McCune-Albright syndrome (MAS) is a rare disease defined by the triad of polyostotic fibrous dysplasia of bone, café-au-lait skin spots, and precocious puberty. No available treatment is effective in changing the course of fibrous dysplasia of bone, but symptomatic patients require therapeutic support to reduce bone pain and prevent fractures and deformities. We report the case of a 27-year-old woman with MAS and severe fibrous dysplasia. She was diagnosed with MAS at 4 years of age and, during follow-up, she had multiple pathological fractures and bone pain refractory to treatment with bisphosphonates, tricyclic antidepressants, and opioids. The pain was incapacitating and the patient required a wheelchair. Intranasal calcitonin was then started, and, 30 days later, the patient already showed significant improvement in pain severity at the affected sites. After 3 months, she was able to walk without assistance. No adverse effects were observed, nor were any significant changes in serum levels of calcium, phosphorus, and alkaline phosphatase. Calcitonin has a well-recognized analgesic effect on bone tissue. Despite the small number of studies involving patients with MAS, calcitonin may be considered a short-term therapeutic option in cases of severe and refractory bone pain. Other hyperfunctioning endocrinopathies may be involved, including hyperthyroidism, growth hormone excess, Cushing syndrome, and renal phosphate wasting. MAS has an estimated prevalence ranging from 1/100.000 to 1/1000.000, affects both sexes equally, and is generally diagnosed in children and young adults [McCune-Albright syndrome (MAS) is classically defined by the presence of fibrous dysplasia (FD),α-subunit of the stimulatory G protein (Gs α). The mutation results in locally increased stimulation of adenylyl cyclase and cAMP overproduction, leading to autonomic secretion in endocrine tissues. At the bone tissue level, FD is characterized by dysplastic lesions consisting of abnormal and poorly organized fibrous tissue, with a lytic or cystic appearance [Patients with MAS have involvement of multiple bone sites (polyostotic FD) that is usually established early in life . FD occupearance . The natpearance , 2.Clinical studies on FD are difficult because this condition is rare and clinically heterogeneous. There are no available medical therapies capable of altering the disease course. Recently, a multidisciplinary workshop, including patients, clinicians, and researchers, discussed the priorities of diagnosis and treatment of patients with FD and MAS. Among these priorities is the management of chronic pain with typical and atypical analgesics as well as adjuvant interventions when necessary . The priWe report here a case of MAS presenting severe FD and refractory bone pain and the effect of short-term treatment with intranasal calcitonin, highlighting the challenges of the management of this uncommon clinical presentation.μg/L, cortisol 400 nmol/L, TSH 1.3 mIU/L, and a prepubertal response to GnRH test. An initial diagnosis of autonomous ovarian follicular cyst was then made and the patient was kept on regular clinical follow-up with expectant management. After 7 months of follow-up, progression of premature thelarche (M2P1) was observed, with serum estradiol levels 38.2 pmol/L and normal prepubertal gonadotropin levels. Bone scintigraphy showed increased radioisotope concentration in the humerus, femur, tibia, and maxilla on the right side. Bone densitometry showed adequate bone mineral density (BMD) for age. The 24 h urine analysis showed a tubular phosphorus reabsorption rate of 88%, with phosphaturia of 16.9 mmol/24 h. Due to the presence of precocious puberty and FD, the patient was diagnosed with MAS.A 4-year-and-8-month-old girl was referred to the endocrinology outpatient clinic in 1993 for evaluation of bilateral development of breast tissue followed by vaginal bleeding lasting 5 days. After 2 months, she had another episode of vaginal bleeding, with duration similar to that of the first episode. She was born from vaginal delivery at term, weighed 2,350 kg, and had adequate motor and cognitive development. She used no continuous medication. On physical examination, the patient had a weight of 15,5 kg (p25), height of 1,02 m (p25), Tanner stage M1P1, and absence of café-au-lait skin pigmentation. Bone age was compatible with chronological age. Pelvic ultrasound showed uterus with 3.2 cc, endometrium of 0.2 cm, right ovary with a volume of 0.5 cc, and left ovary with a cyst measuring 1.5 × 2.3 cm. Laboratory tests showed estradiol 18.3 pmol/L, LH 1.70 IU/L, FSH 2.10 IU/L, prolactin 15 Z-score −0.7 in lumbar spine (BMD 1.091 g/cm2), −1.3 in left total femur (BMD 0.840 g/cm2), and −2.8 in forearm 33% (BMD 0.629 g/cm2). The patient had severe pain, according to a visual scale of pain [At that time, letrozole was not yet available in the country and suppressive therapy was started with 200 mg of intramuscular medroxyprogesterone every 3 weeks, with regression of breast tissue. A bone age X-ray performed at follow-up was compatible with chronological age. No increase in ovarian volume was detected on pelvic ultrasound. She had menarche at age 11, with irregular cycles and facial acne that improved with oral contraceptives. At age 14, due to bone pain in the right thigh and radiographic evidence of cysts, the patient started receiving a treatment protocol with intravenous pamidronate every 6 months, 40 mg/day for 3 consecutive days, for five cycles. The dose was not increased because of patient intolerance to medication. Over the follow-up period, vitamin D levels were monitored and supplementation provided if necessary. Treatment response was assessed by subjective pain intensity, alkaline phosphatase levels, and serial bone scintigraphy. At age 17, the patient had severe spontaneous pain in the hip, and a computed tomography scan showed interruption of the cortex in the right femoral neck related to fracture . The patThe present report shows the favorable outcome of a patient with MAS and severe bone pain after short-term treatment with nasal calcitonin. Calcitonin is a 32-amino acid polypeptide hormone produced by the parafollicular cells of the thyroid gland whose secretion is mainly regulated by serum calcium levels. Its main role is to inhibit bone resorption by reducing osteoclast activity [FD is characterized by the development of fibrous bone lesions that replace normal skeletal structures. Abnormal fibroblast proliferation and defective osteoblast differentiation result in the replacement of cancellous bone and marrow with fibrous connective tissue. There is no cure or spontaneous resolution of FD, but, since bone pain, deformities, and pathological fractures are the main symptoms, the condition often requires treatment . HoweverBenhamou et al.  recentlyBisphosphonates are often used as a medical treatment to reduce the increased bone turnover in the affected bone tissue. Thomsen and Rejnmark  publisheThe use of calcitonin in patients with FD is not new. The first study published by Bell et al.  in 1970 β endorphin levels and effects on central serotonergic or monoaminergic pathways [The analgesic activity of calcitonin has been demonstrated in several trials in patients suffering from different painful skeletal conditions –15. The pathways . Althougpathways .Concerning potential adverse effects of calcitonin, the European Medicines Agency (EMA) recently published a press release stating that the increase in cancer rates with calcitonin varied between 0.7% in studies with the oral formulation and 2.4% in the studies with the nasal formulation . HoweverWe reported the case of a patient with a diagnosis of MAS and incapacitating bone pain refractory to treatment with intravenous bisphosphonate associated with opioids and tricyclic antidepressants. There are only a few references in the literature to the use of calcitonin in MAS, but, in the present case, there was a significant improvement in both bone pain severity and quality of life. The treatment was very well tolerated and no adverse effects were noted. Based on this experience, short-term use of calcitonin may be considered an effective alternative in selected patients with polyostotic FD and severe and refractory bone pain.


