to our understanding of the disease. Currently, three alternative models describe the development of MMR deficiency. One model hypothesizes that, initially, an adenoma forms through the usual mechanisms (han 8 mm . Howeverhan 8 mm  found MMlleagues  detectedlleagues  or occurlleagues . The curSuch conflicting evidence could blend in a unitary theory. LS-associated neoplasms have a peculiar local inflammatory response  with abuThis study is the first on oral microbiota in patients with Lynch syndrome, and this is one major strength. On the other hand, the small sample size of LS patients and the female prevalence in both patient and control groups are limitations of the study.This study describes a microbial signature associated with LS, characterized by a statistically significant increase of Bacteroidetes and Proteobacteria and a significant decrease of Firmicutes at the phylum level and of Ruminococcaceae at the family level in LS fecal samples, similar to that observed in sporadic CRC. These results suggest a possible role of proinflammatory bacteria in tumor development in patients with a genetic predisposition. Indeed, an increase of fecal Proteobacteria is able to enhance the permeability of the sterile inner layer of the intestinal mucus, resulting in bacterial inflammation close to the epithelium . Moreove


Gene expression, Western blot, and immunofluorescence analysis of MMP2, MMP3, and MMP10 and their tissue inhibitors TIMP1 and TIMP2 demonstrated that these enzymes are finely tuned in these tissues. In OMA lesions, all the investigated MMPs and their inhibitors were significantly increased, while DIE expressed high levels of MMP3. Finally, in vitro TNFα treatment induced a significant upregulation of MMP3, MMP10, and TIMP2 in both healthy and eutopic endometrial stromal cells. This study, shedding light on MMP and TIMP expression in endometriosis, confirms that these molecules are altered both in eutopic endometrium and endometriotic lesions. Although further studies are needed, these data may help in understanding the molecular mechanisms involved in the extracellular matrix remodeling, a crucial process for the endometrial physiology.Endometriosis is a condition defined as presence of endometrium outside of the uterine cavity. These endometrial cells are able to attach and invade the peritoneum or ovary, thus forming respectively the deep infiltrating endometriosis (DIE) and the ovarian endometrioma (OMA), the ectopic lesions feature of this pathology. Endometriotic cells display high invasiveness and share some features of malignancy with cancer cells. Indeed, the tissue remodeling underlining lesion formation is achieved by matrix metalloproteinases (MMPs) and their inhibitors. Therefore, these molecules are believed to play a key role in development and pathogenesis of endometriosis. This study investigated the molecular profile of metalloproteinases and their inhibitors in healthy ( Endometriosis is a hormone-dependent pathology that affects up to 5–10% reproductive-aged women, and it is characterized by the presence of endometrial tissue in extrauterine locations, primarily at the pelvic level  and more rarely in other organs and tissues outside the pelvis  . Indeed,Several authors demonstrate an increased level of TNFα in peripheral fluid and endometrium of women with endometriosis and detected a positive correlation between TNFα concentrations and endometriotic lesion size ,9. PeritAbout the endometriosis origin, several theories were formulated but still none seem to be exhaustive . The mosMatrix metalloproteases are a family of zinc-dependent endopeptidases able to degrade the molecules of extracellular matrix (ECM) and basal membrane, and therefore, they are involved in physiological processes that include ECM remodeling, an integral part of tissue growth and differentiation . They arTheir activity and functions are regulating by tissue inhibitors of metalloprotease (TIMPs), and it is of paramount importance to maintain the dynamic equilibrium established between the expression of the MMPs and their inhibitors so that the remodeling of the extracellular matrix occurs correctly. Modifications of this balance are related to pathological conditions . TherefoTaking into account these observations, the present study assessed the expression of MMP2, MMP3, MMP10, TIMP1, and TIMP2 by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blotting in healthy (HE) and eutopic (EE) endometrium, ovarian endometrioma (OMA), and deep infiltrating endometriosis (DIE) lesions. Furthermore, in vitro expression of selected MMPs and TIMPs in primary human endometrial stromal cells (HESCs) from healthy and eutopic endometrium, after TNFα exposure, was investigated.MMP2, MMP3, MMP10, TIMP1, and TIMP2 expression was measured in healthy and eutopic endometrium, in OMA and DIE lesions. To this end, endometrial biopsies were collected from patients who underwent surgery for not endometriotic ovarian cysts  and from women affected by endometriosis undergoing laparoscopic surgery . At the same time, ovarian endometrioma  and deep infiltrating endometriosis  lesions were collected.In order to clarify the role of metalloproteases and their inhibitors in endometriosis development, PPIP–TBP–HPRT1–GAPDH–B2M–ACTB. According to these results, an accurate normalization factor for qRT-PCR data could be calculated by using the three most stably expressed genes, TBP, HPRT1, and ACTB.To perform gene expression analysis, the best reference gene for quantitative expression studies was validated by using geNorm software. This VBA applet ranked the candidate housekeeping according to their stability, from the most to the least stable: MMP2 are significantly increased in OMA (p < 0.001) when compared to the eutopic and/or healthy endometrium. MMP2 expression in DIE lesions seems to be more variable, and for this reason, the highlighted increase does not reach the statistical significance when compared to the eutopic endometrium, whereas its expression appears significantly reduced when compared to OMA (p < 0.01). As shown in MMP3 is significantly increased in OMA if compared to healthy and eutopic endometrium (p < 0.01).As shown in p < 0.01). Finally, our results demonstrated that tissue inhibitors of metalloproteases are overexpressed in OMA; in fact, mRNA levels of both TIMP1 and TIMP2 are significantly increased in OMA when compared to endometrium  or to DIE (p < 0.001) , as well as MMP3 and TIMPs  were significantly correlated.Based on this evidence, we further analyzed the correlation between the expression of In order to confirm the data obtained by gene expression analysis, we analyzed protein extracts prepared from endometrium and from endometriotic lesions by Western blot; the overall relative intensity of bands was normalized with respect to β-Actin.As shown in p < 0.01). Western blot analysis confirms the higher abundance of MMP10 in endometriotic lesions already shown by gene expression analysis  and EE-HESCs (p < 0.05)  , but also when compared to HE-HESCs treated with TNFα at the same concentration (p < 0.05)  B. Interectively) C. When t < 0.05) .This study reports, for the first time, a comprehensive analysis of the overall expression of MMPs and their inhibitors in eutopic endometrium and in endometriotic lesions, in vivo as well as in vitro after treatment inducing an inflammatory response.The main strength of the present study relies on robust inclusion criteria that