Very limited data exist on the role of NLR and risk of BC. The BREOGAN study is a population-based case–control study conducted in Galicia, Spain. We examined the WBC- and NLR-BC relationships. The risk of BC increased with increasing levels of neutrophils percentage (NE%) (multivariable OR for the highest category (95% CI) = 2.14 (1.39–3.32), P-trend < 0.001) and of the NLR (multivariable OR for the highest category (95% CI) = 1.93 (1.26–2.97), P-trend < 0.001). Lymphocytes absolute (L#) and percentage (L%) were associated with a decreased risk of BC (multivariable OR for the highest category (95% CI) = 0.54 (0.35–0.83), and 0.51 (0.33–0.79), P-trend = 0.001 and < 0.001, respectively). The NLR-BC association was more pronounced among Luminal A BC (multivariable OR for the highest category (95% CI) = 2.00 (1.17–3.45), P-trend < 0.001), HER2-negative BC (multivariable OR for the highest category (95% CI) = 1.87 (1.16–3.02), P-trend < 0.001), and those with high total cholesterol and low H It was responsible for 6,421 deaths . Breast cancer is the most frequent cancer in Spain and ranks the fourth in cancer-related mortality. There has been a 30% increase in breast cancer incidence between 2012 and 20181.In 2018, 32,825 new breast cancer cases were diagnosed in Spain, representing 12% of all cancer cases, and 29% of all cancers in women3. Similarly, there is abundant evidence for an adverse prognostic value of neutrophil to lymphocyte ratio (NLR) on breast cancer. Multiple studies have shown that higher NLR was associated with poorer survival9, and a recent meta-analysis found that higher NLR was associated with both worse disease-free survival and overall survival6. Several previous studies have found that higher NLR was also associated with more advanced or aggressive breast cancer11.High concentration of blood neutrophils is seen in patients with advanced cancer and are associated with poor survival13. NLR is higher in patients with more advanced cancers10, and correlates with poor survival in many cancers15. Thus, NLR, a simple and inexpensive biomarker, has been introduced as a significant prognostic factor in many tumour types3. However, it has not been accepted in many clinical settings since neutrophilia can be also the result of elevated granulopoiesis and, therefore, may not be an adverse sign for cancer progression3. Another reason is that neutrophilia is associated with poor clinical outcome in all cancers except one, stomach cancer, in which a high NLR is a marker of good prognosis16. However, the evidence for a detrimental effect of circulating neutrophils and the NLR on cancer prognosis and survival altogether, with only the exception of gastric cancer, is very consistent.For this reason, the ratios between neutrophils in blood and other leukocytes, as the NLR, have been suggested as a prognostic value in cancer18. Two of the three studies compared breast cancer cases with benign breast disease (BBD) controls17, and the third one used healthy controls as reference group18. In one of the studies17, the NLR was significantly higher in breast cancer patients compared with the BBD group, and patients with NLR > 1.67 were related to an increased risk of breast cancer. This finding was consistent with the other two studies18, suggesting that NLR could be an independent risk factor for breast cancer.To our knowledge, only three small studies, conducted in Asian women, assessed the association between NLR and the risk of breast cancerIn the present study, we examined the association of WBC including neutrophils, lymphocytes and monocytes, and the NLR, on the risk of breast cancer overall, and by major subtypes, menopausal status, and grade and morphology, in Spanish women.21, obesity was associated with an increased risk of breast cancer (multivariable OR (95% CI) = 2.02 (1.30–3.17), P-trend = 0.003). Use of oral contraceptives was also associated with an increased risk of breast cancer (multivariable OR (95% CI) = 2.01 (1.22–3.30)). Similarly, ex-smokers and current smokers were associated with an increased risk of breast cancer (multivariable OR (95% CI) = 2.18 (1.14 – 4.14), and 1.86 (0.87–3.93), for ex-smokers and current smokers, respectively). A history of first- and/or second- degree relatives with breast and/or ovarian cancer was associated with an increased risk of breast cancer (multivariable OR (95% CI) = 2.46 (1.49–4.06)). Consistent with previous studies, invasive ductal carcinoma was the most frequent histological type (80%), and the distribution of the Luminal A, Luminal B, HER2 overexpressed, and TNBC subtypes were 63%, 13%, 8% and 15%, respectively. Mean tumor size in cm was 2.1.Table 2O2) (multivariable OR for the highest versus the lowest category (95% CI) = 0.26 (0.13–0.51), P-trend < 0.001).Table Table 2O2 levels. The NLR-breast cancer association was more pronounced for women with high total cholesterol levels (multivariable ORs (95% CI) = 2.79 (1.60–4.91), and 1.41 (0.66–3.04), P-trend < 0.001 and 0.09), for high and low total cholesterol levels, respectively), and low urinary H2O2 levels (multivariable ORs (95% CI) = 1.74 (0.59–5.43), and 3.30 (1.12–10.40), P-trend = 0.31 and 0.056, respectively), for high and low urinary H2O2 levels, respectively).Table We also examined the NLR-breast cancer association by tumour grade, stage, and histology  = 1.68 (1.12–2.58), P-trend = 0.01). We also examined PLR by tumor grade, stage, and histology 17, and only one used healthy controls as comparison group18, as in our study. In one of the studies17, NLR was significantly higher in the breast cancer group compared with the BBD group, and patients with NLR > 1.67 were associated with an increased risk of breast cancer. This finding was consistent with those of the other two studies18, suggesting that NLR could be an independent risk factor for breast cancer. In one study, the best cut-point value for NLR was 2.96 and it was replicated, giving a sensitivity and a specificity of about 80% and 76%, respectively, in patients with breast cancer. In another study, the cut-point value for the NLR was 2.56.There is abundant evidence for the adverse prognostic value of NLR on breast cancer. Multiple studies have shown that higher NLR was associated with adverse survival24. There is evidence for a detrimental role of neutrophils during tumour progression, but there is also solid evidence of an anticancer effect. Activated neutrophils can kill tumour cells in vitro25 and in vivo 26. Neutrophils only possess this antitumor effect after they have been activated. It was shown that a colon adenocarcinoma cell line transfected to express G-CSF (Granulocytic Colony Stimulating Factor) lost tumour activity after massive concentration of neutrophils at the tumour site27. In that study, purified neutrophils from SR/CR mice directly killed cancer cells in vitro and entirely conveyed resistance to recipient wild-type mice27. In addition, the cancer progressively disappeared after penetration of a big amount of neutrophils and a lesser amount of lymphocytes into the rest of the tumour tissues28. Neutrophils from tumour-bearing animals displayed enhanced reactive oxygen species (ROS) such as superoxide anion generation and phagocytosis, which led to reduced tumours and less metastatic foci in lungs30.Neutrophils are the most frequent (40% to 75%) type of white blood cells in mammals, and a crucial part of the innate