G20210A polymorphism (rs1799963) within the prothrombin gene is associated with a higher circulation level of prothrombin, thus increasing the likelihood of developing myocardial infarction (MI). Opinions differ regarding the correlation between prothrombin G20210A genotype and MI risk, which prompted us to conduct a meta-analysis to determine this association. PubMed, EMBASE, Web of Science and CNKI were searched for pertinent reports. A total of 34 studies involving 14 611 MI cases and 84 358 controls were analyzed in this quantitative analysis. We found a statistically significant association between prothrombin G20210A polymorphism and MI in the allele model , heterozygote model  and dominant model . The association remains significant in Caucasians but not in non-Caucasians. Moreover, prothrombin G20210A polymorphism increases MI risk in an age-related manner. A further significant association was found in a subpopulation younger than 55 years . Sensitivity analysis and publication bias analysis revealed stable and statistically robust results. Our meta-analysis demonstrated that prothrombin G20210A polymorphism may represent a risk factor for MI. Myocardial infarction (MI) is a highly lethal disease in developed countries, usually caused by pathological coronary artery occlusion. However, unstable atherosclerotic plaque is not destined to cause MI. Following plaque rupture, coronary thrombosis involving platelet adhesion, coagulation cascade activation and subsequent thrombus formation plays a fundamental role in the clinical progression to MI. Over-activated procoagulation, acquired from complex patterns of inheritance or environmental risk factors, has more propensity to induce localized coronary occlusion. One of the most concerning etiological factors is single-nucleotide polymorphisms (SNPs) in coagulation and fibrinolytic systems.1. Numerous studies have demonstrated that a G20210A variant (rs1799963) of the prothrombin gene (i.e. a G to A substitution at position 20 210 in the 3′-untranslated region) is associated with elevated circulation levels of prothrombin4 and risk of venous thromboembolism6. Nevertheless, the detrimental role of prothrombin G20210A is controversial in MI8. The reason for this confounding phenomenon may be diversity of the ages of patients recruited in the previous studies. Inherited defects in coagulation may contribute more to MI in the young than in the elderly9. In comparison to MI in the elderly, premature myocardial infarction might be more predisposed to a SNP such as prothrombin G20210A because of relatively less environmental exposure. To draw a conclusion about the clinical impact of the prothrombin G20210A with regard to MI risk within a given age stratum, we gathered data from a number of published studies and performed a meta-analysis.Prothrombin, also called factor II, is the precursor of the thrombin, a key enzyme acting as a procoagulant, through platelet activation and the generation of fibrin and factors Va, VIIIa, and XIII39.As depicted in Fig. 6 and 33 cross-sectional case–control studies. Twenty-two studies included Caucasian patients39, 9 included non-Caucasians32, and 3 did not depict ethnicity of subjects31. As the study population overlapped in two reports39, Maren’s data39 were evaluated only in the >55-year-old subgroup analysis and not in the overall analysis.There was one prospective study27, which consequently was included in the allele model analysis only. Because there was only one eligible study19 of homozygote and recessive models in non-Caucasian subgroup analysis, homozygote and recessive analyses were not performed in the non-Caucasian subgroup.A total of 14 611 cases and 84 358 controls were included in the meta-analysis. Main characteristics, including first author, publication year, study country, ethnicity, age category of MI patients, source of controls, genotyping method, genotype frequencies, and minor allele frequencies are presented in Supplementary Table Overall, a meta-analysis of 13 488 cases and 77 085 controls in 33 studies was performed. The mean age was 48.41 years in cases and 47.16 years in controls. The association between prothrombin G20210A variant adenine (A) allele distribution and MI risk was calculated in comparison with wild-type guanine (G) allele in the allele model (A vs. G). Similarly, the association between specific genotype frequency and MI risk was assessed in the dominant model (AG + AA vs. GG), recessive model (AA vs. AG + GG), homozygote model (AA vs. GG), and heterozygote model (AG vs. GG), respectively. A statistically significant association was found in the allele model  but not in non-Caucasians . Table 28 (on 5 294 patients and 8 149 controls) provided data in subjects younger than 55 years. Four studies39 recruited 2 174 MI patients and 8 261 MI-free controls older than 55 years, respectively. The mean age was 41.75 years in cases and 42.03 in controls in the subgroup ≤55 years old, and 64.33 years in cases and 56.96 in controls in the subgroup >55 years old. Subgroup meta-analysis revealed that a significant association was found in three genetic models between prothrombin G20210A and MI in patients younger than 55 years  but not in individuals older than 55  , 0.62 (homozygote model), 0.13 (heterozygote model), 0.13 (dominant model), and 0.62 (recessive model) in Begg’s funnel plot Fig.  and 0.272. The prothrombin G20210A SNP has been correlated with increased levels of prothrombin in circulation and is clearly associated with an increased risk of venous thrombosis2. Substantive studies over the past decade have been making efforts to unmask the correlation between the G20210A SNP and MI. However, previous conflicting reports failed to draw a consistent conclusion, thereby making the association between the G20210A SNP and MI elusive39. The variance may result from different study designs and distinct ethnic backgrounds of subjects. Furthermore, several studies have reported that the observed association may be affected by other risk factors, such as age at onset. For instance, Butt et al.13 observed that the risk of MI was not elevated in prothrombin G20210A carriers beyond 50 years old, whereas carriers younger than 50 had a 5.6-fold higher risk of MI than age-matched non-carrier control subjects (p = 0.04). Thus, additional risk factors may influence the risk associated with this prothrombin G20210A variation. More importantly, inconsistency in the age of analyzed subjects may also cause discrepancy, as MI events in genetically vulnerable subjects occur much earlier than in normal counterparts40. Two previous systematic reviews41 analyzing the association between G20210A polymorphism and MI risk have drawn similar conclusions, namely that G20210A has no significant association with MI risk in the overall population at all ages, but does have a significant association with MI risk  or risk tendency  in persons younger than 55 years. To analyze whether MI is susceptible to prothrombin G20210A polymorphism, we performed this meta-analysis of 34 published studies to assess the association between prothrombin G20210A polymorphism and MI risk.Myocardial infarction is pathologically caused by coronary thrombotic occlusion following rupture of atherosclerotic plaques. Prothrombin is the precursor of thrombin, which plays a pivotal role in the physiological process of coagulation. Located on chromosome 11, the G20210A variant (rs1799963) substitutes a single base of adenine (A) for guanine (G) at position 20 210 in the 3′-untranslated region of the prothrombin geneIn the current systematic review we extracted allele and genotype frequency of the prothrombin G20210A polymorphism in MI patients and corresponding controls, and estimated the pooled ORs and 95%CI in five genetic models. Our integrated results showed that the G20210A polymorphism was significantly associated with MI risk in the allele model, heterozygote model, and dominant model. The tendency was similarly observed in Caucasians but not in non-Caucasians. The point estimates of the ORs in non-Caucasians, while not significant, are actually higher than those in Caucasians in three models. In subgroup analysis stratified by age, the MI risk in allele, heterozygote and dominant models was even higher in subjects ≤55 years old than in the overall population, but was not statistically significant in those older than 55.41. Boekholdt et al.41 evaluated 4 studies and concluded that G20210A polymorphism was not significantly associated with MI risk . Burzotta et al.7 assessed 13 studies and produced similarly insignificant results . This inconsistency between prior and present outcomes may have at least two possible explanations. Firstly, the two previous meta-analyses enrolled 4 and 13 studies, respectively, whereas 34 studies are included herein. These additional studies with larger numbers of subjects may have enhanced the soundness of the conclusion. Secondly, there may be an age discrepancy between the present and previous meta-analyses. For instance, MI patients younger than 55 years accounted for 16.9% of the total (624/3 687) in Burzotta’s meta-analysis7, whereas in the present study this proportion was 39.2% (5 294/13 488). According to age-related subgroup analysis in previous reviews41 and the present study, prothrombin G20210A polymorphism markedly increases MI risk in younger people. The discrepancy in age may help to explain the disagreeing conclusions.In the present meta-analysis of 34 studies we drew the conclusion that prothrombin G20210A SNP was significantly associated with overall MI risk at all ages, contrasting with results from two previous independent meta-analysis reports7. Premature myocardial infarction (PMI) commonly indicates a first onset of myocardial infarction before the age of 55 years in males and 65 years in females. Considering less exposure to long-term environmental risk factors, MI events in young individuals are more likely attributable to genetic susceptibility than those in elderly counterparts40. The dominant role of acquired risk factors in MI, such as hypercholesterolemia and smoking, attenuates the effects of genetic prothrombin G20210A in the elderly9. Several genetic mutations affecting coagulation proteins have been suggested as PMI risk factors19.An important conclusion from the present research is that G20210A polymorphism correlated with MI in young patients but not in the elderly, consistent with the previous meta-analysis42 of 33 cases of fatal MI and 165 controls showed a noticeable trend toward an association between the GA genotype and fatal MI . (2) Statistically significant association between prothrombin G20210A and MI risk was not observed in the homozygote model  and the recessive model , which seemed to conflict with the findings in the allele model , heterozygote model  and dominant model . The reason for this conflict may be that AA genotype frequency in the overall cases and controls was extremely low (0.07% and 0.04%), which resulted in a population inadequate for researching the association with MI risk. Although two independent researchers searched the databases for published articles and recorded data from eligible studies, it was possible that some articles might not be included in the present study because: (a) the possibility of publication bias could not be eliminated even though no evidence of publication bias was detected through Begg’s funnel plot and Egger’s linear regression method; (b) the language was restricted to English and Chinese. The relatively small sample size and number of included studies for gene-susceptibility investigation might distort the stability of the results regarding