are crucial for the accurate and conclusive analysis of results and thus allow a deeper understanding of MMPs and TIMPs role in endometriosis etiology. Indeed, the results of this study are normalized for uterine cycle phase, since all tissue biopsies are scheduled in proliferative phase, 2–3 days after the menstrual bleeding, thus overcoming criticisms evident in several published studies, where no attention was paid to the phase of the menstrual cycle during sample collection, which makes the results quite unreliable and difficult to analyze . This crMoreover, another strength of this study was the identification of the best housekeeping genes to be used for gene expression analysis in endometrium and in endometriotic lesions. To the best of our knowledge, no data in the literature are available on the validation of suitable housekeeping genes for normalization in endometriotic tissues. These tissues display remarkable morphological and molecular differentiation; therefore, a proper normalization is strongly required in order to guarantee a precise and correct gene expression analysis.In light of these information, here we demonstrate that different MMPs are expressed at different levels in healthy and pathological tissues, in accordance with data reported by most of the studies focused on this disease ,25,27.MMP2 gene expression detected in eutopic endometrium, the corresponding protein seems to be upregulated. This may be due to post-transcriptional modifications or increased protein accumulation, as also suggested by several studies demonstrating that endometriotic tissues display impaired mechanisms of post-transcriptional regulation [The role of MMP2 in endometriosis is still matter of debate: the available data on its expression in uterine endometrial tissue in women with and without endometriosis remains contradictory . This stgulation ,29. Moregulation . In thisAnother interesting finding of the present work is the proven increase in MMP2 expression in OMA, that confirms data reporting higher activity of MMP2 in ectopic lesions in comparison to uterine endometrium ,30,31. AAs regard to stromelysins MMP3 and MMP10, their expressions significantly increased in eutopic endometrium and in endometriotic tissues, in agreement with data reported in the literature, suggesting that, like in cancer cells, stromelysin’s upregulation might contribute to the high invasive potential of endometriotic cells ,33. In pAccording to their spectrum of activity, MMPs modulation has to be carefully controlled to prevent deleterious side effects; therefore, the importance of the regulation of their inhibitors TIMP1 and TIMP2 in reproductive tissue is evident, delineating the key role of this molecule in extracellular matrix homeostasis, the physiologic process maintaining the appropriate balance between deposition and degradation of extracellular matrix components. In this respect, this work demonstrated an increase in TIMP1 and TIMP2 expression levels in OMA. These results led us to hypothesize that an increased expression of TIMPs in these pathological tissues may be correlated with the higher expression of MMP2 and MMP3 in the same lesions as confirmed by the significant correlation demonstrated by this study. In fact, other published studies demonstrated a fine balance between the expression of MMPs and TIMPs in the processes of growth, differentiation, and destruction of tissues ,35.MMP10 and TIMP2 in both healthy and eutopic HESCs. These data are relevant, since it is known that this pro-inflammatory cytokine has been implicated in the pathophysiology of endometriosis [Finally, aiming to shed light on the mechanisms underlying MMP derangement in endometriotic tissues, this work underlined that TNFα treatment is able to induce a significant upregulation of etriosis . TNFα exetriosis , and itsetriosis , while aetriosis ,40.Indeed, altered MMP expression is crucial for the physiology of the reproductive system, where these molecules play a crucial role in ECM remodeling during embryo implantation based on fine-tuning between MMPs activation and inhibition . ImpairmMoreover, TNF-α may also play a role in the pathogenesis of endometriosis, as it has been shown that the adhesion of endometrial fibroblast to mesothelial cells is increased in vitro and specific polymorphisms in the TNFα gene are related to the predisposition to endometriosis .It is well known that many MMPs and TIMPs are regulated at the transcriptional level by a variety of growth factors, cytokines, and chemokines ,44, and n = 10) and deep infiltrating endometriosis  lesions were collected from the 19 patients suffering from endometriosis.For this prospective study, endometrial and endometriotic tissue samples were collected at the Obstetrics and Gynecology clinic of University Hospital of Siena, from January 2018 to October 2019. The study cohort consisted of 34 women including (i) 15 healthy women (age range between 22 and 38 years old) who underwent surgery for not endometriotic ovarian cysts and (ii) 19 endometriotic patients (age range between 21 and 40 years old) who underwent laparoscopic surgery for endometriosis. At the time of surgery, endometrial biopsy was collected from 15 healthy women and from 19 endometriotic patients; at the same time, ovarian endometrioma  (cyst diameter ranging between 42 and 68 mm) were carefully stripped from the inner cyst wall and the lack of contamination with ovarian tissues was confirmed by histological evaluation. The DIE lesions (n = 9) were located in the posterior compartment . Tissues were immediately submerged in liquid nitrogen to allow subsequent RNA and protein extraction, paraffin embedded for immunohistochemistry or immediately treated to set up primary cell cultures of HESC.A complete medical history, physical examination, and transvaginal ultrasound evaluation, carried out by the same examiner with experience in transvaginal ultrasound scans for endometriosis, was performed for each patient. According to the American Society of Reproductive Medicine (ASRM) classification (1985), all endometriosis patients enrolled in this study had stage IV of disease; this stage was histologically confirmed by pathologists. Exclusion criteria were: prior or current infections, endocrine disorders or hormonal treatment within the past 3 months and the concomitant presence of OMA and DIE lesions. All the specimens were collected during laparoscopic approach in proliferative menstrual phase, 2–3 days after the menstrual bleeding. Endometriotic specimens from OMA . All patients provided their informed consent before being enrolled in the study.Frozen samples were homogenized with TissueLyzer  and RNA was extracted by using the RNeasy Mini kit  according to the manufacturer’s instructions. RNA concentration was assessed using an ND-1000 Nanodrop Spectrometer , and its integrity was evaluated by an electrophoretic run on agarose gel with FlashGel System . 