immune system32. In resting neutrophils cells, NADPH oxidase is inactive. Neutrophils migrate from blood to tissue and it is at the tissue level where they become activated through release of ROS such as H2O2, and then subsequently kill cancer cells. As neutrophils-induced ROS have not just been found extracellularly but also intracellularly33, accumulating that they participate as signalling molecules in cellular pathways such as apoptosis of tumour cells34.To do their anti-cancer effect, neutrophils have a remarkable ability to produce a high amount of ROS which is the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidaseThis anticancer, pro-apoptotic, effect of activated neutrophils in tissue underlies our finding of an elevated risk of breast cancer with increasing levels of blood NE% and NLR. Higher neutrophil levels in blood, where they are inactive, may indicate a low corresponding concentration in tissues, where they are active. Inactive neutrophils are unable to release ROS or cause ROS-induced apoptosis of tumour cells. Neutrophils leave their patrol zone in the blood and go to the tissue where they commit suicide after becoming activated by releasing ROS and inducing apoptosis.17. In another study of ovarian cancer cases, total neutrophil counts and WBC were significantly increased in premenopausal women who were Jewish35, and premenopausal women who had a family history of breast or ovarian cancer also had higher NLR and lower lymphocyte counts35.In the present study, we found NE%- and NLR-breast cancer associations to be more apparent when restricted to postmenopausal women. The mechanism underlying this finding is unknown. One possibility is reduced statistical power in premenopausal women, since, although we did find an increased risk in both, postmenopausal and premenopausal women, our study had smaller sample size and therefore limited precision for the latter . Another possibility is that a different mechanistic pathway may exist between breast cancer patients and healthy controls by menopausal status, however, we found no evidence in the literature in support of this. A previous study conducted in Chinese women using BBD as controls examined the NLR-BC association by menopausal status and found that intermediate and high NLR levels significantly increased breast cancer risk compared to very low NLR levels in both, pre- and postmenopausal women2O2, by neutrophils is called oxidative burst and it may be responsible for their anticancer effect. We measured urinary hydrogen peroxide H2O2 in these patients and found that the detrimental effect of NLR on breast cancer was more pronounced among women with low levels of H2O2 in urine, as expected (multivariable ORs (95% CI) = 3.30 (1.12–10.40), P-trend = 0.056, and 1.74 (0.59–5.43), P-trend = 0.31 for low and high levels of H2O2, respectively).The rapid release of ROS such as superoxide radical and hydrogen peroxide, H2• − , which are transformed into another ROS such as H2O2. It has been found that one of the consequences of an increase of total cholesterol amount is an inhibition of the activity of NADPH oxidase in the presence of proinflammatory signals. This would imply a modification of the response to the signalling regulation36.We also found that total cholesterol may modify the NLR-breast cancer relationships. Specifically, we found that the NLR effects on breast cancer risk were more pronounced in women with high levels of total cholesterol. One possible reason is that cholesterol inhibits NADPH oxidase, the crucial enzyme for ROS production and ROS-induced apoptosis of the cancer cell. NADPH oxidase catalyses a very intense production of superoxide ions O38. In another study including young European women and men (median ages 25 years), neutrophil counts were observed to be higher in women39. Higher neutrophil counts in women were found in a study of Africans of 18–55 years of age40. In another study of healthy adults of several races, females had higher granulocyte counts but lower monocyte counts41.Breast cancer is much higher in women than men. Several prior studies have reported higher blood neutrophils rates in females than males37. In a prior study, it has been shown that in healthy women aged around 50 years of age, there is an important drop of NE% and a significant rise of LY%, with a subsequent substantial fall in the NLR; however, in women older than 70 the NLR increased again37.Early age at menopause is an established protective factor for breast cancer, and, in healthy women, menopause decreases the NLR43. Estradiol has been shown to inhibit neutrophil apoptosis44, and decrease lymphocyte production in the bone marrow46. Therefore, in healthy women after menopause, the significant reduction of estradiol levels will likely result in higher neutrophil apoptotic rates and increased lymphocyte production, leading to reduced NE% and risen LY%, and reduced NLR compared to healthy premenopausal women. The neutrophil-apoptotic pathway could be one underlying mechanism behind the significant drop of NE% and substantial rise of LY% in women of around 50 years of age and that women of < 50 years of age have significantly higher NE% and lower LY% than women of 51–70 years of age from that study37. These findings, shown by this37 and other studies47, are also consistent with our own study results, as our mean neutrophil and NLR values in healthy women (controls) are higher in premenopausal than postmenopausal women .In healthy women, estradiol levels dramatically fall (by ~ 70%) during menopause, which commonly occurs around 50 years of age48. HER2 over-expression occurs in approximately 15–30% of breast cancers49, and it is strongly associated with poor prognosis50. However, we found a more pronounced risk from NLR among HER2-negative breast cancer.We also found an increased risk among both HER2-negative breast cancer, and, less pronounced, HER2-positive breast cancer. In a study, HER2 expression, among other factors (increased tumour size and PLR), was significantly associated with an elevated NLRWe will next describe the relationships between NLR, ROS and stem cell divisions. Peripheral blood neutrophils could be a marker of baseline apoptosis levels in tissues. Decreased peripheral blood neutrophils may indicate increased baseline apoptosis level in tissues, as a marker of the innate levels of ROS-induced apoptosis.Among the mechanisms of tumour cell killing, activated neutrophils clearly have the potential of directly killing tumour cells, through ROS that can directly kill tumour cells. In addition to this direct anti-cancer effect, another cell killing mechanism relates to the immune system, also through the rapid release of ROS or the stimulation of T-cell response.51. Mesenchymal stem cells (MSCs) have been shown to suppress neutrophil activation through the inhibition of ROS release and subsequent apoptosis. Therefore, NE% and/or NLR could be a marker of the number of stem cell divisions. An increased number of stem cells divisions would imply a decreased activation of neutrophils and elevated numbers in blood52.It has been shown that the number of endogenous mutations resulting from stem cell divisions correlates with cancer incidence53. PD-L1 functions as an immunomodulatory molecule, and it is broadly expressed in a variety of immune cells including neutrophils, T cells, B cells, dendritic cells, and monocytes. Increased NLR