homozygote and recessive models. The true detrimental role of homozygote genotype AA in MI may need to be proved in studies with a larger population. (3) Moreover, a potential age discrepancy existed in the overall population analyzed, as there was a greater proportion of young individuals. Subpopulations younger than 55 years were available in 20 studies (containing 5 294 cases and 8 149 controls), whereas those older than 55 were from only 4 studies (containing 2 174 cases and 8 261 controls). The mortality rate of MI in young patients is far less than that in elderly patients, demonstrating the possibility that more elderly patients are lost through fatal MI43.The association between prothrombin G20210A polymorphism and susceptibility to MI has been carefully investigated. Notwithstanding, in addition to the inevitable clinical heterogeneity in most systematic reviews, we must acknowledge some limitations of this meta-analysis. (1) We cannot exclude the potential for a survival bias. Most of the included studies were designed to recruit survivors of MI as a case group. It cannot be excluded that patients who had died during the acute phase of fatal MI more often carried the prothrombin G20210A allele mutation. A necropsy studyIn conclusion, this comprehensive meta-analysis of 34 studies shows evidence supporting a causal association between prothrombin G20210A SNP and vulnerability to myocardial infarction. The risk was higher in the population younger than 55 years, but was not observed in the subjects older than 55. Larger population studies are urgently needed to validate our current findings and address the pathogenic role, which remains poorly understood.44. PubMed, EMBASE, Web of Science and China National Knowledge Infrastructure (CNKI) databases were searched from inception to 30 April, 2016 without language restriction. We used the combination of the search terms:  AND (“polymorphism” OR “polymorphisms” OR “mutation” OR “gene polymorphism” OR “SNP” OR “Single Nucleotide Polymorphism” OR “genotypes” OR “genotype” OR “variants” OR “variant”) AND (”Prothrombin” OR “Factor II”). The searches were complemented by checking the references of the retrieved articles.The PRISMA statement was followed in this meta-analysisEligibility criteria included the following: (1) case-control studies focused on association between prothrombin G20210A and myocardial infarction; (2) at least 30 patients were recruited in the case group; (3) when recruited patients overlapped with those in another study, the study with the largest number of individuals was selected; (4) if a study did not meet the inclusion criteria but a subgroup of the subjects qualified, only the subgroup was included; (5) studies that did not provide sufficient data following contact with authors by e-mail were excluded; (6) case reports and reviews were excluded. There was no notable deviation from Hardy–Weinberg equilibrium (HWE) in the control group.45. Studies that were awarded 5 stars or more, considered as of medium to high quality, were included in the present study. The following information in studies was extracted by two independent researchers: first author, year of publication, ethnicity, country, age of MI patients, source of controls, genotyping method, genotype frequencies, number of cases and controls analyzed, mean value of age.Two independent researchers (J.S. and S.L.) separately assessed the quality of the included studies using the Newcastle–Ottawa Scale (NOS) criteriaR programming language (version 2.13.1) was used to analyze the selected data. The pooled ORs were calculated for the allele model , dominant model (AG + AA vs. GG), recessive model (AA vs. AG + GG), homozygote comparison (AA vs. GG), and heterozygote comparison (AG vs. GG), respectively. After adjusting for multiple testing with Bonferroni’s method, a p value of any comparison less than 0.01 (=0.05/5) was considered statistically significant. When calculating the OR in one genetic model, we excluded studies with no events in either MI or control groups. When only one group contained no events, we added 0.5 to each cell of the 2 × 2 table for the trial before analysis. HWE for each study was determined by Chi-square test.2 statistics and Cochran’s Q statistics for each meta-analysis. I2 more than 50% or a p value less than 0.1 was considered heterogeneous among included studies. In such a case, the REM was used to summarize the values for each study, otherwise the FEM was applied. Begg’s and Egger’s tests were performed to assess publication bias, whereby a p value of less than 0.05 indicates an existence of publication bias among included studies.Tests for study heterogeneity were performed using Higgins’ ISupplementary Information


Tuberculous (TB) lymphadenitis can mimic cervical node metastasis from papillary thyroid carcinoma (PTC) since the distribution and appearance of affected lymph nodes are similar. We present the case of an asymptomatic 50-year-old Filipino who sought consult for a gradually enlarging anterior neck mass and a single palpable cervical lymph node. Preoperative workup suggested a thyroid malignancy with nodal metastasis. He underwent total thyroidectomy with node dissection where histopathology confirmed follicular variant- (FV-) PTC. Lymph node examination, however, revealed TB lymphadenitis, and the patient was given standard antimycobacterial therapy. This is the first documented case in Southeast Asia, a high TB burden region. This is also the first report involving FV-PTC, which has features between those of conventional PTC and follicular thyroid carcinoma. The case suggests that, in endemic areas, TB should be a differential in the etiology of cervical lymphadenopathy in PTC patients. In developed countries, this differential diagnosis is also valuable because of the increasing incidence of HIV and TB coinfection. Proper preoperative evaluation is important and needs to be highlighted in the formulation of local guidelines. Papillary thyroid carcinoma (PTC) is sometimes associated with cervical lymphadenopathy on presentation , 2. TubeA 50-year-old Filipino male presented at the outpatient clinic with a seven-year history of a gradually enlarging anterior neck mass. Prior to this consult, he was apparently healthy with good functional capacity. On physical examination, a firm, nontender mass measuring 5 × 4 × 2 cm was palpated at the left thyroid lobe without any bruits. An associated 1 × 1 × 0.5 cm rubbery, nontender lymph node was also palpated at the left cervical area. The rest of the examination was otherwise unremarkable. The patient denied any hypo- or hyperthyroid symptoms or compressive symptoms such as dyspnea, dysphagia, or odynophagia. Likewise, he denied symptoms of chronic cough, night sweats, weight loss, or fever. There was no personal history of prior treatment or known exposure to TB, no previous head and neck irradiation, and no family history of thyroid disease.μIU/mL (NV 0.3–3.8 μIU/mL). A thyroid ultrasound (US) showed a 6.5 × 5 × 3 cm solid mass at the left lobe with a singular 1.2 × 1 × 0.5 cm cervical lymph node.Laboratory testing revealed a normal hemogram and serum biochemistry. Thyroid function was normal with free thyroxine (fT4) levels at 21.3 pmol/L (NV 12–24 pmol/L) and thyroid stimulating hormone (TSH) levels at 0.59 Preoperative fine-needle aspiration biopsy (FNAB) was performed for the thyroid mass revealing PTC. The patient underwent total thyroidectomy with neck dissection, with an unremarkable perioperative course. Histopathological examination confirmed follicular variant- (FV-) PTC, measuring 6.5 cm in widest dimension at the left lobe with extension to the isthmus. There was no extension beyond surgical margins or into extrathyroidal tissue .Histopathology of the lymph node, however, yielded findings of chronic granulomatous inflammation with caseation necrosis and Langhans type giant cells consistent with a tuberculous etiology . A chestThe patient was followed up at the outpatient clinic six weeks after surgery. The surgical site had satisfactorily healed and antimycobacterial therapy was well-tolerated. He underwent adjuvant high-dose (100 mci) radioactive iodine treatment with no untoward complications and is currently on levothyroxine suppression therapy.PTC is the most common type of thyroid malignancy, constituting over 80% of all thyroid cancer cases . It prefThe coexistence of PTC and TB lymphadenitis has been described in several case reports from India, Korea, Japan, and the US , 6–8. InOur patient initially presented as a straightforward case of PTC manifesting as an asymptomatic gradually enlarging anterior neck mass with an associated solitary cervical lymph node. Due to the documented malignancy plus the proximity of the lymph node, our primary consideration for the lymphadenopathy was metastatic spread. Although US is a reasonably sensitive diagnostic method, it is difficult to truly ascertain the etiology of lymphadenopathy as the distribution and sonographic appearance are virtually identical in TB adenitis and cervical node metastasis. Both preferentially occur in the supraclavicular area or posterior triangle of the neck , appeariIn a surveillance report by the WHO-Western Pacific Region, the Philippines ranks ninth among the highest TB-burdened countries in the world. TB adenitis is the most common manifestation of extrapulmonary TB, which has an estimated prevalence of 1.1% in the country . A histoConsidering the high TB prevalence in Southeast Asia, this is the first documented case of its kind in the region. Moreover, in contrast to other reports dealing with either conventional type PTC (C-PTC) or mixed conventional and macrofollicular PTC, this is also the first case involving pure FV-PTC, the most common PTC subtype. Our case is significant in the sense that FV-PTC has a unique clinical behavior, representing an intermediate entity with features that are between those of C-PTC and follicular thyroid carcinoma (FTC). As compared to C-PTC, FV-PTC has been shown to have a lower rate of lymph node metastasis and a higher rate of distant metastasis .The case illustrates that cervical lymphadenopathy in a patient with PTC may not always indicate metastatic spread from the disease. In developing countries, TB should be considered an important differential in the etiology of cervical lymphadenopathy in a patient with PTC. Likewise, in developed countries, this differential diagnosis is also valuable because of the increasing incidence of HIV and TB coinfection . Proper