300ng of extracted RNA were reverse transcribed into cDNA using the iScript Reverse Transcription kit .MMP2, MMP3, MMP10, TIMP1, and TIMP2 were determined in triplicate by qRT-PCR on a CFX connect Real-Time PCR Detection System  using SsoFast EvaGreen Supermix . The cycling conditions were as follows: 5 min at 95 °C, 30 s at 96 °C, 1 min at 58 °C, 5 min at 4 °C, and 5 min at 90 °C.Gene-specific primer sets used in this study  are listed in +++ buffer for Sodium Dodecil Sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Pooled samples were obtained by mixing equal protein amounts  of 5 individual samples that were extracted in the same lysis buffer. The protein concentration in pooled samples was re-estimated using the appropriate quantification protocol. About 25 ng per sample were run on 12% SDS-PAGE for electrophoresis separation and then transferred to nitrocellulose membranes in a mini Trans-Blot apparatus . Membranes were subsequently blocked with 3% w/v non-fat dry milk in 10 mMTris–HCl (pH: 7.5), 0.15 M NaCl, for 1 h at room temperature and then probed overnight at 4 °C with primary polyclonal antibody as reported in Endometrial and endometriotic tissues were suspended in RIPAHuman endometrial stromal cells (HESCs) were isolated from healthy and eutopic endometrial biopsies immediately after collection. Tissue samples were minced into small pieces and incubated for about 2 h at 37 °C in a shaking water bath, in 10 mL of DMEM/F12 (Invitrogen) supplemented with 0.1% collagenase and DNase (0.01 mg/mL). Stromal cells were then separated by filtration with filters of 70 μm pore diameter and subsequently of 40 μm, and then they were centrifuged for 8 min at 1200 rcf and finally suspended in complete DMEM/F12 medium supplemented with 10% charcoal-stripped calf serum (Sigma-Aldrich). The lower passage number (P0–P4) of cells was used for experiments to avoid changes in phenotype and gene expression. The medium was changed to a serum-free DMEM/F12, 24 h before each treatment, performed in duplicate. To determine the effect of TNFα on gene expression, the cells were stimulated for 48 h with recombinant human TNFα  at a final concentration of 20µM or with free medium alone (control condition). Cells were collected with 350 µL RLT and stored at −80 °C until RNA extraction.For immunofluorescence analysis, cells were grown on coverslips, washed with phosphate-buffered saline solution (PBS), and fixed for 15 min in 4% paraformaldehyde and then permeabilized in 0.5% Triton X-100 in PBS for 10 min. Coverslip slides were immunolabeled using an indirect procedure. After blocking in 5% BSA in PBS for 30 min, cells were incubated 1 h at 25 °C with the primary antibodies see , washed p < 0.05. Correlation was determined by using Spearman’s correlation analysis.Statistical analysis was performed using the GraphPad Prism 5.0 . Statistical significance was evaluated by using nonparametric tests. Differences among groups of data were tested by the Kruskal–Wallis test or the Mann–Whitney test. Statistical significance was set at The systematic methodological approach set up in this study allows to overcome the conflicting results still present in the literature, thus clarifying the pivotal role of MMPs and their inhibitors in this pathology. Indeed, although further studies are needed to fully understand the molecular mechanisms involved in these processes, the present data confirm that investigated MMPs and their inhibitors are altered both in eutopic endometrium and endometriotic lesions. These findings demonstrate that the deregulation of ECM remodeling is a crucial process for the endometrial physiology, and it may lead to an increase in the ability of endometrial cells to implant and to originate an invasive growth of the endometrial tissue. We would like to disclose that, at this time, the data reported in this study does not allow the use of endometrial expression of MMPs and their inhibitors expression in the diagnostic process of endometriosis. Indeed, further research on the role of MMPs and TIMPs in the onset and development of the endometriosis in a larger study groups would be required before clinical application. Based on our preliminary data, we may hypothesize that the timely detection and the fine-tuning of endometrial MMPs activity should be effective in endometriosis diagnosis and management, thus providing new targets for the clinical treatment of endometriosis.


The clinical behaviour and outcome of young patients with gastroesophageal tumours (GET) is surmised to differ from older patients, yet data on the comparison of these two patient subgroups is scarce. This study focuses on the investigation of the clinical characteristics and survival outcome of younger-age people with GET, when compared to older patients.Patients diagnosed with GET at the Medical University of Vienna between 2004 and 2016 were included in this study. Clinical parameters and the overall survival (OS) were compared between young (≤ 45 years) and elderly (≥ 65 years) patients.p < 0.05). In a non-metastatic setting younger patients showed a significant longer OS than older patients . Furthermore, young patients with extensive metastatic disease (2 or more metastatic site) had a significantly poorer OS than elderly patients .Among 796 patients, who were eligible for this analysis, fifty-eight patients (7%) were ≤ 45 years at the initial onset of the disease. These 58 young patients were then matched to elderly patients based on the gender, tumour stage, histology and tumour location. The number of metastatic lesions was significantly higher among young patients (These results indicate that young patients might be diagnosed very late, which might lead to the development of a more aggressive disease compared to older patients, but a relatively long OS when diagnosed and treated in a non-metastatic setting. Thus, screening methods for younger patients might be considerable to enhance the outcome of young patients with GET. Gastroesophageal tumours (GET), including stomach, gastroesophageal junction and oesophagus, is the fourth most common cancer, and the second leading cause of cancer-related deaths worldwide  and adenocarcinomas were included. Upper gastrointestinal cancer was defined as cancer located in the oesophagus, gastrointestinal junction and stomach.All patients had a pathologically confirmed tumour specimen, either from biopsy or from surgical resection, reviewed by an experienced pathologist. Following clinical data were routinely collected and obtained from the patient database of the General Hospital Vienna, Austria: nicotine intake status, histopathological data of the tumour specimen including Her2 status, grading, staging, tumour location, metastatic status, treatment of the tumour , laboratory findings of circulating tumour marker (carcinoembryonic antigen [CEA] and CA19-9 [carbohydrate antigen 19-9]) recurrence and survival outcomes. Men and women older than 18 years of age are included.Young age was defined as ≤ 45, whereas old age was identified to be ≥ 65 years. Young and old patients were matched manually by exploring the collected data in a Microsoft Excel list. No specific software was used for the matching process. The patients were matchedbased on gender, location of the tumour , histology (adenocarcinoma/squamous cell carcinoma) and metastatic status at the initial presentation of the tumour. If a single young patient had potentially more than one old matching partner, the index patient was matched with an old patient having a similar survival time. This study was approved by the ethics committee of the Medical University of Vienna under the reference number 2267/2016.Patients who were administered an anti-tumour treatment received a computed tomography every 3 months within the first year of the diagnosis. The amount of tumour shrinkage was investigated based on computed tomography images and the tumour response was classified in accordance with RECIST  and fluorescence in-situ hybridization . For FISH, HER2 gene copy number and centromere enumerator probe 17 (Cep17) were investigated. The pathologists reported average copy numbers of Her2 and Cep17. The diagnosis criteria were based on Hofmann and colleagues  were censored at the date that they were last known to be alive. Overall survival (OS) was calculated from the date of initial diagnosis of gastric cancer to the death of patient or patient’s last follow-up date. Kaplan–Meier survival estimates with log rank test and Cox regression analyses of OS were done. All reported values are two sided and p = 0.4). Gender distribution had a tendency of having more female patients among the young group, which again was not statistically significant . Younger patients had significantly less proportion of oesophageal carcinoma as location of the tumour when compared to other patients . Most probably as a consequence of having fewer oesophageal cancer patients, younger patients had a slightly lower proportion of squamous cell carcinoma .In our institution, 885 patients were diagnosed with a GET between 2004 and 2016. 