has been reported to be an independent poor prognostic indicator and its normalization following treatment has been found to predict progression-free survival in cancer patients undergoing treatment with PD-1/PD-L1 inhibitors54.Human tumour macrophages express PD-L1Results of this study must be interpreted in light of its limitations. First, measurement of WBC can be difficult, and misclassification can occur. Our WBC levels were measured in standard clinical hospital laboratories, so measurement error is expected to be reduced. Also, any misreporting of blood WBC is unlikely to be influenced by tumour subtype. Residual confounding from alcohol intake, mammographic density, and female steroid hormones such as HRT can also influence the WBC-BC association. Also, for some breast cancer subtypes, such as HER2 overexpressing tumours, we lacked precision. Another limitation of our study was the lack of pre-diagnostic data from the cases to be able to conclude that NLR is positively related to breast cancer. Another limitation is lack of data on TILs in the tumors of our patients, which would strengthen the mechanistic pathway of apoptosis being one mechanism responsible for neutrophils anti-cancer effect. Another limitation is that we did not have enough follow-up on breast cancer survival or recurrence and thus could not explore the NLR-breast cancer effect on survival and/or recurrence, although this association has been confirmed by multiple studies. Conversely, strengths of our study are the direct measurements of WBC in our study population, and the available information on HER2, in addition to hormonal receptor status.2O2, and among grades II and III than among grade I breast cancer. Neutrophils, which are inactive in blood, must migrate to tissue to become activated and liberate ROS to kill tumour cells, after which, they commit suicide and die. If there are increased neutrophils in blood it may indicate that they are not being sufficiently activated and therefore not releasing ROS, implying low ROS levels in tissues. Our finding that increased NE% and NLR in blood is associated with increased risk of breast cancer may indicate that neutrophils are not being sufficiently activated in breast tissue to induce neutrophil-induced apoptosis. Increased peripheral blood neutrophils could be a marker of decreased baseline or innate apoptosis levels in tissues.We found elevated NLR to be associated with increased risk of breast cancer, principally Luminal A and HER2-negative breast cancer. To our knowledge, this is the first study examining the association between NLR and risk of breast cancer in non-Asian women, overall and by major breast cancer subtypes. The NLR-breast cancer associations appeared to be more pronounced among women with high levels of total cholesterol and low levels of H57. Cases comprised 1,766 women with invasive breast cancer diagnosed and treated between 1997 and 2014 at the Clinical University Hospitals of Vigo (CHUVI) and Santiago (CHUS), as described in previous studies. Controls were 1,205 women free of cancer, except non-melanoma skin cancer, living in the same population health area as cases. Response rates were 98% and 99% for cases and controls, respectively. Ethics approval for this study was obtained from the CEIC, Comité Ético de Investigación Clínica de Galicia , responsible for the oversight of both university hospitals, CHUVI and CHUS, and family, primary clinics from where all participants were recruited. All participants provided written informed consent. The study was conducted in accordance to the Helsinki Principles of 1975, as revised in 1983.The Breast Oncology Galician Network (BREOGAN) includes a population-based case–control study conducted in the cities of Santiago de Compostela and Vigo, Spain, within a geographically defined health region that covers approximately one million inhabitants. Data collection methods have been previously described56, risk factor information was collected through a risk factor questionnaire adapted from the Ella Binational Breast Cancer Study59 to meet the needs of the population in Spain. Clinical and histopathological information was abstracted from computerized medical records by trained physicians. The following variables were recorded: level of education , lifetime breastfeeding , age at menarche, age at first full-term pregnancy, parous (categorized as never vs. ever pregnant), number of pregnancies , age at menopause , menopausal status at diagnosis , oral contraceptive use , hormone replacement therapy , smoking status , family history (categorized as none vs. one or more first and/or second degree relatives with breast and/or ovarian cancer). Alcohol consumption was evaluated by the number of alcoholic drinks consumed regularly per week in last year before reference date. The alcohol categories correspond to the CDC definition  of light/moderate and heavy drinking among women.Similar to previous studiesData on circulating WBC including neutrophils, monocytes, and lymphocytes before date of diagnosis for cases and date of interview for controls were abstracted from Ianus, the Galician universal computerized medical history. WBC count was done through automated machine counting of cells. Briefly, withdrawal of peripheral blood by venipuncture was performed during fasting. Total WBC and differential counts were performed on the peripheral blood samples by using the ADVIA 2120 Hematology analyzer . We calculated neutrophil to lymphocyte ratio (NLR) by dividing the number of neutrophils by number of lymphocytes from peripheral blood sample.56, histopathological information was abstracted from computerized medical records by trained physicians. Immunohistochemistry (IHC) analyses on paraffin-embedded material have been previously performed following standard procedures in Galician hospitals to determine the status of ER and PR. In every tumour, 4-μm histological sections were cut and stained with haematoxylin and eosin for histopathological examination according to the criteria of the World Health Organization60. Histological grading was evaluated using the Nottingham modification of the Bloom-Richardson system61. Similar to previous studies56, immunohistochemistry (IHC) analysis on paraffin-embedded material was performed using a universal second antibody kit that used a peroxidase-conjugated labelled dextran polymer , with antibodies for ER , PR . Negative and positive controls were concurrently run for all antibodies with satisfactory results. Cells were considered immunopositive when diffuse or dot-like nuclear staining was observed regardless of the intensity of the staining,only nuclear immunoreactivity was considered specific. The number of positive cells was counted by two different observers independently. Whenever necessary, a consensus was reached using a double-headed microscope. ER and PR were considered positive when the percent of immunostained nuclei was ≥ 10%.Similar to our previous studies56, IHC analyses were performed to determine HER2 status (Dako). No immunostaining (0) or weak membrane immunostaining (1 +) was considered low HER2 expression (HER2). Strong membrane immunostaining (3 +) was considered HER2 overexpression (HER2 +). Moderate membrane staining , grade , histology type , and tumour size (cm). As previously described in previous studies21, of the 1766 women who participated in the study, 100 had unknown