Patients with ulcerative colitis (UC) frequently present with psychological disturbances as well as dysfunctions of autonomic nervous system (ANS). Salivary alpha-amylase (sAA) secretion is predominantly controlled by sympathetic nervous activity, while salivary fluid secretion is by parasympathetic nervous activity. Thus, it is speculated that alterations of salivary secretion may be addressed in UC populations. AMY1) copy number, respectively. The psychological disorders, anxiety and depression, were evaluated by the scoring system of Hospital Anxiety and Depression Scale (HADS) for each participant.  Thirty-five UC patients as well as 32 age- and sex-matched healthy controls were enrolled. Saliva samples before and after citric acid stimulation were collected from each participant, and salivary flow rate (SFR) was calculated accordingly. Western blotting and quantitative PCR were applied to measure the sAA level and sAA gene (p < 0.001) as well as depression (p < 0.001) in UC patients relative to controls. Interestingly, we detected elevated basal (p = 0.015) and stimulated (p = 0.021) sAA levels in the UC populations compared to controls. However, no differences were found for basal (p = 0.643) or stimulated (p = 0.402) SFR between the two study groups. Besides, AMY1 gene copy number was comparable between UC patients and controls.  We observed robustly increased prevalence of anxiety ( Our results reveal an overactivity of the sympathetic nervous system and a normal activity of the parasympathetic nervous system in the UC population. Ulcerative colitis (UC) is a major form of inflammatory bowel disease and is characterized by chronic inflammation of the colon and rectum and by alternating periods of flares and remissions. Patients typically present with abdominal pain, diarrhea, and blood in the stools . UC has UC has been long claimed to be of psychosomatic origin since a half decade ago. It is been widely documented that UC patients apparently have higher prevalence of psychiatric disturbances than normal controls –7. For eThe autonomic nervous system (ANS) is an important regulator of intestinal inflammatory activity in an animal model . SpecifiAMY1 [AMY1 copy number in adults [AMY1 gene shows extensive variations in the copy number, with a range from 2 to 15 diploid copies [Salivary alpha-amylase (sAA) is one of the most abundant proteins in saliva . GeneticAMY1 . The sAAn adults , 28. AMYd copies , 30. sAAd copies . Mechanid copies –34. Reced copies . Unlike d copies , 36. TogConsidering that (1) UC patients present with altered ANS activities as well as psychological disorders, (2) the ANS dysfunction serves as one of the pathophysiological link between the psychological disorders and UC, and (3) salivary secretion is mainly regulated by ANS activity, we thus expected alterations of salivary secretion in UC patients, which might further support the altered ANS activities in the population.The study was conducted adhering to the Declaration of Helsinki. Procedures involving human participants were reviewed and approved by the Academic Ethics Committee of Guangxi Traditional Chinese Medical University. A written informed consent was given by each participant.The UC sample consisted 35 consecutively recruited patients with a confirmed diagnosis based on clinical, endoscopic, and histopathological evidence according to the standard diagnostic criteria . These pg for 15 min, followed by collection of the supernatant and the remaining precipitate. The supernatant was subjected to determination of sAA amount (μg/ml), while the precipitate (containing cheek cells) was used for AMY1 gene copy number analysis.Saliva samples were collected in the morning. Participants were instructed not to eat or drink (except water) or do exercise before collection. For collection of basal saliva sample, we used the passive drooling method as described by Navazesh . We thenμg/ml) was determined by Western blotting. Briefly, saliva samples of equal quantity of total protein were prepared by solubilizing with the SDS-PAGE loading buffer and heating at 100°C for 10 min. For quantification purpose, a human sAA protein sample (Sigma-Aldrich) of known quantity was also run on each gel. Proteins were separated by SDS-PAGE and transferred onto a nitrocellulose membrane. The membrane was then blocked by 5% milk at room temperature, followed by incubation with rabbit anti-sAA antibody (Abcam) at 4°C overnight. After washing with PBST, the membrane was subjected to incubation with goat anti-rabbit IgG-horseradish peroxidase conjugate (R&D Systems) at room temperature for 1 hour. After washing, the membrane was then exposed to Pierce™ ECL Western Blotting Substrate (Thermo Scientific) for 1 min. A ChemiDoc XRS+ Chemiluminescence system (Bio-Rad) was used for detecting the sAA protein band. The test sAA amount (μg/ml) was estimated by comparing with the respective standard sAA protein of known quantity.sAA amount  gene was amplified as an internal control. Primers for amplifications of AMY1 and TP53 were provided by Perry et al. [AMY1 diploid copy number was calculated by using a standard curve constructed from a reference DNA sample  [Genomic DNA was extracted from cheek cells by using DNAiso Reagent (Takara). Quantitative PCR (qPCR) was performed to determine the diploid y et al. . Determiy et al. . AMY1 diitories) .t-test was applied to compare means between groups for age, SFR, sAA amount, and AMY1 gene copy number. A chi-square test was used to compare sex composition and the prevalence rate of anxiety and depression between the two study groups. A p value of less than 0.05 was considered as statistically significant.Graph preparations as well as the statistical analyses were carried out by GraphPad Prism 5 (GraphPad Software). Data were expressed as mean ± standard deviation (SD), otherwise indicated. Unpaired Student's χ2) was not detected between the two study groups , which was consistent with previous reports [In the current study, we enrolled 35 UC patients as well as 32 healthy controls. Difference of age or sex .For basal SFR, we found extensive variations, ranging from 0.11 ml/min to 1.1 ml/min and 0.11 ml/min to 0.99 ml/min in the controls and UC patients, respectively . After gμg/ml). Similar to salivary fluid secretion, we observed extensive variations for the basal sAA level, ranging from 46 μg/ml to 322 μg/ml and 66 μg/ml to 350 μg/ml in the controls and UC patients, respectively  as well as a higher level of stimulated sAA  in the UC population relative to controls , which indicated that the AMY1 gene copy number variations might not contribute to the altered sAA secretion in UC patients.The difference of the sAA level may be explained by riations , 28; we AMY1) copy number were evaluated/determined accordingly. In the UC population, we detected significantly higher prevalence of anxiety as well as depression compared to controls. Interestingly, we observed elevated basal and stimulated sAA levels in UC patients relative to controls. However, no difference was found for SFR before and after citric acid stimulation between the two groups. Besides, AMY1 gene copy number was comparable between UC patients and controls. Our findings might implicate an altered ANS activity in UC patients, which was consistent with previous studies [In the current study, we collected 35 UC patients and 32 age- and sex-matched healthy controls. The psychological disorders (anxiety and depression), salivary fluid secretion, sAA level, and sAA gene . We argued that the basal sAA level is capable of reflecting the synthetic capacity of the salivary glands, while the stimulated sAA level is more likely to test the sympathetic nervous tension/sensitivity to stimulation. In the present study, UC patients presented higher basal and stimulated sAA levels relative to control, both pointing to an overactivity of the sympathetic nervous system due to the fact that the sAA synthesis as well as its secretion is tightly controlled by the sympathetic nervous system .AMY1 gene copy number [AMY1 gene copy number variations in the pathogenesis of UC. Interestingly, we observed extensive variations of the AMY1 gene copy number within each group, which was consistent with previous studies [AMY1 gene copies in the current population might suggest that a significant proportion of the participants' ancestors may undergo positive selection for increased AMY1 gene copy when they traditionally fed on starch-based foods.Because sAA production is mainly determined by the y number , we thus studies , 40. MorSalivary secretion can be affected by a myriad of factors, which has been largely elucidated . Thus, tIn conclusion, we detected an elevated sAA secretion in UC patients relative to controls, which might implicate sympathetic overactivity in the UC population. Together with previous studies, our findings corroborate the importance of ANS dysfunction in the pathogenesis of UC.


To predict severe fibrosis, the AUROC of eLIFT was lower than that of APRI  and FIB-4 . To predict cirrhosis, the AUROC of eLIFT was also lower than that of APRI  and FIB-4 . The eLIFT is not a good non-invasive test for the diagnosis of liver fibrosis and cirrhosis in CHB patients.Recently, the easy Liver Fibrosis Test (eLIFT), a sum of points attributed to age, gender, gamma-glutamyl transpeptidase, aspartate transaminase, platelets, and prothrombin time, was developed for diagnosing advanced fibrosis and cirrhosis in chronic liver disease. We aimed to evaluate the performance of eLIFT to predict liver fibrosis and cirrhosis in patients with chronic hepatitis B (CHB). Histologic and laboratory data of 747 CHB patients were analyzed. The performance of eLIFT for diagnosing liver fibrosis and cirrhosis was compared with that of aspartate transaminase to platelet ratio index (APRI) and fibrosis index based on the 4 factors (FIB-4). To predict advanced fibrosis, the AUROC of eLIFT was comparable with that of APRI (0.66  A recent study from China showed that HBV prevalence in men in rural China has changed from highly endemic into intermediate endemic in the past two decades, but the absolute number of HBV-infected men and the susceptible population is still large2. Another recent study from China also showed that the hepatitis B surface antigen (HBsAg) positive rate was 6.1%3. Chronic hepatitis B (CHB) can lead to a progressive accumulation of liver fibrosis which progressively evolves to cirrhosis, hepatocellular carcinoma (HCC), liver failure, and death4. Both the prognosis and the management of CHB patients are closely linked to the level of liver fibrosis. In CHB patients, the clinically relevant endpoints are: (1) detection of advanced fibrosis, which indicates that patients should receive antiviral treatment immediately to prevent further progression to cirrhosis and its complications; (2) detection of cirrhosis, which indicates that patients should not only potentially be treated for longer duration but also monitored for complications related to large esophageal varices and regularly screened for HCC1.Chronic hepatitis B virus (HBV) infection is very common: worldwide, 240 million have chronic HBV infection5. However, the FibroScan device is expensive and only accessible in specialized centers in developing countries, and not all CHB patients can be referred to the specialized centers. Therefore, the development of tests that can be easily used by all physicians to detect liver fibrosis and cirrhosis is greatly urgent.Liver biopsy is the reference procedure for liver fibrosis evaluation; however, limitations of this procedure include invasive nature, cost, and risk of serious complications. These limitations of liver biopsy make it unsuitable as first-line test to screen liver fibrosis in CHB patients. Recently, assessments of liver fibrosis by non-invasive tests have been developed. The FibroScan has been used for the non-invasive evaluation of liver fibrosis in clinical practice, and provides an exciting alternative to liver biopsyet al. developed a novel algorithm-the easy Liver Fibrosis Test (eLIFT), a sum of points attributed to age, gender, gamma-glutamyl transpeptidase (GGT), aspartate transaminase (AST), platelet count, and prothrombin time, for the diagnosis of advanced fibrosis and cirrhosis in the training set of 2503 patients with chronic liver disease (CLD)6. In the validation set of 1251 patients with CLD, eLIFT and FIB4 had the same sensitivity  but eLIFT gave less false-positive results , and was thus more suitable as screening test6. In a word, the eLIFT was identified as a novel noninvasive algorithm, which was able to estimate advanced fibrosis and cirrhosis in CLD patients. However, in the study by Boursier et al., the main causes of CLD were chronic hepatitis C (45.5%) and nonalcoholic fatty liver disease (NAFLD) (34.2%), and the prevalence of CHB was only 3.2%6. As the authors conclude, the study is limited by the presence of a subgroup with undetermined diagnosis, and further works will be needed to see if eLIFT can provide a precise diagnosis in the subgroup of CHB patients6.In January 2017, Boursier At present, there is a lack of data about the performance of eLIFT for the diagnosis of liver fibrosis and cirrhosis in CHB patients, and clinical research is needed urgently to see if eLIFT can provide a precise diagnosis of liver fibrosis and cirrhosis in CHB patients. In this study, we evaluated the diagnostic performance of eLIFT for advanced fibrosis, severe fibrosis, and cirrhosis, and compared with that of APRI and FIB-4 in 747 CHB patients.7. Exclusive criteria: (1) alcohol consumption ≥20 g/day (n = 103); (2) coexistence with NAFLD defined as the presence of more than 5% steatosis of hepatocytes and the clinical records indicated that the patient was felt to either be totally abstinent or to consume less than approximately 20 g of alcohol daily (n = 128); (3) hepatitis C virus (HCV), hepatitis D virus (HDV), or HIV co-infection (n = 87); (4) coexistence with autoimmune liver disease (n = 40); (5) the history of antiviral therapy (n = 147); (6) prothrombin time unavailable (n = 75). Finally, seven hundred and forty-seven CHB patients were included. Figure Thirteen hundred and twenty-seven consecutive CHB patients who underwent liver biopsies and routine laboratory tests in Shanghai Public Health Clinical Center, Shanghai, China, between January 2010 and January 2017 were retrospectively screened. CHB was defined as the persistent presence of HBsAg for more than six monthsAll patients signed the informed consent before liver biopsy, and all clinical procedures were in accordance with the Helsinki declaration. The ethics committee of Shanghai Public Health Clinical Center approved the study protocol, and experiments, including any relevant details. All experiments were performed in accordance with relevant guidelines and regulations.8. For this study, we defined none/mild fibrosis as METAVIR F0-1, advanced fibrosis as METAVIR F2-4, severe fibrosis as METAVIR F3-4, and cirrhosis as METAVIR F4.Liver biopsy was performed under ultrasound localization in all enrolled patients, and a minimum of 15 mm of liver tissue with at least six portal tracts is considered sufficient for histological scoring. Histological scoring was performed by two liver pathologists who were blinded for the data of non-invasive tests. In case of discrepancies, slides were reviewed by a third senior expert specialized in hepatology. The METAVIR scoring system was adopted as the gold standard of liver fibrosis, which was classified into five stages: F0, no fibrosis; F1, portal fibrosis without septa; F2, portal fibrosis with rare septa; F3, numerous septa without cirrhosis; and F4, cirrhosis6: age (years): <40 = 0, ≥40 = 3; gender: female = 0, male = 1; AST (IU/L): <35 = 0, 35–69 = 2, ≥70 = 4; GGT (IU/L): <35 = 0, 35–89 = 1, ≥90 = 2; platelet count (109/L): ≥250 = 0, 170–249 = 1, <170 = 4; prothrombin time (%): ≥97 = 0, 84–96 = 2, <84 = 4;eLIFT is the sum of age, gender, GGT, AST, platelet count, and prothrombin time9 = (AST/ULN of AST)/platelet count ×100;APRI10 = (age ×AST)/(platelet count ×(ALT)1/2).FIB-4The data available enabled the calculation of three blood fibrosis tests according to published formulas as follows:p < 0.05 was considered statistically significant. All statistical analyses were carried out using the SPSS software version 15.0  and MedCalc Software version 16.1 .Normality tests of baseline data were performed by Kolmogorov-Smirnov test. The baseline characteristics of patients are presented as follows: normal distribution data as mean ± standard deviation, non-normal distribution continuous data as median (interquartile range), and categorical variables as number (percentage). The correlations between noninvasive markers and METAVIR fibrosis scores were analyzed using Spearman test. The ROC curve analysis was performed to evaluate the performances of non-invasive tests for the diagnosis of advanced fibrosis, severe fibrosis, and cirrhosis, respectively. Two sets of cut-offs were calculated: (1) maximizing Youden’s index (sensitivity + specificity−1), which is an optimized balance between sensitivity and specificity, or (2) obtaining a sensitivity of at least 90%. Diagnostic accuracy was evaluated by sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV), positive likelihood ratio (+LR), and negative likelihood ratio (−LR). All significance tests were two-tailed, and 9/L (IQR = 159–215), and 88% (IQR = 82%–96%), respectively. The median APRI and FIB-4 was 0.40 (IQR = 0.28–0.61) and 0.82 (IQR = 0.61–1.19), respectively. The METAVIR inflammation stage was distributed as follows: A0 = 114 (15.3%); A1 = 349 (46.7%); A2 = 186 (24.9%); and A3 = 98 (13.1%). The METAVIR fibrosis stage was distributed as follows: F0 = 74 (9.9%); F1 = 438 (58.6%); F2 = 106 (14.2%); F3 = 54 (7.2%); and F4 = 75 (10.1%). Among 747 patients, 235 (31.5%), 129 (17.3%), and 75 (10.1%) were classified as having advanced fibrosis, severe fibrosis, and cirrhosis, respectively.Baseline characteristics of enrolled patients were presented in Table vs 34 years, p < 0.001), AST , GGT , APRI , and FIB-4  levels, but lower platelet count  and prothrombin time  than patients with none/mild fibrosis. No significantly differences were seen in sex, HBeAg status, HBV DNA levels, ALT levels, and METAVIR inflammation stages between patients with advanced fibrosis and patients with none/mild fibrosis (Table Patients with advanced fibrosis had higher age (39 p < 0.001), male , AST , and GGT  had positive correlation with METAVIR fibrosis stages; and platelet count  and prothrombin time  had negative correlation with METAVIR fibrosis stages. The METAVIR fibrosis stages were positive correlated with eLIFT scores , with higher correlation coefficient than any maker alone. The association between METAVIR fibrosis stages and noninvasive markers was presented in Fig. The correlations between noninvasive markers and METAVIR fibrosis stages were showed in Table vs 0.71, p = 0.095) and FIB-4 . To predict severe fibrosis, the AUROC of eLIFT was lower than that of APRI  and FIB-4 . To predict cirrhosis, the AUROC of eLIFT was lower than that of APRI  and FIB-4 .The diagnostic performances of eLIFT, APRI, and FIB-4 for liver fibrosis and cirrhosis were presented in Table Diagnostic thresholds of eLIFT for liver fibrosis and cirrhosis were presented in Table 11. However, a systematic review of 118 studies showed that two thirds of patients with cirrhosis were diagnosed belatedly when complications appeared12. This demonstrates an urgent need for non-invasive tests to screen patients with advanced fibrosis and cirrhosis, who require antiviral management immediately for preventing the disease progress. In this setting, the non-invasive diagnosis of liver fibrosis has been a hot field of research. A study by Jia et al. showed that liver elastometry got high AUROCs of 0.82, 0.88, and 0.90 for the diagnoses of advanced fibrosis, severe fibrosis, and cirrhosis, respectively13. Recently, the study by Shi et al. showed that magnetic resonance elastography technique had almost perfect AUROCs of 0.97 and 0.98 for the diagnoses of advanced fibrosis and cirrhosis, respectively14. Although liver elastometry and magnetic resonance elastography have been increasingly recognized as the excellent tools for the diagnosis of liver fibrosis, they are currently available only in specialized tertiary centers because of the devices were expensive. Simple, inexpensive, and accurate fibrosis tests are urgently needed for screening CHB patients for liver fibrosis and cirrhosis.The early management of CHB patients had been recommended by all international guidelines6. Compared to APRI and FIB-4, the eLIFT has two main advantages6. First, while the APRI and FIB4 need a computer for calculation, eLIFT is very easily calculated at-a-glance and in one’s head, which makes eLIFT easier and faster to use in clinical practice than APRI and FIB46. More importantly, the eLIFT gave less false-positive results in the validation set of 1251 patients with CLD , and was thus more suitable as screening test6.The blood fibrosis tests such as APRI and FIB-4 have been used widely because of the noninvasive procedure, comprising only inexpensive laboratory tests, and available in primary care. The eLIFT is a new, user-friendly, at-a-glance fibrosis test available to all physicians, whether specialized in hepatology or not, who manage CLD patients in their daily clinical practice, as it is based on parameters commonly assessed in CLD15. In this retrospective study of 747 CHB patients, we observed that the eLIFT did not get higher performance than APRI and FIB-4, which have been recommended by the WHO HBV guidelines16.Given that the eLIFT was developed in patients with CLD-the vast majority of them were HCV (45.5%) and NAFLD (34.2%) and just a small number of them were HBV (3.2%), it would be necessary to evaluate the diagnostic performance of eLIFT for liver fibrosis and cirrhosis in CHB patients before it can be popularized widely in this populations. The eLIFT needs further evaluation in subgroup of CLD because of the different pathogenesis, patterns of fibrosis, and patterns of regression have been observed in different causes of CLDThe AUROCs of eLIFT for diagnosing advanced fibrosis and cirrhosis were unsatisfactory . The optimal cut-off values for excluding and confirming advanced fibrosis were 3 and 9, respectively: and these cut-offs were not useful either for excluding or confirming presence of advanced fibrosis (negative likelihood ratio 0.38 for cut-off 3 and positive likelihood ratio 2.60 for cut-off 9). Based on 31.5% pre-test probability of advanced fibrosis, the post-test probability of having advanced fibrosis increased to only 49% with positive test (eLIFT ≥ 9). Similarly, the optimal cut-offs for excluding and confirming cirrhosis were 5 and 10, respectively: and these cut-offs were also not useful either for excluding or confirming presence of cirrhosis (negative likelihood ratio 0.57 for cut-off 5 and positive likelihood ratio 3.50 for cut-off 10). Based on 10.1% pre-test probability of cirrhosis, the post-test probability of having cirrhosis increased to only 25% with positive test (eLIFT ≥ 10).17. Second, there was no validation of noninvasive tests such as Hepascore, Fibrotest, or Fibrometer, because these tests were all protected by patents, and some laboratory tests which are necessary for the calculations of these tests were costly and difficult-to-obtain in our hospital. Third, this study has been conducted in tertiary referral centres with a higher proportion of patients with liver fibrosis and cirrhosis than in the general population. Thus, it might produce bias when the performance of eLIFT was extrapolated to the general CHB population.The limitations of this study were difficult to avoid. First, the retrospective design of this study might have caused selective biasIn conclusion, eLIFT, which showed application prospect in identifying advanced fibrosis and cirrhosis in CLD patients, was not a good non-invasive test for the diagnosis of liver fibrosis and cirrhosis in CHB patients. The larger sample, prospective, multi-centre studies will be necessary to validate the performance of eLIFT in CHB patients further.The funding organizations are public institutions and had no role in the design and conduct of the study; collection, management, and analysis of the data; or preparation, review, and approval of the manuscript.