89 patients were presented at the outpatient clinic only once, where only sparse information of the disease course was available. Among 796 patients with sufficient hospital data, 58 (7%) were diagnosed with a GET before the age of 45. Among the young patients, the percentage of cases with metastatic disease at the initial disease presentation (stage IV) was slightly higher than the entire cohort, which however did not reach the statistical significance (25 patients (43%) in young group versus 274 patients (37%) within the rest; p = 0.59), respectively.The 58 patients, whose age was younger than 45, were matched with GET patients older than 65 based on the gender, tumour location, histology and metastatic status at the initial onset of the disease. Baseline characteristics of the patient population are described in Table n = 36, 62%) compared to older patients  (p = 0.48). Among the young patients’ group, 9 (16%) and 16 (27%) patients presented with one and two metastatic sites at the initial presentation, respectively, whereas this was 17 (30%) and 8 (14%) among the older group (p = 0.02). Interestingly, young patients developed statistically more lymph node and peritoneum metastases, whereas older patients had a tendency to generate metastasis to the liver (p = 0.03). Helicobacter pylori and human epithelial growth factor receptor 2 (Her2) findings were similar in both patient groups.The cases with the histology of adenocarcinoma and SCC were identical in both groups with 52 (90%) and 6 (10%) patients, respectively. In both groups, 25 patients (43%) had metastatic disease already at the initial presentation of the cancer. Younger patients had a slightly higher proportion of poorly differentiated tumours  in the young group and 523 days  in the old group.p = 0.5) Due to the retrospective nature of this study, not all side effects of the chemotherapy could be obtained from the hospital chart data. Based on the available data of the palliative chemotherapy setting, the most observed side effects were nausea, blood count changes and diarrhoea. The distribution of the side effects between young and old patients with upper GI-carcinoma was almost identical.Type of treatment and the type of chemotherapy regimen did not differ in both groups Table . The medp = 0.139; HR 1.367, 95% CI 0.902–2.072; Fig. p = 0.028; HR 1.954, 95% CI 1.065–3.584; Fig. p = 0.931; HR 0.975, 95% CI 0.542–1.752; Fig. The overall survival (OS) did not differ statistical significantly in both groups when all patients of the young and old groups are included , but not between adenocarcinomas (p = 0.484).Furthermore, there was a statistically significant difference between the overall survival of young and old patients with squamous cell carcinomas , tumour location , Helicobacter pylori infection , nicotine , grading , stage  and Her 2 status .There was no statistically significant difference between young and old patients when separated by gender . 3- and 5-year survival was also similar in both groups with 20 (35%) and 10 (17%) and 18 (31%) and 8 (14%) patients, respectively .50 (86%) and 48 (82%) patients survived more than one year in young and old patients’ groups, respectively  and Her 2 status  were associated statistical significantly with the OS, whereas age , gender , tumour location , histology , grading , stage , helicobacter pylori infection  as well as nicotine  were not.Within the older cohort only initial metastatic disease , initial metastatic disease , grading  and stage  were also associated statistical significantly with the OS, whereas the parameters age , gender , tumour location , Helicobacter pylori infection , Her 2 status  and nicotine  were not.Within the young cohort the parameters histology . Interestingly, although not fulfilling the significance criteria, older patients tended to show increasing tumour marker levels after anti-tumour therapy, whereas the concentration of the tumour markers were decreasing among younger patients. Among these patients, cox regression analysis did not reveal any association of pre- or post-treatment concentrations of CEA or CA19-9 with the survival.Serum concentrations of CEA and CA19-9 were available in 39 patients before initiation of any kind of anti-tumour treatment and 6 months after the first established treatment. Concentrations of CEA and CA19-9 did not significantly change after initiation of the therapy in both groups  who are treated at the Department of Oncology, Medical University of Vienna and compares demographic, clinical and pathological data as well as outcomes of older GET patients who are treated at the same clinic. The definition of “young” patients varies among literature, where different cut-off values have been identified Chaytors . HoweverThe portion of young patients was reported to be between 2 and 15% among all GET patients  patients concerning Her2 status, in 47 (26 young) patients concerning Helicobacter pylori status, in 13 (7 young) patients concerning grading and in 38 (17 young) patients concerning nicotine consumption.Concerning the OS, 91 of the patients  were already dead at the time of this analysis. The other patients were either still alive or lost to follow-up.Although 796 patients with sufficient hospital data compose a large European cohort with upper gastrointestinal cancer, the matched cases were only 58 patients per group. This rather small sample size has to be considered when interpreting the obtained data.Thus, to confirm the results of this study, a prospective study should be conducted to minimize missing data points.In conclusion, this retrospective survey demonstrates clinical characteristics of young patients with GET and describes a tendency of relative unfavourable outcomes in a metastatic setting and favourable outcomes in a non-metastatic setting for these patients when compared to older ones. Older patients within this group tolerated the anti-tumour treatment regimen in the same way as the younger patients and had comparable outcomes, which makes this group of patients potential candidates for the full programme of the anti-tumour treatments in metastatic settings. Furthermore, these retrospective findings again underline the importance of the early diagnosis of gastroesophageal cancer in young patients to possibly find more favourable disease conditions which might have an impact on the outcome.