ER status, 114 had unknown PR status, and 340 had unknown HER2 status. In the same manner, one hundred and eighty-four women had unknown grade, 14 had unknown histological type and 144 had unknown tumour size21. Sixty-two women had unknown age at menarche, and 48, out of 1,443 parous women, had unknown lifetime breastfeeding21. Urinary H2O2 was measured by ELISA test using K034-F1 Hydrogen Peroxide Fluorescent Detection Kit, de Arbor, 339,00 Arbor Assays DetectX® Hydrogen Peroxide Fluorescent Detection Assay, with all 4 °C stable reagents.Similar to previous studies2O2, were calculated based on distribution among controls. Briefly, cut points for NLR, based on tertile distribution among control group, were comparable to cut points based on previous studies on the effect of the NLR on breast cancer survival and/or prognosis13. Similarly, cut points for PLR were based on tertile distribution among control group, which were comparable to cut points based on previous studies on the effect of the PLR on breast cancer62. Cut points for serum total cholesterol levels were based on tertile distribution among the control group , which are equivalent to standard levels of normal and borderline/high total cholesterol levels . Cut points for urinary H2O2 levels were also based on tertile distribution among the control group . All statistical analyses were performed using the R statistical software version 3.3.3. All reported test significance levels  were two-sided.The association of breast cancer with circulating WBC and NLR was measured by odds ratios (ORs) and corresponding 95% confidence intervals (CIs) using unconditional polytomous logistic regression. Analyses were initially adjusted for the following established risk or protective factors for breast cancer: reference age (age at diagnosis for cases and age at interview for controls), age at menarche, parity, breastfeeding, menopausal status, weight, height, oral contraceptive use, hormone replacement therapy and family history of first-degree relatives with breast and/or ovarian cancer. Results were virtually unchanged after adjustment for all these variables or only age, age at menarche, parity, menopausal status, and BMI, therefore we present results adjusted for the latter. Outcome (dependent) variables were breast cancer subtypes defined by ER, PR, and HER2 status ), compared to controls (comparison group), and explanatory variables were circulating WBC . Cutoff points for subgroup analysis, i.e., NLR, PLR, total cholesterol and urinary HSupplementary information.


An upgraded understanding of factors (sex/estrogen) associated with survival benefit in advanced colorectal carcinoma (CRC) could improve personalised management and provide innovative insights into anti-tumour mechanisms. The aim of this study was to assess the efficacy and safety of cetuximab (CET) versus bevacizumab (BEV) following prior 12 cycles of fluorouracil, leucovorin, oxaliplatin, and irinotecan (FOLFOXIRI) plus BEV in postmenopausal women with advanced KRAS and BRAF wild-type (wt) CRC.Prospectively maintained databases were reviewed from 2013 to 2017 to assess postmenopausal women with advanced KRAS and BRAF wt CRC who received up to 12 cycles of FOLFOXIRI plus BEV inductive treatment, followed by CET or BEV maintenance treatment. The primary endpoints were overall survival (OS), progression-free survival (PFS), response rate. The secondary endpoint was the rate of adverse events (AEs).p=0.007); Median PFS was 10.7 months  for CET vs. 8.4 months  for BEV . Dose reduction due to intolerable AEs occurred in 29 cases .At a median follow-up of 27.0 months (IQR 25.1–29.2), significant difference was detected in median OS (17.7 months [95% confidence interval [CI], 16.2–18.6] for CET vs. 11.7 months  for BEV; hazard ratio [HR], 0.63; 95% CI, 0.44–0.89; CET tends to be superior survival benefit when compared with BEV, with tolerated AEs. Colorectal cancer (CRC), an aggressive disease, is historically the prominent cause of cancer-related deaths worldwide , 2. DespThe assessment of mutational status in the RAS and BRAF genes gained increasing importance for treatment of CRC . For advThis retrospective cohort study was performed in postmenopausal women with advanced KRAS and BRAF wt CRC, aiming to balance a favourable effect of survival benefit and adverse events (AEs) in these patients who underwent CET or BEV maintenance treatment following prior FOLFOXIRI plus BEV.Data regarding general condition, drug delivery, and survival status for postmenopausal women with advanced CRC were retrieved from institutional prospectively maintained databases from Jan 3, 2013, to Jan 15, 2017. These data were coded in all participating organizations with a standard protocol using the International Classification of Diseases (ICD) v10.0 , adequatA retrospective, multi-centre study was performed in which eligible postmenopausal women underwent up to 12 cycles of FOLFOXIRI plus BEV inductive treatment , followeGenomic DNA was purified from formalin-fixed, paraffin-embedded samples of primary tumor tissue, and was extracted using the Qiamp FFPE DNA kit  following the manufacturer’s instructions. Assessment for KRAS and BRAF mutational status was centrally performed for each individual included in the study by a pyrosequencing approach, as previously described . All assThe primary endpoints were OS, PFS, and response rate. OS was defined as the interval from the beginning of maintenance to death from any cause or final follow-up. PFS was defined as the time from the beginning of maintenance to clinical or radiological progression or death from any cause, whichever occurred first. The response and progression were assessed in accordance with RECIST v1.1. The secondary endpoint was the AE rate. The severity of AEs was graded using the US National Cancer Institute’s Patient-Reported Outcomes Version of the Common Terminology Criteria for Adverse Events  and appropriate 95% confidence intervals (CIs) were calculated using a Cox proportional hazard model. All statistical tests were two-sided, and the significance level was set at 0.05. All data were analysed using SPSS software, v 26.0 .n=100, mean age 64.2 years [SD 9.5] and BEV: n=104, 64.5 years [SD 8.7]) who were eventually included for study eligibility   and the control rate (p < 0.001) between groups.At final follow-up, significant difference was detected in median OS . The difference in OS corresponded to an HR of 0.63  due to vomiting, nausea, or fatigue. AEs occurred earlier in the CET group, with nausea having a longer duration in the CET group than in the BEV group (median time to first occurrence 5 days [IQR 3–11] vs. 11 days [p=0.030). The most frequent grade 4 AE was diarrhoea .With regard to the safety profile, the most frequent drug-related AEs at final follow-up are summarized in Table  11 days , 21–29 f 11 days  vs. 5 daOur study demonstrates that CET maintenance treatment is inferiorly tolerated but has a moderate, if any, survival benefit when compared with BEV maintenance treatment. The superiority of CET over BEV in the clinical setting tends to be positive, which does not deviate from prior studies involving patients with advanced KRAS and BRAF wt CRC , 22, 23.p=0.048); the median PFS was 10.9 months  for CET plus chemotherapy versus 7.7 months (95%CI 7.4–9.0) for chemotherapy alone , and confirms the benefit of adding CET to first-line