Citrobacter rodentium-induced inflammation, effects that can be attenuated with probiotic Lactobacillus reuteri. This study assessed the structure of the colonic mucosa-associated microbiota in mice exposed to a social stressor , as well as non-stressed control mice, during challenge with the colonic pathogen C. rodentium. Mice were exposed to the social stressor or home cage control conditions for six consecutive days and all mice were challenged with C. rodentium immediately following the first exposure to the stressor. In addition, mice received probiotic L. reuteri, or vehicle as a control, via oral gavage following each stressor exposure. The stressor-exposed mice had significant differences in microbial community composition compared to non-stressed control mice. This difference was first evident following the six-cycle exposure to the stressor, on Day 6 post-C. rodentium challenge, and persisted for up to 19 days after stressor termination. Mice exposed to the stressor had different microbial community composition regardless of whether they were treated with L. reuteri or treated with vehicle as a control. These data indicate that stressor exposure affects the colonic microbiota during challenge with C. rodentium, and that these effects are long-lasting and not attenuated by probiotic L. reuteri.Stressor exposure significantly affects the colonic mucosa-associated microbiota, and exacerbates  The human gastrointestinal (GI) tract is the site of many chronic inflammatory illnesses including the inflammatory bowel diseases (IBD), i.e., ulcerative colitis and Crohn’s disease34Lactobacillus and Parabacteroides. These previous observations were made in healthy, uninfected mice even though studies indicate that stressor-induced changes in the microbiota impact the colonic inflammatory response to C. rodentium613C. rodentium changed gut microbiota composition and increased subsequent colonic inflammatory responses to C. rodentiumC. rodentium resulted in an exaggerated colonic inflammatory response compared to germfree mice that received microbiota from non-stressed donorsC. rodentium infection. However, because inflammation in the intestines can significantly change microbial community compositionC. rodentium-induced colonic inflammation, and whether the effects of the stressor are evident in probiotic-treated animals.Psychological stressor exposure affects GI functioning and symptoms in both healthy and diseased individuals. For example, psychological stress is associated with elevated inflammation, bleeding, and pain in both IBD and enteric infections6791011Lactobacillus reuteri is an immunomodulatory probiotic that can ameliorate the severity of colonic infectionC. rodentium-infected mice, as well as abrogate the heightened colonic pathology in stressor-exposed miceL. reuteri can act directly upon host immunity, such as by modulating phagocytosis and cytokine release by macrophages and monocytes or intestinal epithelial cells13141920L. reuteri prevents the exacerbating effects of stressor exposure on C. rodentium-induced intestinal inflammation by preventing stressor-induced dysbiosis. The purpose of this study was to determine whether the effects of stressor exposure on microbial community composition were evident throughout the course of C. rodentium infection and extend beyond termination of the stressor. A secondary objective was to determine whether the effects of the stressor on microbial community composition were evident in probiotic-treated animals.Probiotic bacteria, as defined by the World Health Organization, are living microbes that can confer a health benefit upon a host when given in adequate numbers. ad libitum and kept on a 12:12 hour light:dark cycle, with lights on from 0600 to 1800 hr. All procedures were carried out in accordance with guidelines by Office of Laboratory Animal Welfare at the National Institutes of Health and were approved by the Animal Care and Use Committee at the Ohio State University.Male C57Bl/6 mice (age 6–8 weeks) were obtained from Charles River , housed three to a cage, and allowed to habituate in an approved Ohio State University vivarium for one week upon arrival. Mice were given food and water Citrobacter rodentium, DBS120, was grown for 18 hr at 37 °C in lysogeny broth. Prior to infection, C. rodentium was diluted to a final stock concentration of 3–5 × 107 CFU/mL in PBS. To measure C. rodentium in shed stool pellets, stool was homogenized in a slurry in PBS, then plated in serial dilutions in MacConkey Agar with 40 μg/mL of kanamycin added. Lactobacillus reuteri, ATCC 23272, was grown for 18 hr at 37 °C at 5% CO2 in MRS broth. Lactobacillus reuteri was prepared to a stock concentration of 1 × 109 CFU/mL. Each mouse received a total inoculum of 1 × 108 CFU of L. reuteri.C. rodentium (Cr) immediately following the first cycle of SDR. Each mouse received 100 μl of the C. rodentium stock for a total of 3–5 × 106 colony-forming-units (CFU)/mouse. All infected mice had food and water removed for two hours post infection. In addition, following each of the six cycles of SDR, half of the SDR and HCC mice received 1 × 108 CFU of L. reuteri (Lr), while the other half of the SDR and HCC mice received PBS vehicle (Veh). In sum, there were four experimental groups: HCC-Cr-Veh, HCC-Cr-Lr, SDR-Cr-Veh, and SDR-Cr-Lr.Test mice were exposed to social disruption stress (SDR), wherein an aggressive CD-1 retired breeder male mouse is placed in a cage with the smaller and younger test mice. The aggressive intruder attacks and defeats the test mice over the course of two hours as previously described152223Cr-Veh, HCC-Cr-Lr, SDR-Cr-Veh, and SDR-Cr-Lr) were sacrificed at 1, 6, 12, and 24 days post infection (DPI). Colons were collected for Illumina sequencing analysis, while stool was collected for the purpose of C. rodentium quantification. Colons were snap frozen in liquid nitrogen and stored at −80 °C until DNA was isolated for sequencing. An initial experiment was performed, as well as three experimental repeats, for four total experimental runs. The experimental design is shown in Mice from the four experimental groups (HCC-t) method as described by the manufacturer.Total RNA was isolated from the distal portion of the colon using Trizol reagent as per manufacturer’s instructions , and RNA was reverse transcribed to make complementary DNA using a commercially available kit . Real-time PCR primers and probes were synthesized by Applied Biosystems with the sequences as previously reportedDNA was extracted from the proximal section of the colon (~10 mg tissue) using a QIAgen DNA Mini Kit, following manufacturer’s instructions with slight modifications. In summary, colon contents were removed via direct excision, and colon tissues were briefly washed in a PBS bath, so as not to disturb the mucosal layer. Tissues were incubated for 45 mins at 37 °C in lysozyme buffer , then bead-beat for 150 sec with 0.7 mm zirconia beads. Samples were incubated at 56 °C for 2 hr with Buffer ATL and Proteinase K, then incubated at 56 °C for 30 mins and 95 °C for 10 mins upon addition of Buffer AL. From this point, the Qiagen DNA Mini Kit isolation protocol was followed from the ethanol step forward. Samples were quantified with the Qubit 2.0 Fluorometer  using the dsDNA Broad Range Assay Kit. Samples were standardized to at least 5 ng/μl before being sent to the Molecular and Cellular Imaging Center (MCIC) in Wooster, OH for library preparation. The V1–V3 hypervariable region of the 16S rRNA gene was targeted in this study. To amplify and sequence the V1–V3 hypervariable region of the 16S rRNA gene, we used primers that contain a heterogeneity spacer in line with the targeted sequence. Four sets of spacers of different lengths were used to compensate for the low nucleotide diversity of the amplicons; since accurate base-calling on Illumina platforms and generation of high-quality data requires sequence diversity at each nucleotide position before the clustering occurs. For the targeted region, we used well-known universal primers that were modified to include degenerate bases for maximal inclusivenessLibraries were prepared in two rounds of PCR amplification. The first round amplified the locus of interest and added a portion of the Illumina adapter sequence; and the second round completed the Illumina adapter sequence which contained a unique dual combination of the Nextera indices for individual tagging of each sample. Twenty nanograms of each genomic DNA was used as input for the first PCR reaction and 3 μl of the clean PCR 1 product was used as input for PCR 2 reaction. PCR amplifications were carried out as follows: initial denaturation at 96 °C for 3 min, followed by 25 (PCR 1) or 8 (PCR 2) cycles each of 96 °C for 30 sec, 55 °C for 30 sec and 72 °C for 30 sec, and a final extension at 72 °C for 5 min. The PCR products were purified after each PCR amplification using the Agencourt AMPure XP beads . All the steps for library preparation and cleaning were carried out on the epMotion5075 automated liquid handler . The purified amplicon libraries were quantified and pooled at equimolar ratios before sequencing. The final pool was also purified using the Pippin Prep size selection system  to discard the presence of any primer dimers.2 and the libraries were sequenced using a 300PE MiSeq sequencing kit with the standard Illumina sequencing primers. Image analysis, base calling and data quality assessment were performed on the MiSeq instrument.The amplicon libraries were sequenced at the Molecular and Cellular Imaging Center (MCIC) in Wooster, OH using the MiSeq sequencing platform (Illumina) at a final concentration of 15.4 pM. A genomic library of well-known diversity previously sequenced in the lab was combined with the pool of amplicon libraries for the sequencing run (expected at 20%). The run was clustered to a density of 1131 k/mmhttp://github.com/najoshi/sabre), with 1 allowed barcode mismatch. Barcodes were removed and sequences were trimmed for equal lengths using FastX Trimmer (website: http://hannonlab.cshl.edu/fastx_toolkit). Sequences were joined with Fastq-Join, with 10% allowed differences within the overlap region. Quality filtering was performed with the following parameters: quality score of 20, 0 allowed N characters, 1.5 allowed barcode errors, 3 consecutive low quality bases allowed. qiime_tools (website: http://github.com/smdabdoub/phylotoast) was used for closed reference OTU picking against the 13_8 GreenGenes databaseForward and reverse ends were demultiplexed using Sabre  and C. rodentium infection  were associated with significant differences in microbial community composition, whereas probiotic gavage with L. reuteri did not affect the microbiota structure in the overall community . However, the adonis statistic also indicated that microbial community composition was significantly different across each of the experimental runs, regardless of treatment condition . Therefore, in order to visualize the effects of experimental treatments on community composition using a PCoA plot, a custom axis for experimental run was added at the expense of the third PCoA axis. This strategy indicated that exposure to the stressor impacted community composition based on sample clustering in experimental runs 2, 3, and 4 (Alpha diversity was estimated using the Shannon Diversity Index and Chao1. There was a significant effect of DPI on Shannon Diversity Index (p < 0.05), with post-hoc testing indicating that 24 DPI was higher than 12 DPI   (2 = 0.0478). Probiotic gavage no longer significantly associated with changes in the microbiota. The effect of the SDR stressor was observed at 12 DPI  and 24 DPI   . In orde0.05243) , while s0.05725) , despiteFirmicutes were reduced across the different DPI, as indicated by a significant main effect in the ANOVA (p < 0.001). This main effect was due to significant reductions on 6, 12, and 24 DPI compared with 1 DPI (p < 0.01). In contrast, Bacteroidetes were increased across the different DPI (p < 0.001) with significant increases observed on 6, 12, and 