Breastfeeding is related to maternal health. However, the association of women’s breastfeeding duration with cognitive function in their later life is limited and inconsistent. The aim of this study was to accurately evaluate the association in Chinese postmenopausal women. We analyzed the data from Zhejiang Ageing and Health Cohort Study including 5487 postmenopausal women. Cognitive impairment was assessed via the Mini-Mental State Examination. Data on breastfeeding duration was collected in the reproductive history section within the questionnaire. Generalized additive models (GAMs) and logistic regression models, controlled for an extensive range of potential confounders, were generated to examine the associations. A U-shaped association was identified between breastfeeding duration and cognitive impairment based on GAM. The nadir with lowest odds of cognitive impairment was ascertained by quadratic model as 12 months. The logistic models showed that compared with women breastfeeding 12 months per child, the fully adjusted odds ratios (ORs) were 1.50 : 1.20–1.88), 1.58 (95% CI: 1.29–1.93), 1.33 (95% CI: 1.06–1.68), 2.08 (95% CI: 1.64–2.65) for those averagely breastfeeding <6, 6-<12,>12–18,>18 months, respectively. Furthermore, we did not observe significant effect modification of the association. Future longitudinal studies are needed to confirm the association. The prevalence of dementia and AD in Chinese population aged 65 years and older were 5.14% and 3.21% respectively2, and the situation of cognitive impairment would be more serious.Cognitive impairment, a common symptom of dementia and its most common cause Alzheimer’s disease (AD), is becoming an important health issue in the elderly. Based on estimation, more than 35.6 million people lived with dementia worldwide in 2010; this number is likely to be doubled by 2030, and tripled by 20503. It leads to various hormonal changes, and consequently affects the health of mothers. Several systematic reviews and meta-analyses demonstrated that breastfeeding reduced the risk of diseases in mothers, including breast cancer4, ovarian cancer5, type 2 diabetes mellitus6, hypertension7, etcetera. These studies provided evidence that breastfeeding had the capacity to influence disease risk by altering hormone profiles. Meanwhile, accumulating evidence showed that variation of sex hormones exposure in reproductive period may modify the risk of cognitive impairment and AD in women’s later life9. However, the epidemiological data on association between breastfeeding duration and risk of cognitive impairment or AD is limited and inconsistent. To our best knowledge, only two previous studies focused on this topic. Heys and colleagues10 reported that shorter average duration of breastfeeding per child was associated with better cognitive function. On the contrary, another study11 found that longer breastfeeding duration corresponded to lower AD risk. Both of the two studies tested the linear association regardless the possibility of curvilinear relationship, which may partially explain the inconsistency. Moreover, World Health Organization (WHO) recommends exclusive breastfeeding up to 6 months of age with continued breastfeeding up to 2 years or beyond12, which is not a short period. For maternal cognitive function, whether an appropriate or optimal duration of breastfeeding exists is worth further study.Breastfeeding is recognized as the normal method for feeding infants worldwideThus in this study, we utilized the cross-sectional data from a large population-based cohort study to investigate the linear or curvilinear association of breastfeeding duration with risk of cognitive impairment among Chinese postmenopausal women, and the possibility of optimal breastfeeding duration for cognitive function.By using participants selection criteria mentioned above, we initially identified 5635 postmenopausal women with at least one parity from an accumulation of 10801 participants. After checking for data completeness, we excluded women with missing data on cognitive impairment (n = 17) and covariates (n = 131). The final total of women included in the analysis was 5487. The general characteristics of included women are presented in Table Among the 5487 participants, 1053 were indicated with cognitive impairment by MMSE, and the prevalence of cognitive impairment was 19.2% (95% CI 18.2~20.2%). The mean MMSE score of participants was 22.8, and detail information on MMSE scores is presented in Table Descriptive statistics for reproductive characteristics of included women are shown in Table The results of GAMs and quadratic models suggested a U-shaped association between mean breastfeeding duration and risk of cognitive impairment. Adjusted ORs for cognitive impairment using 12 months of mean breastfeeding duration as the reference demonstrated higher odds with longer as well as shorter mean breastfeeding duration -promoting activity20, while REMS was associated with better cognitive function22. Consequently, prolactin release during lactation may lead to a benefit on cognition. In summary, the secretion of oxytocin and prolactin during breastfeeding may have beneficial effect on cognitive function, and estrogen deprivation contributes to detrimental effects, while progesterone presented beneficial or detrimental effects. The variation of different hormones exposure with their potential interactions may result in the non-linear relationship found in this study. Further research is warranted to elucidate the mechanism of breastfeeding’s effect on cognitive function.It remains unclear about the mechanisms responsible for the effects of breastfeeding duration on cognitive function. The endocrine change of lactation is complex. Longer breastfeeding duration usually suppresses ovulation longer and results in suppression of gonadotropins, which causes depressed production of plasma estradiol23. Moreover, the association of breastfeeding duration with cognitive impairment might be affected if the error rate of recalling and summing over the breastfeeding duration of each child was higher among women with more severe forms of cognitive impairment. Second, considering the cross-sectional design, the reverse causation cannot be excluded on the occasion that women’s onset age of cognitive impairment was earlier than that of breastfeeding children. This situation may rarely occur because age is a major risk factor of cognitive impairment, which is not prevalent before the age of 65. Prospective studies are needed to ascertain the temporal relationship. Third, pregnancy interval, which was not concerned in the questionnaire, is also likely to influence the breastfeeding duration, with shorter interval between pregnancies resulting in shorter breastfeeding duration with the arrival of another child. Forth, we did not take genetic factors into consideration, such as apolipoprotein E (APOE)24 carrier status. Fifth, some factors which might probably affect the cognitive function were not measured in this study. Psychiatric history (e.g. schizophrenia) as well as other current psychiatric status (e.g. anxiety) may also result in cognitive decline. Meanwhile, we