chemotherapy. Consistent with our findings, a distinct separation of OS curves favouring the continuation of CET rather than the switch to BEV was detected. Consistent results have also been reported in the CECOG/CORE 1.2.002 study [Our findings align with those obtained from published studies , 24 and 02 study , which s02 study .In a randomized clinical trial , 1137 enFrequent debate has occurred over the influence of postmenopausal women who tend to be even less responsive to the present treatment regimen , 36. TheAs predicted, the safety profiles of CET and BEV were manageable. In light of our findings, 54% of cases underwent more than 2 drug-related AEs, the majority of which were grade 1 to 2. The frequency of AEs was in line with the known safety profiles of CET and BEV. BEV seems to be safer than CET. The more frequent grade 3 or 4 AEs in response to CET than to BEV were mainly attributed to skin AEs, which was generally controllable.Several limitations should be acknowledged in this study. First, the retrospective nature has inherent limitations, and potential confounding variables  could not be processed very well. Second, generalisability is lacking because the study population involved only postmenopausal women with advanced CRC. Replication of these findings in a prospective cohort trial is required to clarify the generalisability of results. Third, our data were gathered from different institutions, in which diagnostic procedures may be different. Nonetheless, the multicentre data were merged using standardised methods, which provides reliability across these institutions.The results reported in the current study might echo a growing body of evidence showing that for postmenopausal women with advanced KRAS and BRAF wt CRC treated with prior 12 cycles of FOLFOXIRI plus BEV, CET appears to be inferiorly tolerated but has a moderate, if any, survival benefit compared with BEV. Future clinical trials of the efficacy and safety of CET versus BEV in an analogous setting might benefit from stratification of analyses according to the study population.


Asiadodis yunnanensis was characterized in this study. The circular molecule is 15,416 bp in length (GenBank accession no. MN037794), containing 13 protein-coding genes (PCGs), two ribosomal RNA (rRNA) genes, 22 transfer RNA (tRNA) genes. The nucleotide composition is asymmetric , with an overall A + T content of 75.3%. The gene arrangement of A. yunnanensis is identical to that observed in other praying mantises. Seven PCGs are initiated with typical ATN start codons, Four genes  begin with TTN, nad1 begin with GTT, and nad5 use CTT, as initiation codon. Twelve PCGs stop with complete termination codon TAA and TAG, whereas nad5 uses incomplete termination codon (T––). Twenty reading frame overlaps and seven intergenic regions are found in the mitogenome of A. yunnanensis. The phylogenetic relationships based on 13 PCGs show that A. yunnanensis clusters closest to the species of Mantidae.The complete mitochondrial genome of the praying mantises  Asiadodis was established by Roy  and stored in the praying mantises specimen room of College of Plant Medicine, Qingdao Agricultural University with an accession number 01-XXBNJH.d by Roy  to distiA. yunnanensis is a circular molecule of 15,416 bp in length (GenBank under accession no. MN037794) and contains 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (rrnL and rrnS). The nucleotide composition of A. yunnanensis mitogenome is asymmetric , with an overall A + T content of 75.3%. The AT-skew and GC-skew of this genome were 0.049 and −0.223, respectively. The gene organization of A. yunnanensis is similar to that observed in other praying mantises , Glu (E), and Trp (W) are most frequently used. Moreover, the A + T content were also reflected further in the codon usage: the relative synonymous codon usages showed that A. yunnanensis used more NNA and NNT codon. The rrnL was 1313 bp in length with A + T content of 79.3%, and rrnS was 767 bp in length with A + T content of 74.9%.The A. yunnanensis mitogenome start with a typical ATN (ATA and ATG) codon, four genes begin with TTN , nad1 uses GTT and nad5 use CTT as initiation codon. Twelve PCGs stop with complete termination codon (TAA and TAG), whereas nad5 uses incomplete codon (T––) as termination codon. Based on the Maximum-likelihood analyses, we constructed the phylogenetic relationships of A. yunnanensis and 23 other praying mantises based on the 13 PCGs amino acids using RAxML. Cryptocercus kyebangensis was used as an outgroup. The results have shown that A. yunnanensis is closely clustered with other species in family Mantidae (Seven PCGs in Mantidae , which a


Low level viremia (LLV) often occurs during antiretroviral therapy (ART) against HIV-1. However, whether LLV increases the risk of virologic failure (VF) is controversial because of the non-uniform definitions of LLV and VF.A long-term first line regimen ART cohort from 2002 to 2018 from Shenyang, northeast China, was retrospectively studied. All participants were followed up every 3 to 6 months to evaluate the treatment effect. The high-risk LLV subgroups leading to VF (with strict standards) were explored with Cox proportional hazards model and linear mixed-effect model. The association factors of high-risk LLV were further explored using multivariate logistic regression analyses.HLLV = 5.93, and HRHLB = 2.84, p <  0.05 respectively). Moreover, HR increased with prolonged duration of LLV. Independent factors associated with high-risk LLV included the zenith baseline viral load (VL) above 6 log copies/ml , nadir baseline CD4 + T cell counts below 200 cells/mm3 , Manchu , ART over 60 months , AZT + 3TC + NVP  or DDI-based regimen , and subtype B′ infection .A total of 2155 HIV-1 infected participants were included; of these, 38.7% showed LLV. Both high level LLV (HLLV) and any other level LLV coupled with high level blip (HLB) showed higher risk of VF (hazards ratios, HRIn case of VF with strict standards, high-risk LLV leading to VF includes VL above 400 copies/ml, occurring at least once. Serious laboratory indicators or advanced stage of infection, long term ART and subtype B′ infection might also predict the occurrence of high-risk LLV. Human immunodeficiency virus type 1 (HIV-1) rapidly replicates mainly in CD4 + T cells and is released into the blood. Antiretroviral therapy (ART) can limit virus replication to less than 50 copies/ml and effectively prolong the life of HIV-1 infected patients , 3. The The impact of LLV on clinical prognosis is still controversial. An earlier national observational cohort study from USA found that an LLV above 400 copies/ml could not predict AIDS or death . HoweverAlthough most studies suggest that LLV contributes to VF, the potential risk factors of LLV have not been sufficiently investigated. Several HIV cohort studies from developed countries where subtype B was predominant found that the high zenith baseline VL and low nadir baseline CD4 + T cell counts were associated with the risk of LLV , 22–25. The aims of the current study, which used clinical data from a long-term first line regimen ART cohort in China, were to determine the characteristics of high-risk LLV associated with VF, and to elucidate the factors contributing to this kind of LLV, by considering both the level and the duration of LLV. The results of this study might provide new evidence for the