24 DPI compared with 1 DPI (p < 0.01). Interestingly, the relative abundances of these phyla were also impacted by stressor exposure. There was a main effect of stress in the ANOVA (p < 0.01) indicating that stressor-exposure significantly reduced the relative abundance of Firmicutes. Similarly, stressor exposure significantly affected Bacteroidetes  and Enterobacteriaceae (p < 0.05) were significantly increased in infected mice, whereas Prevotella (p < 0.05) and Parabacteroides (p < 0.005) were reduced in those mice. Post-hoc testing indicated that S24-7 was increased on 6, 12, and 24 DPI compared with 1 DPI (p < 0.001), whereas the Enterobacteriaceae were increased on 6 and 12 DPI compared to 1 DPI. Parabacteroides levels were reduced on 6 compared to 1 DPI and reduced on 12 DPI compared to 1 and 24 DPI (p < 0.001) (Prevotella levels were significantly reduced at 12 DPI (p < 0.001)  . Prevote< 0.001) .L. reuteri treatment on the relative abundance of bacterial taxa were tested, it was evident that stressor exposure, but not L. reuteri treatment impacted the microbiota , but this difference did not remain significant after correction for multiple tests. Stressor exposure had a more predominant effect on S24-7 relative abundance which was significantly increased in SDR-Cr mice across the duration of the experiment regardless of whether they were treated with vehicle or with L. reuteri . Parabacteroides and Lactobacillus levels were also significantly reduced by stressor exposure throughout the experiment , and there was a tendency for L. reuteri treatment to normalize Parabacteroides ) (When the effects of stressor exposure and of crobiota . Mice exrisons)) .S24-7 OTUs, and no stress identified with Bacteroides, Parabacteroides, and Adlercreutzia OTUs. Clostridiales had a negative association with the temporal variable of DPI  using CANOCO further highlighted these results. OTUs with <10 total observations were removed for RDA analysis. DPI , Stress , and Infection  were all significant on the RDA plot. OTUs that accounted for greater than 20% of the variance explained by the two axes were included on the RDA triplot, wherein SDR associated with e of DPI .L. reuteri had only a modest effect on alpha diversity and failed to significantly impact beta diversity of the gut microbiota of SDR-Cr-Lr, C. rodentium load and markers of colonic inflammation were assessed to verify that exposure to the SDR stressor enhanced the colonic inflammatory response to C. rodentium, and that administration of probiotic L. reuteri attenuated the effects of the stressor on colonic inflammation as we have previously reported. Consistent with our previous studies, exposure to the stressor significantly increased the amount of C. rodentium in the infected mouse stool (C. rodentium levels were significantly increased at 12 DPI in SDR mice over HCC (p < 0.005) (Cr-Veh mice had significantly more C. rodentium than non-stressed HCC-Cr-Veh control mice treated with vehicle (p < 0.005). SDR-Cr-Lr mice had marginally increased pathogenic burden over HCC-Cr-Lr mice that received the probiotic (p = 0.06).Because se stool . In the < 0.005) . LikewisC. rodentium levels are associated with increases in C. rodentium-induced colonic pathology, and that L. reuteri can attenuate the effects of the stressor on colonic pathologyL. reuteri attenuated the effects of the stressor in the current study. We previously found that colon mass strongly correlates with colonic histopathologyCr-Veh mice; stressor-exposed SDR-Cr-Lr mice did not have elevations in iNOS mRNA levels in comparison to non-stressed controls on any day post-challenge. Stressor-exposed mice also had increases in TNF-α gene expression on 12 DPI (p < 0.0001); SDR-Cr-Lr mice had lower levels of TNF-α than did SDR-Cr-Veh mice, but this difference was not statistically significant (data not shown).We have previously demonstrated that stressor-induced increases in C. rodentium-induced colonic inflammation was greater in germfree mice that were colonized with microbiota from donor mice exposed to a prolonged restraint stressor when compared to germfree mice colonized with microbiota from non-stressed control donorsC. rodentium challenge.Exposure to psychological stressors can alter microbial community structures, turning a normally stable microenvironment into a dysbiotic profile of volatility910C. rodentium challenge peaked at 12 DPI. However, the prolonged effects of the stressor on the microbiota on 24 DPI is not likely due to the C. rodentium infection, because C. rodentium levels were below the level of detection on 24 DPI and markers of colonic inflammation were not elevated in stressor-exposed mice on 24 DPI. Thus, the effects of the stressor on microbial community composition extended past effects of the stressor on colonic inflammation. This finding has important implications, because many physiological and behavioral effects of the SDR stressor that have been associated with changes to the microbiota are evident up to 14 days after termination of the stressor. For example, both enhanced immune system activity and anxiety-like behavior are evident up to 14 days after termination of the stressor2322The current results confirm previous studies demonstrating that the SDR stressor can impact microbiota composition during the 6 day period of stressor exposure10C. rodentium challenge (Shannon Diversity) and by L. reuteri treatment (Chao1). Alpha diversity was significantly different between 12 and 24 DPI, which overall is the peak of colonic inflammation (12 DPI) and the return to baseline (24 DPI). The effects of L. reuteri on alpha diversity were transient, and were only evident on 1 DPI. L. reuteri treatment ended on 5 DPI, and by 6 DPI (as well as later time points), Chao1 measures were not different in mice that had been treated with L. reuteri. This study also corroborated an earlier study that showed no change in the alpha diversity of the colonic mucosa-associated microbiota as a function of SDR353739Measures of alpha diversity were affected over the 24 day Parabacteroides, Adlercreutzia, and Bacteroides, whereas SDR stressor associates with increased S24-7. In addition, consistent with previous studiesParabacteroides and Lactobacillus. Both genera have anti-inflammatory properties819C. rodentium infection. To better understand how SDR-induced disruptions in the microbiota adversely affect host immune responses, the involvement of these microbial groups in immune maintenance and why they are specifically targeted by social stress must be explored in future studies. The importance of Adlercreutzia and Bacteroides for maintaining health are not completely understood, however some species of Bacteroides, namely B. fragilis, are known to promote Foxp3+ T regulatory cells that can limit intestinal inflammationBacteroides in non-stressed control mice contribute to increased intestinal homeostasis during pathogen challenge.The RDA plot indicated that the HCC group associates with L. reuteri had little effect on the composition of the gut microbiota. Administering probiotic L. reuteri only affected microbial populations at 1 DPI, even though L. reuteri administration was repeated through 6 DPI. The repeated L. reuteri administration was not sufficient to significantly impact microbial community structure. This result is surprising, because severe inflammation within the GI microenvironment is known to lead to dysbiosis16L. reuteri reduced colonic inflammation in animals exposed to the stressor during C. rodentium challenge. As with our previous studyL. reuteri significantly attenuated the exacerbating effects of stressor exposure on markers of C. rodentium-induced colonic inflammation including colon mass , as well as systemic diseases, such as diabetes and obesity. And, there are a growing number of studies indicating that the symptoms of many of these same illnesses can be exacerbated during stressful periods. As an example, low social support, perceived distress, the experience of the disease as traumatic, and low quality of life were predictive of disease exacerbation in IBD patientsHow to cite this article: Galley, J. D. et al. Stressor exposure has prolonged effects on colonic microbial community structure in Citrobacter rodentium-challenged mice. Sci. Rep.7, 45012; doi: 10.1038/srep45012 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


There is sufficient evidence supporting a relationship between increased body mass index (BMI) and an increased risk for breast cancer among postmenopausal women. However, most studies have found a decreased risk for premenopausal breast cancer. This study was conducted to find out the different effects of BMI on the risk of breast cancer among premenopausal and postmenopausal women, and explore the potential factors that influence the associations.A dose-response meta-analysis with 3,318,796 participants from 31 articles was conducted. Cohort studies that included BMI and corresponding breast cancer risk were selected through various databases including PubMed, Medline, Web of Science, the China National Knowledge Infrastructure (CNKI) and Chinese Scientific Journals (VIP). Random effects models were used for analyzing the data.2) the RR in total postmenopausal women were1.03 (95% CI: 1.02–1.05) per 1 kg/m2 increment. However, no statistically significant association among total premenopausal women was detected. In subgroup analysis among European premenopausal women, the summary RR was 0.79(95%CI: 0.70–0.88). The non-linear relationship showed a negative non-linear association between BMI and breast cancer risk among European premenopausal women. When compared to the mean level of the normal BMI category, the RRs were 0.98 (95%CI: 0.96–1.00) per 1 kg/m2 increment, respectively.The summary relative risks (RRs) were 1.33 (95%CI: 1.20–1.48) and 0.94(95%CI: 0.80–1.11) among postmenopausal and premenopausal women, respectively. The dose-response meta-analysis indicated a positive non-linear association between BMI and breast cancer risk among postmenopausal women, and compared to the mean level of the normal BMI category  contains supplementary material, which is available to authorized users. Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer death among females worldwide. It accounts for 25% of all cancer cases and 15% of all cancer deaths among females [. However, studies among premenopausal women are inconsistent and unclear. Some studies have shown a decreased risk for premenopausal breast cancer [There is sufficient evidence supporting a relationship between increased body mass index (BMI) and an increased risk for breast cancer among postmenopausal women –5. Howevt cancer , while ot cancer , 8, 9. Tt cancer  currentlWe searched PubMed, Medline, Web of Science, and Chinese academic databases including the China National Knowledge Infrastructure (CNKI) and Chinese Scientific Journals (VIP) databases for publications on the associations between BMI and breast cancer in humans, through December 31, 2015. The keywords were (obesity OR obese OR adiposity OR fat OR fatness OR “body mass index” OR BMI OR “body size” OR weight OR overweight) AND (“breast cancer” OR “breast carcinoma” OR “breast neoplasm”). References from reviews and meta-analyses were also searched for additional publications.Original article publication.Study design was a prospective cohort study.Study had BMI categories of no fewer than three, with a relative risk (RR) or hazard ratio (HR), and corresponding 95% confidence interval (CI) for the breast cancer presented for each BMI category.The studies provided the cases and person-years number for each BMI category.Studies were included according to the following criteria:From the selected studies the information extracted was: first author’s last name; publication year; country of origin; duration of follow-up; intervals of each BMI category; number of cases and cohort size of each category; relative risks and the 95% CI for each BMI category; and variables adjusted for in the multivariable analysis. The results that were adjusted