could not exclude the probability that cognitive impairment was caused by tumor, endocrine disorders, hormonal dysfunction, vitamin deficiency, vascular lesions, previous and anamnestic neurological disorders, etc. Participants did not undergo dementia protocol such as thyroid function, neurological tests and neuroimaging. Additionally, many of medication could have a negative effect on the cognitive performance. Finally, the results of our study should be interpreted cautiously. Although the lowest odds of cognitive impairment was found at 12 months of average breastfeeding, our finding does not mean 10 or 14 months of average breastfeeding is harmful, yet 12 months is protective. On one hand, 1 or 2 months error margin often exists when women recall breastfeeding duration, which was mentioned before. On the other hand, our results do support the protective effect of breastfeeding on maternal cognitive function, while appropriate duration was preferable (neither too short nor too long).Several methodological issues and limitations should be mentioned. First, the self-report of retrospective information on breastfeeding history may cause recall bias. One study found that under- and over-reporting occurred with equal frequency, and 55% of women were able to recall mean breastfeeding duration accurately within 1 month and 71% within 2 monthsDespite these limitations, this study has a number of strengths. (1) With the application of GAMs and quadratic models which can more naturally describe the shape of curvilinear relationship, our study have originally identified the U-shaped association between breastfeeding duration and cognitive impairment. (2) Our study was based on a large community-based sample, which guaranteed the power of statistics. (3) A wide range of covariates including other aspects of reproductive characteristics were adjusted in multivariable analysis to minimize the potential confounding bias.In conclusion, this study suggested a U-shaped association between breastfeeding duration and cognitive function among Chinese elderly women. Our findings are important, and have several implications for practice and cognitive impairment prevention. It supports the beneficial effects of breastfeeding for maternal health. Moreover, our results help mothers to decide the duration of breastfeeding children with regard to their cognitive function in later life. Future longitudinal studies are needed to confirm the association.25, a community based cohort study focusing on ageing and health among elderly in Zhejiang province, China. It was conducted by Zhejiang Provincial Center for Disease Control and Prevention. Seven counties were randomly selected from a total of 90 counties in Zhejiang province. One town in each county and several communities in each town were then randomly selected. All permanent residents aged ≥60 years old in these selected communities were expected to be included in the study. The baseline survey was finished at 2015, and 10911 elderly were recruited and completed the survey. One year later, the second round survey was finished in which 10801 elderly completed the survey .The data is from Zhejiang Ageing and Health Cohort StudyA face to face interview based on a self-designed questionnaire was performed by trained research assistants for each participant both at baseline survey and second round survey. The questionnaire included the information of demographic characteristics, family status, reproductive history, medical disease, behavioral habits, diet habits, injury, depressive symptoms, self-care ability and cognitive function. Data were primarily checked by staff at Zhejiang Provincial Center for Disease Control and Prevention. Missing data and logical errors were fed back to the initial interviewer who would try to complete the dataset by reinvestigating the participants. In the questionnaire of second round survey, questions about breastfeeding duration were added into the reproductive history section, which were not involved in the questionnaire of baseline survey. Therefore, this study utilized the dataset of second round survey (n = 10801).The study population was restricted to the postmenopausal women with at least one parity, which was selected from the dataset mentioned above. Female participants who answered “yes” to the question “Do you have menopause already?” and not “0” to the question “How many parity do you have?” were recognized as eligible participants. Data completeness was checked subsequently, and participants were excluded if they had any missing values on cognitive function and each reproductive factors.26 within the questionnaire. The maximum score is 30 points, with higher MMSE scores indicating better cognitive function. The widely accepted cut-off score of cognitive impairment in China is education-specific27: 17/18 for illiteracy, 20/21 for people with primary education level, 24/25 for people with higher than primary education level. We defined cognitive impairment according to this standard.Cognitive function was assessed using the Chinese version of the Mini-Mental State Examination (MMSE)Data on breastfeeding duration was collected in the reproductive history section within the questionnaire. Our analysis focused on the mean breastfeeding duration, the ratio between total months spent on breastfeeding and number of full-term pregnancies. Total months spent on breastfeeding was determined from the response to the question “For how long do you breastfeed your children totally?” Number of full-term pregnancies was obtained from the response to the question “How many full-term pregnancies (live births) do you have?” In our analysis, mean breastfeeding duration were considered as both continuous variable and categorical variable. The method of categorizing will be described in ‘statistical analyses’ section.2, continuous variable), physical activity (“yes” and “no”), hypertension (“presence” and “absence”), diabetes (“presence” and “absence”), coronary heart disease (“presence” and “absence”), Activities of Daily Living Scale scores (continuous variable), Patient Health Questionnaire-9 scale (continuous variable). Moreover, following reproductive characteristics which might also affect the cognitive function were included for further adjustment: reproductive period , menstrual cycle regularity (“regular” and “irregular”), age at first birth , parity (continuous variable), incomplete pregnancies (continuous variable), and oral contraceptive use (“ever” and “never”). Detailed information on some of these variables were described as follows.Based on findings reported in the literature, general characteristics described below were considered as potential confounders and were included in our analysis: age , race (“Han ethnicity” and “minority”), education level , marital status , family income , smoking , alcohol drinking , body mass index (kg/m2). (4) The self-care ability was evaluated by the Barthel index of Activities of Daily Living Scale (ADL)28. It consists of self-feeding, bathing, dressing, toilet hygiene and functional mobility, with the total score of 100. (5) Depressive symptoms was evaluated using the Patient Health Questionnaire-9 scale (PHQ-9)29, nine-question version of the Primary Care Evaluation of Mental Disorders measured by self-reporting. Total score for the nine items ranges from 0 to 27, with greater values indicating increased severity. (6) Reproductive period, an indicator of endogenous estrogen exposure, was calculated as age at menopause minus age at menarche.