occurrence and contribution of risk factors of LLV, and thereby help to optimize ART treatment in resource-limited setting.This retrospective study was based on an open HIV-1 infection treatment cohort in a big comprehensive hospital in Shenyang, northeastern China between 2002 and 2018. All subjects received first-line ART regimen according to Chinese guidelines for diagnosis and treatment of HIV/AIDS . This inThe inclusion criteria for this study were as follows: (1) participants received first-line ART regimen in China (2 NRTIs and 1 NNRTI); (2) VL had been controlled to less than 50 copies/ml after 6 months treatment; (3) participants were followed-up for at least 12 months. Patients were excluded from this study if their VLs were incomplete. This study was approved by the ethics committee of the First Affiliated Hospital of China Medical University. In addition, informed consents were obtained from all participants.In this study, we used the definition of LLV based on the WHO guideline ; LLV wasp-value of less than 0.05 was considered statistically significant.The CD4 lymphocyte count and the results of viral load were included as quantitative variables. The K-S normal test method was used to evaluate the normality of variables. The comparisons were made between LLV categories by log-rank tests. Chi-squared test was used for comparisons among other variables. The incidence of VF was calculated by the total number of VF occurrence/total follow-up time * 100% , 33. The3  of participants experienced at least one episode of LLV. In terms of the LLV level, participants belonging to LLLV, MLLV, HLLV, and HBL groups accounted for 52.1% (435/835), 18.0% (150/835), 14.3% (119/835), and 15.7% (131/835), respectively. In terms of the duration of LLV, participants with Blip, LLV lasting for 3–6 months, 6–12 months, and above 12 months accounted for 72.1% (602/835), 11.4% (95/835), 11.4% (95/835), and 5.1% (43/835), respectively  between LLV level and duration categories. Compared with the control group (incidence rate (IR): 0.52/100 person years), those with > 400 copies/ml  and LLV duration of > 12 months  had the least favorable Kaplan–Meier curves and higher risk of VF  and HBL  were associated with significantly increased risk of subsequent VF compared with the control group .The VF rate increased along with increasing level of LLV . In addition, any level LLV (> 3 months) coupled with HBL also showed increased risk of VF . In contrast, no significant increase of VF was observed among MLLV and LLLV groups, even in those that lasted for > 12 months .Based on LLV duration analyses, we found that the risk of VF increased in the HLLV group for any duration; moreover, the HRs increased with prolonged duration . LLLV showed no increase of VF in both models  and lower nadir baseline CD4 + T cell counts  showed increased risk of high-risk LLV. Other associated factors included Manchu , ART for over 60 months , receiving AZT + 3TC + NVP regimen  or DDI + NRTI+NNRTI regimen  and subtype B′ infection .We further assessed the socio-demographic information, transmission route, clinical data, and laboratory test results to explore the factors associated with high risk LLV. Using chi-square tests, we found that the different groups of zenith baseline VL, nadir CD4 + T cell counts, and other demographic information, clinical data, and laboratory tests had varied frequencies of high risk LLV Table . MultiplIn this study, we observed LLV from 38.7% of participants in a local long-term ART cohort from northeastern China. With a strict definition of VF, we found that the subgroup of high level LLV of above 400 copies/ml and any level LLV coupled with blip of above 1000 copies/ml were high-risk LLV associated with VF. Moreover, we found that the duration of LLV also played an important role in contributing to the risk of VF. The association factors included high zenith VL, low nadir CD4 + T cell counts, long term ART, Manchu, and subtype B′ infection.The occurrence of LLV has not been reported in China, where uniform ART is provided freely; however, there is a lack of individual monitoring and treatment. In this study, we observed LLV from 38.7% of participants, and this was much higher than the LLV occurrence 15–20%) in resource-rich settings with similar LLV definition criteria (50–1000 copies/ml) , 37. Mul0% in resUsing this new VF definition, we revealed that both level and duration of LLV were factors associated with VF. In this study, only LLV above 400 copies/ml could increase the risk of VF, which differed from several recent studies that implied a load of as low as 20 copies/ml might lead to VF , 21. SucMoreover, we also found that any level of LLV coupled with HLB increased the VF risk. In previous studies, HLB (> 1000 copies/ml) was usually defined as VF. However, in our study, 6.1% 131/2155) participants  showed spontaneous control of viral replication in the follow up time. This phenomenon is more common in the cohort of South Africa (26%), which we speculate is caused by irregular compliance or accident 155 parti, 20; howOur current study implied that MLLV or LLLV were not associated with higher risk of VF, and therefore such cases should be only kept under observation without changing treatment. In contrast, a previous study suggested that patients with such level of LLV should be switched to other regimes or given stronger treatments immediately.Some previous studies have considered both level and duration of LLV, which introduced a new parameter of viremia copy-years (VCY), combining the level and duration of virus replication, defined as the area under a patient’s longitudinal VL curve. These studies found that the risk of VF rose with increasing VCY, which is consistent with the findings of the current study. However, no uniform threshold had been determined, because of the varied definitions of both LLV and VF , 25. In According to this study, for patients with the baseline characters, such as high zenith VL, low nadir CD4 + T cell counts, Manchu, and subtype B′, who were more likely to develop high-risk LLV, a more frequent follow-up should be recommended. The patients with LLV levels above 400 copies/ml, including both HLLV and HLB, and MLLV persisting longer than 12 months should be recommended to undergo drug resistance testing and blood drug concentration testing. On the other hand, for the patients with LLV levels below 200 copies/ml, follow-up can be continued.Our study has some limitations. First, as a retrospective study, there may be some bias in participant selection. Second, 90% patients in this study self-reported good drug compliance, but the drug concentration in their plasma was not detected; thus, we were unable to assess the reliability of self-reported compliance. Third, about 5–10% of the social demographic and laboratory information were unavailable in this study, which might impact the results.In conclusion, we observed the common occurrence of LLV among a local long-term ART cohort in China, among which, some LLV demonstrated with higher risk of VF and should be treated immediately. Our study also identified prognostic factors that can help determine the potential occurrence of high risk LLV. The results of this study provide a basis for development of differential treatment of LLV at various levels.Additional file 1: Table S1. Baseline characteristics of the included participants (N = 2155).