by the most possible confounding factors were extracted. If data were reported by age category, hormone replacement therapy (HRT) use or other factors in one publication, they were considered as different studies. Two investigators (Cai and Wang) independently retrieved the data. The disagreement was resolved by mutual discussion.1375 studies were duplicated or irrelevant to our study according to titles or abstracts.18 studies were reviews or meta-analyses.35 studies were case-control studies.29 studies were excluded because the data needed were not available.Figure Using the Newcastle-Ottawa Scale (NOS), the quality of the included studies was assessed independently by two reviewers (Cai and Wang). This scale ranges from 0 to 9 stars and awards four stars for selection of study participants, two stars for comparability of studies, and three stars for the adequate ascertainment of outcomes. Scores of 0–3, 4–6, and 7–9 are viewed as low, moderate, and high quality, respectively.Because the incidence of breast cancer is low, HR is mathematically similar to RR. The results were reported as RR. The associations between BMI and the risk of breast cancer were quantified by comparing the highest versus the lowest categories. For the dose-response meta-analysis, the dosage value corresponding to each BMI was the median of the upper and lower boundaries. For the studies that did not report a median, the midpoint of closed categories was used. In the case of open ended highest category or lowest category, we assumed that the boundary had the same amplitude as the adjacent category , 12.The least squares estimation was conducted to account for the correlation with the logRR estimates across the BMI , 14. Theχ2 test. The quantification of heterogeneity was assessed by the I2 statistic. An I2 above 50% indicated high heterogeneity. A random effect model was implemented. Publication bias was identified with the Begg’s rank correlation test and Egger’s regression test [P < 0.05, except where there was a publication bias or heterogeneity when P < 0.10.Heterogeneity among studies was assessed using the ion test , 17. Theion test  were estAdditional file Among the studies, 12 were conducted in Europe , 41, 45,P = 0.01, I2 = 49%). Both Begg’s regression test (P = 0.705) and Egger’s correlation test (P = 0.347) showed no publication bias. Another 26 studies reported a link between BMI and breast cancer risk among postmenopausal women, with a summary RR of 1.33(95%CI: 1.20–1.48) . The Begg’s correlation test (P = 0.113) and Egger’s regression test (P = 0.603) showed no publication bias for these studies, as shown in Table In this study, we selected the RRs corresponding to the highest BMI categories as the highest dose, and the RRs corresponding to the normal BMI categories as the lowest dose. Eighteen studies reported on the relationship of BMI with premenopausal breast cancer risk, the summary RR was 0.94(95%CI: 0.81–1.11) see Fig , and hetst P = 0.47 showedP = 0.68, I2 = 0). When subgroup analysis was done for different geographical locations, it showed significant associations between BMI and breast cancer in Asia  and America , but poor association in Europe . Interestingly, for premenopausal women, an inverse significant association between BMI and breast cancer was detected in Europe , but no significant associations in America  or Asia .The subgroup analyses are shown in Table Additionally, we conducted a subgroup analysis stratified by follow-up year, the number of cases in the studies with shorter follow-up is lower, and consequently the power is reduced. In postmenopausal women, there was a significant association between BMI and breast cancer risk when the follow-up was above 5 years, while no significant association was detected when the follow-up was below 5 years. In premenopausal, there was no significant association between BMI and breast cancer risk in both subgroups.2. In terms of premenopausal women, a dose-response analysis including 18 studies with 25 comparisons was conducted, the non-liner relationship between BMI and breast cancer risk showed no statistical significance among total premenopausal women (χ [P = 0.168). In the subgroup analysis of premenopausal women, high BMI showed a decreased breast cancer risk in European women, and it showed a 2.3% decreased breast cancer risk per 1 kg/m2 compared to 21.5 kg/m2. The detailed information of the dose-response meta-analysis results and charts of premenopausal and postmenopausal women as well as the subgroup analyses are shown in Table For postmenopausal women, 26 studies including 62 comparisons reported RRs for corresponding BMI categories. The dose-response meta-analysis indicated a positive non-linear association between BMI and breast cancer risk among postmenopausal women. When compared to the mean level of the normal BMI range, it showed a 3.4% increased breast cancer risk per 1 kg/mP = 0.046). After using a trim-and-fill analysis, no study showed bias, and the results were unchanged.No publication bias was observed for any of the studies or the subgroup studies, except for studies on postmenopausal women in Europe using the Egger’s test  to 0.98(95%CI: 0.84–1.15), and the result was not influenced by any single study. Similarly, the sensitivity analysis in postmenopausal women showed stable results, which ranged from 1.30 (95%CI: 1.17–1.45) to 1.36 (95%CI: 1.23–1.51).2 increment of BMI in postmenopausal women.In this meta-analysis, there was a significant positive association between BMI and breast cancer risk among postmenopausal women but no correlation among total premenopausal women. Non-linear dose-response analysis showed that the breast cancer risk increased by 3.4% for every 1 kg/mI2 = 0) when stratified by ER status, suggesting ER status may be an interactive factor for the relationship between BMI and breast cancer risk. The lastest WCRF 2017 report showed that in postmenopausal women BMI was significantly positively associated with ER+ breast cancer, but not ER– breast cancers, it was consistent with our study.Heterogeneity was observed among the studies included in our meta-analysis. The diversity most likely comes from differences in study design, sample source and size, reference categories, different BMI cut-offs, different weight distribution, length of follow up, racial difference, ER status, HRT use or other potential confounders. We conducted a subgroup analysis to explore the heterogeneity and potential factors influencing the associations. The heterogeneity was zero  between studies remained, and the relationship between BMI and BC risk was not statistically significant. In WCRF 2017 report, dose-response meta-analyses for postmenopausal breast cancer by geographical location showed a statistically significant increased risk in North American and European studies, and a stronger association in Asian studies. The AICR&WCRF 2017 report includes 19 cohort and case-control studies of Europe (I2 = 75%), while 9 cohort studies of Europe (I2 = 71%) were included in this study. Potential confounding factors such as the difference in fat distribution, life styles or a possible interaction effect might be the source of the high heterogeneity. It necessitates further studies in order to provide more insights into such effects among European women.When stratified by geographical location among postmenopausal women, a significant non-linear dose-response relationship between BMI and breast cancer risk existed in American and Asian women, but not among European women. These results were inconsistent with the previous report conducted by Xia Xiaoping, et al.  Xia showFor premenopausal women, the AICR&WCRF report summarized the epidemiologic evidence available up to 2017 and concluded that there is a substantial dose response relationship for the inverse association between BMI and risk of breast cancer among premenopausal women. However, the non-significant association of BMI with risk of premenopausal breast cancer observed in this study is not consistent with the WCRF report. This may be because the studies included in this study differed from those included in AICR&WCRF 2017 report. The studies included in this article were all cohort studies which were more convincing, however, the number of studies included in AICR&WCRF 2017 report were more than this study, and more recent studies may be included. Therefore, the sample source and size, different fat distribution, follow-up year, race, ER status, HRT use or other confounders are possibly the reasons of differences.2. In American premenopausal women, there was no association between BMI and breast cancer. These results are comparable to those observed in a systematic review by Amadou et al. [2, which was similar to the report of Amadou et al. These results suggest that ethnicity or geographical location may be a factor influencing the relationship between BMI and breast cancer risk, but this conclusion is speculative. In this meta-analysis, breast cancer risk could be influenced by overweight or obesity according to different geographical locations, irrespective of menopausal status. The difference may be related to different genetic susceptibility among different ethnic groups, as demonstrated for breast cancer susceptibility genes (BRCA1 or BRCA2) mutations previously [A lack of data prohibited many subgroup analyses from being carried out. However, stratification by geographical location demonstrated an inverse significant association between BMI and breast cancer among European premenopausal women but not among Asian and American premenopausal women. This conclusion is consistent with the AICR&WCRF 2017 report when stratified by geographical location. When compared to women with a normal BMI , women with a higher BMI were at a decreased risk of breast cancer risk by 2.3%. In Asian women, the results showed a positive non-linear relationship between BMI and breast cancer when BMI was above 25 kg/mu et al. , which eu et al. , in addieviously .BMI, adult weight gain, body weight and fat distribution may play a role in the prognosis of breast cancer separately or together in combination. IARC working group , 60 repoThe studies included in this meta-analysis were all cohort studies, with large sample sizes, and adequate subgroup analyses. Moreover, the confirmation of the breast cancer cases was done by diagnosis, hospital medical record or linkage data. Thus, the results were persuasive, and were less likely to suffer from recall bias and selection bias. However, some limitations also exist for this meta-analysis; the measurement of BMI was determined mostly by questionnaire investigation or self-reported, and people tended to report their weight lower and height higher than reality. The adjustments factors of each study were not the same, thus the RRs we extracted from the studies were possibly affected by potential confounders even after selecting the adjusted RRs. In addition, the categorization of BMI in a number of articles included in this study was not in accordance with the WHO standard, thus misclassifications may be caused when we conduct the highest versus the lowest BMI meta-analysis.s. In postmenopausal women, breast cancer risk increased along with increasing BMI. In premenopausal women, there was no signification between BMI and breast cancer. This is inconsistent with the 2017 report of AICR&WCRF which showed higher BMI is defined as probably decreasing breast cancer risk. However, most conclusions stratified by various factors in this study are consistent with AICR&WCRF.In conclusion, BMI had different effects on premenopausal and postmenopausal breast cancer riskSubgroup analyses suggested that the geographical location or genetic factors may influence the relationship between BMI and breast cancer. Further research is needed to provide more continuous updating insights.Additional file 1:Study characteristics of published cohort studies on body mass index(BMI) and breast cancer risk. (DOC 725 kb)Additional file 2:The related data and materials in this study. (ZIP 1785 kb)