(1) Physical activity was assessed on the basis of the response (yes or no) to a single question about doing exercise regularly. (2) Medical disease section of the questionnaire contained the items on the presence or absence of 16 common diseases, which supposed to be formally diagnosed. Hypertension, diabetes and coronary heart disease (CHD) were considered in this study. (3) Body mass index (BMI) was calculated by dividing weight (kg) by the square of height  were considered as potential confounders.30 was used for dependent variable (mean breastfeeding duration), and link function was Logit due to the binary outcome. Three knots were set at the 5th, 50th and 95th percentile31 according to the distributions of mean breastfeeding duration. As GAM analysis suggested a U-shaped association, quadratic regression was performed to determine the nadir where the risk is lowest: Pr (Cognitive impairment | mean breastfeeding duration) = exp [−α + β1 (mean breastfeeding duration) + β2 (mean breastfeeding duration)2], with parameters α, β1, and β2 estimated using maximum likelihood methods. If β1 and β2 are both nonzero and statistically significant, then the estimated level of mean breastfeeding duration at which risk of cognitive impairment is lowest equals −(β1/2β2). The value of nadir was used as reference for displaying the overall trend of ORs of cognitive impairment through the range of mean breastfeeding duration. Based on the quadratic regression, β1 and β2 were estimated as 0.049 and 0.002, respectively; both P values were less than 0.05. As a consequence, mean breastfeeding duration associated with lowest risk was identified as 12 months.Generalized Additive Models (GAMs) were performed to explore the linear or curvilinear association between mean breastfeeding duration as continuous variable and risk of cognitive impairment, adjusted for all potential confounders. Restricted cubic spline smoothing techniqueMeanwhile, this value (12 months) was used as a cut-off point for categorizing the mean breastfeeding duration. Individuals above and below the cut-off point were both stratified into 2 subgroups. Finally, individuals were divided into 5 groups: <6, 6-<12, 12, >12–18, >18 months. Then logistic regression models were performed to assess the risk of cognitive impairment with mean breastfeeding duration as categorical variable. Crude odds ratios (ORs), 95% confidence intervals (CIs) and corresponding P values of cognitive impairment associated with mean breastfeeding duration were calculated by binary logistic regression in model 1. Adjustment for general characteristics identified above was performed in model 2, then model 3 was generated by further including related reproductive characteristics.8, we suspected that BMI and other reproductive characteristics  might modify the effect of breastfeeding duration on risk of cognitive impairment. Menstrual cycle regularity and oral contraceptives use were not considered in this step due to the large difference of sample size between variable levels. The continuous variables among these factors were transformed into dichotomous variables for further stratification using the median as cut-off points. Subgroup analyses stratified on these factors were conducted for both continuous variable (in GAMs) and categorical variable (in logistic regression models) of mean breastfeeding duration. Furthermore, interactions between breastfeeding duration and these factors were checked through the addition of cross-product terms in the corresponding main-effects models. All statistical analyses were performed by SAS 9.3 , and the significance level was at two-tailed probability <0.05.Based on previous literatureThe protocol was approved by the Ethics Committee of Zhejiang Provincial Center for Disease Control and Prevention, and written informed consents were obtained from all participants prior to the research. All methods were performed in accordance with the relevant guidelines and regulations.Supplementary information.


Brachionus plicatilis for delivery of BMCs to corals of the species Pocillopora damicornis. Epifluorescence microscopy combined with a live/dead cell staining assay was used to evaluate the viability of the BMCs and monitor their in vivo uptake by the rotifers. The rotifers efficiently ingested BMCs, which accumulated in the digestive system and on the body surface after 10 min of interaction. Scanning electron microscopy confirmed the adherence of BMCs to the rotifer surfaces. BMC-enriched rotifers were actively ingested by P. damicornis corals, indicating that this is a promising technique for administering coral probiotics in situ. Studies to track the delivery of probiotics through carriers such as B. plicatilis, and the provision or establishment of beneficial traits in corals are the next proof-of-concept research priorities.The use of Beneficial Microorganisms for Corals (BMCs) to increase the resistance of corals to environmental stress has proven to be effective in laboratory trials. Because direct inoculation of BMCs in larger tanks or in the field can be challenging, a delivery mechanism is needed for efficient transmission of the BMC consortium. Packaged delivery mechanisms have been successfully used to transmit probiotics to other organisms, including humans, lobsters, and fish. Here, we tested a method for utilizing rotifers of the species  Coral reefs are increasingly impacted by global climate change, which raises the mean sea surface temperature (SST) and the incidence of marine heatwaves . Due to Selection and manipulation of specific members of the resident coral microbiome to mitigate the effects ofthermal stress on the animal health was proposed by Lactobacillus species (Artemia), rotifers, and copepods can be used as vectors for transmitting probiotic bacteria to larger animals in aquaculture, such as fish . Rotifers were cultured in natural seawater, at a temperature of 25°C, salinity of 3.2%, in the dark. Water circulation and oxygenation were produced by bubbling air with an air pump. Using standard culture conditions, rotifers were fed daily with 100 mL of a culture of the alga Tetraselmis gracilis containing 106 cells mL–1. Conversely, before starting the experiment, rotifers were transferred to 1 L containers with oxygenation produced by bubbling air with an air pump. The container was kept in the dark to avoid the growth of photosynthetic organisms. Rotifers were kept under starvation by culturing them in 0.22-μm daily filtered seawater, for a 7-day period.