Camellia sinensis) is a kind of unique wild tea resources in southwest China, containing sizeable amounts of theacrine  and having a special bitter taste both in fresh leaves and made tea. Theacrine has good healthy function locally. But the molecular mechanism of theacrine metabolism in Kucha was still unclear. In order to illuminate the biosynthesis and catabolism of theacrine in Kucha plants, three tea cultivars, C. sinensis ‘Shangyou Zhongye’ (SY) with low-theacrine, ‘Niedu Kucha 2’ (ND2) with middle-theacrine and, ‘Niedu Kucha 3’ (ND3) with high-theacrine, were used for our research. Purine alkaloid analysis and transcriptome of those samples were performed by High Performance Liquid Chromatography (HPLC) and RNA-Seq, respectively. The related gene expression levels of purine alkaloid were correlated with the content of purine alkaloid, and the results of quantitative real-time (qRT) PCR were also confirmed the reliability of transcriptome. Based on the data, we found that theacrine biosynthesis is a relatively complex process, N-methyltransferase (NMT) encoded by TEA024443 may catalyze the methylation at 9-N position in Kucha plant. Our finding will assist to reveal the molecular mechanism of theacrine biosynthesis, and be applied to selection and breeding of Kucha tea cultivars in the future.Kucha ( Camellia sinensis) with bitter taste in both fresh leaves and made tea, was firstly found in Yunnan province, most of which located in the adjoin regions between Guangxi, Guangdong, Hunan and Jiangxi provinces in China2. Kucha plants are usually semi-arbor trees with large leaves, which are difficult to be discriminated from cultivated tea varieties based on morphology observation. However, the leaves of Kucha are remarkably characterized with strongly bitter taste and special aroma compared to other tea cultivars. In the Kucha growing regions, local residents popularly use its leaves as a medicine to treat wound, inflammatory and diarrhea in daily life.Kucha, a unique kind of tea varieties  of theacrine in dry Kucha leaves, while no detection in tea leaves of Cocoa tea (C. ptilophylla) and Longjing tea (C. sinensis)6. Jin et al. identified 36 mg/g of theacrine from a Kucha plant, while null of theacrine in other tea plants such as C. crassicolumna, C. sinensis and C. sinensis var. pubilimba3. At present, high purity of theacrine could be separated by HPLC and LC-MS, and obtained by ultrasonic reflux extraction8. As a unique, naturally occurring purine alkaloid presented in Kucha, more and more studies have been focused on its pharmacology and health function. A few of research showed that theacrine had the benefit effects on anti-inflammation, detoxifying, analgesic and promote sleep11.The tea plant has rich amount of purine alkaloids, such as caffeine and theobromine. Most of tea plants generally contain 25.0 mg/g ~ 45.0 mg/g caffeine, and theobromine has a widely variation range from 0.5 mg/g ~ 46.7 mg/g13. Theacrine is an important purine alkaloid converted from caffeine by hydration, oxidation and methylation. In usual, large quantities of purine alkaloids such as caffeine and theobromine, and little theacrine are synthesized in C. sinensis plants. Zheng et al. used radioactive 14C isotope trace method to explore the metabolic pathway of theacrine, which indicate that theacrine was synthesized from caffeine in what is probably a three-step pathway with 1,3,7-methyluric acid acting an intermediate14. This was the first demonstration about theacrine biosynthesis pathway in Kucha. However, why the theacrine were highly synthesized and concentrated in Kucha plant? And it still remains unclear about the molecular mechanism of theacrine metabolism in Kucha plant.The typical route of caffeine biosynthesis in tea leaves is a ‘xanthosine → 7-methylxantosine → theobromine → caffeine’ pathwayIn order to determine the molecular mechanism of theacrine metabolism, RNA-Seq was performed using three tea cultivars with different theacrine content. They are Shangyou Zhongye (SY) with minor theacrine, Niedu Kucha 2 (ND2) with medium theacrine and Niedu Kucha 3 (ND3) with high theacrine content. In this study, genes related to theacrine metabolism were explored and identified based on transcriptomes analysis. It will assist to explain the theacrine mechanisms in Kucha plant.The total purine alkaloids were relatively stable in tested samples, they were no significant difference between SY and ND3, especially. However, the significant differences of theacrine and caffeine were found between them. The theacrine content were 8.04 mg/g and 10.45 mg/g in ND2 and ND3 respectively, significant higher than SY (0.83 mg/g). While the variation of caffeine content was opposite to theacrine between the tested samples  were identified between two group of ND2 vs SY and ND3 vs SY Fig. . These g15. In the MF category, the molecular function, the catalytic activity and the binding were the top three enriched GO terms of two groups. In the CC category, DEGs were enriched in terms of the cellular component, the cell and the cell part. And in the BP category, the biological process, the metabolic process and the cellular process were the top terms.GO (Gene Ontology) is an international standard database for gene functional classification, which contains three categories, molecular function (MF), cellular component (CC) and biological process (BP)S-adenosylmethionine-dependent methyltransferase activity and the N-methyltransferase activity were enriched in 23, 11 and 7 genes in two groups, which may play a key role in purine alkaloid biosynthesis. The top 20 enriched terms of GO classification are shown in Fig. In ND2 vs SY, 1327, 150, and 1465 DEGs were significant enriched into 106, 15 and 48 subcategories of BP, CC and MF, respectively. And in ND3 vs SY, 492, 295, and 840 DEGs were significant enriched into 41, 8 and 25 subcategories of BP, CC and MF, respectively. Moreover, the terms of the methylation, the 16. Results showed that 192 and 182 metabolic pathways in ND2 vs SY and ND3 vs SY group, respectively. These