–2 s–1, and water recirculation by circulation pumps. Twenty percent of the water in the coral tank system was exchanged every 7 days. Corals were fed daily with 100 mL of a culture containing 200 rotifers mL–1 under standard culture conditions .Corals were kept in tanks containing 100L of seawater at 25°C, salinity of 3.2%, light intensity of 200 μmol mPseudoalteromonas spp., isolated from P. damicornis; plus one strain of Cobetia marina and one strain of Halomonas taeanensis, isolated from the artificial seawater surrounding P. damicornis. The accession numbers of each isolate and their classifications according to their 16S rRNA gene sequences are provided in The seven strains of bacteria used in the experiment were isolated by 7 cells mL–1. The cells were collected by centrifugation at 12,000 × g for 5 min at 4°C and washed twice under the same conditions with sterile saline (2.5% w/v NaCl) to remove the culture medium. All cells from each culture were homogenized together, resuspended in a final volume of 40 mL of saline solution (2.5% w/v NaCl), and stored at 4°C until used in the experiment, which occurred on the same day of the preparation of the cells.To prepare the BMC consortium assembled by Fluorescence and differential interference contrast (DIC) images were obtained with a Zeiss Axio Imager D2 microscope . Fluorescence filters used were: (i) Filter set 10 ; and (ii) Filter set 00 .TM Bacterial Viability Kit  was used to stain BMCs, following the manufacturer’s protocol. BMCs were washed in sterile seawater to remove excess stain prior to delivery to the rotifers. For BMC-rotifer interactions, the stained BMC homogenized consortium (100 μL of a suspension containing approximately 107cells) was inoculated into a 2-mL polypropylene tube containing 900 μL of seawater and a concentration of 70 rotifers mL–1. Promptly after inoculation (around 10 min), a 20-μL aliquot of BMC-rotifer suspension was mounted on a glass slide, alongside a control with unstained bacteria, and both preparations were observed by means of fluorescence microscopy.Based on initial trials, rotifers were starved for 7 days before use in experiments, to limit their autofluorescence by food ingestion. The viability of the rotifers after 7 days of starvation was determined by observing their mobility and cilia beating, using DIC microscopy. The LIVE/DEAD2 critical-point dried, metallized with gold, and observed in an EVO MA10 scanning electron microscope  with a voltage between 1 and 30 kV.The interactions between rotifers and BMCs were also observed using scanning electron microscopy (SEM). Images were produced from two periods: 4 and 16 h following incubation of the BMC consortium and the rotifer cultures. Starved rotifers without BMC inoculation were used as a control treatment, and performed in parallel with the rotifer-BMC interaction treatments. BEEM-modified capsules (pre-shaped polyethylene molds with hinged lids) with a 100-μm polyester mesh filter were used to initiate the sample treatment procedure for SEM. The rotifers were fixed in 2.5% glutaraldehyde and 0.1 M sodium cacodylate buffer for 1 h and then washed three times with 0.1 M sodium cacodylate buffer solution and sterile seawater. Samples were post-fixed using 1% osmium tetroxide for 1 h at room temperature and washed three times with 0.1 M sodium cacodylate buffer solution and sterile seawater. A series of dehydration washes were performed in ethanol  for 10 min each, with the final 100% ethanol step repeated three times. Samples were COP. damicornis was transferred to a 1-L aquarium containing 0.22 μm filtered natural seawater at 25°C, salinity of 3.2%, and a magnetic stirrer to circulate the water. To normalize the concentration of rotifers/mL, the culture was filtered using 100 μM, and reinoculated in 50mL of 0.22 μm filtered natural seawater. The concentration of rotifers was calculated by counting the number of rotifers in 3 × 1 mL replicates of water samples from the previously concentrated samples, using a stereomicroscope . Appropriate dilutions were performed to achieve the desired final concentration of 200 rotifers mL–1. Before added to the coral aquarium, 100 mL of the culture, containing ∼20,000 rotifers, was washed twice in 0.22-μm filtered seawater to clean the maximum of autofluorescence particles that could interfere with the results. Also, the 10-cm coral nubbin acclimatized in the aquarium for 20 min before the rotifers were inoculated. Video and still images to search for in vivo evidence of rotifer uptake by the corals were taken with a USB digital microscope .To monitor uptake of rotifers by corals, one nubbin of Brachionus plicatilis rotifers cultured under standard conditions  and fed the alga Tetraselmis gracilis displayed autofluorescence signals due to uptake of the algae, which contain photosynthetic pigments that are naturally fluorescent  can be visualized with under SEM and show no bacteria interacting with rotifers . ContrarP. damicornis. Using the digital microscope, polyps were seen capturing and ingesting the rotifers containing BMCs  contain enzymes involved in nitrogen fixation or sulfur cycling (The bacteria selected to be part of the BMC consortium consisted of five strains of scenario . Strainsic cells . For exa mixture . Here, ansortium . Many Psrganisms , includiolecules . The Pseeratures . All BMCeratures . Six of  cycling , metabol cycling .P. damicornis corals readily ingesting the rotifers, representing a promising directed-delivery system for bacteria that provide putative beneficial functions for corals. The rotifer itself also provides a nutritional benefit to stressed corals. For example, Acropora cervicornis increased its feeding rate of the rotifer B. plicatilis under high CO2 conditions, and this raised the coral’s total lipid content, which can be a proxy for coral health (Beneficial Microorganisms for Coral studies are targeted toward delivering microbial communities with traits that promote coral health. Delivery of these BMCs through prey items such as rotifers has proven to be possible, with l health . The nexP. damicornis. The rotifers freely ingested the BMC consortium, which accumulated in the digestive tract and on the surface. The rotifers were also captured and ingested by the coral polyps, demonstrating the efficacy of using rotifers as a BMC delivery system. These observations represent a step forward in the administration of coral probiotics, which has the potential to increase the persistence and resistance of corals. These findings are also important for probiotic delivery to a wide range of aquaculture species for which rotifers are used as live feed. Further studies to test this approach in the field and to determine whether BMCs delivered via rotifers are able to establish within the coral microbiome will advance our knowledge of the proper administration of beneficial microorganisms in situ.In summary, this study demonstrated that BMC-enriched rotifers can serve as direct vectors for delivering BMCs to the coral The original contributions presented in the study are included in the article/RP, DB, LH, FA, and JMA conceived and designed the study. JMA, RFV, GD, and RV maintained the coral and rotifer cultures. JMA, IT, and FA performed the microscopy experiments. RP, JMA, FA, HV, and AB drafted the manuscript. RP provided financial support. All authors were involved in critical revision.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.