pathways contain flavonoid biosynthesis, phenylpropanoid biosynthesis, carotenoid biosynthesis, ABC transporters and steroid hormone biosynthesis were significant enriched in both group  in the adenine donor synthesis, TEA015791, TEA028050, and TEA028052  were highly expressed in SY with high caffeine concentration. TEA006735 and TEA015661  in the methyl donor synthesis were up-regulated in ND2 which contains the most purine alkaloid among three samples. The expression levels of TEA024443  were up-regulated in ND2 and ND3 which had a highly content of theacrine compare with SY. We also identified the key genes of purine alkaloid degradation, TEA027082 and TEA011804  which involved the degradation pathway of “AMP → Adenosine” and “Guanosine→GMP” were up-regulated in ND2 and ND3 compared with SY. TEA002149  was highly expressed in ND3 with high theacrine concentration. From the KEGG enrichment analysis, 5 and 6 DEGs which annotated in cytochrome P450 family were obtained in SY vs ND2, and SY vs ND3, respectively. TEA010267 and TEA004815  were highly expressed in high caffeine concentration sample.The expression patterns of key genes in theacrine synthesis such as ant Fig. . TEA0152TCS), TEA015248 (APRT) and TEA000333 (5′-NT) were negatively correlated with the content of theacrine (P < 0.05). Meanwhile, TEA028050 (TCS) and TEA028052 (TCS) were positively correlate with caffeine content (P < 0.05), especially TEA018201 (APRT) presents the significantly positive correlation (P < 0.01). It was speculated that the proteins encoded by these genes were involved in the regulation of theacrine and caffeine content in the Kucha plants.Pearson correlation coefficient was used to evaluate the correlation between the gene expression level and purine alkaloid content Table . The resCYP1A2) and TEA010267 (CYP1A2), were generally consistent with RNA-Seq which further indicated the reliability of the transcriptome date  PCR analysis, and their primers are listed in Table C. sinensis var. kucha Zhang et Wang18; Du et al. defined Kucha as C. sinensis var. kuiea Du et Li based on methods of chemical classification and digital taxonomy19; Min studied the morphological characteristics of Kucha cultivars, indicated that Kucha should be classified into C. sinensis var. assamica, not be defined as a new tea variety20; Chen et al. considered that Kucha should be classified into C. sinensis var. assamica, according to the systematic study of tea resources and the comprehensive investigation of tea plants in original place21; Wang et al. determined biochemical components of 24 Kucha resources, indicated that Kucha germplasms were similar to C. sinensis var. assamica in terms of catechin composition which was reflected the degree of tea plant evolution22. For Kucha classification, further study should be focused on genomic level by comparing Kucha plant with other tea species.As a kind of special tea germplasm, the classification of Kucha still remains being under discussion. In Zhang Hongda classification method, Kucha was defined as 25. In our study, High level of purine alkaloids in Kucha cultivars is conducive to the processing of high-quality black tea, and it also provides a new way to develop product of natural purine alkaloid and multi-purpose exploitation of Kucha.Meanwhile, the exploitation and utilization of bitter tea resources are also an important problem faced by breeders. Jianghua Kucha (a bitter tea) has been studied and utilized deeply which was widely promoted in the area of broken black tea at Hunan province, because of its suitable for black tea22. Ye et al. found that the content of theacrine was stable with season change in young leaves of Kucha. While the theacrine was significantly higher in immature leaves than that in mature leaves and aged leaves4. At present, metabolic pathway of purine alkaloid and the related genes of biosynthesis have been basically determined which lay the foundation for our research of theacrine metabolism27. In our study, RNA-Seq was used to discovered the transcriptome of three tea cultivars, DEGs involved in purine alkaloid metabolism were discovered in two comparison groups ND2 vs SY and ND3 vs SY. Contents of theacrine and caffeine among three cultivars were obviously different, theacrine in ND2 and ND3 was higher than SY, while the caffeine content was lower.Theacrine is an advantage purine alkaloid second only to caffeine in Kucha plant generally, which affect quality and flavor of Kucha products significantly. Previous studies indicated that the theacrine varied in different Kucha cultivars, and less affected by environmentsS-adenosylmethionine synthetase (SAMS), tea caffeine synthase (TCS), N-methyltransferase (NMTs) and Adenine phosphoribosyltransferase (APRT). In general, SAMS is the key enzyme in purine alkaloid metabolism pathway, of which S-adenosylmethionine act as methyl donor in theacrine and other purine alkaloids biosynthesis28. Our results show that the gene expression levels of SAMS were up-regulated in two groups, which the total amount of purine alkaloids was higher than that of control variety. TCS is a class of N-methyltransferase that catalyzes the methylation of N-3 (theobromine synthase) and N-1 (caffeine synthase) in tea plant. Previous studies point that TCS1 plays a crucial role for methylation of xanthosine at 1-N position in caffeine biosynthesis, while TCS1 alleles with low transcription level or encoded proteins with no TCS activity29. TEA015791, encoded TCS1, was down-regulated in ND2 and ND3, it may be the reason that why the lower content of caffeine in two Kucha samples. But no research indicated that TCS has the ability to catalyzed methylation at 9-N position. It still needs the further research that whether TCS involved in the final step of theacrine biosynthesis. TEA024443, encoded NMTs, was highly expression in high-theacrine sample which was positively correlated with the content. NMT may play a significant role in theacrine synthesis which dominant the pathway of “1,3,7-trimethyl xanthine (caffeine)→1,3,7-trimethyluric acid→1,3,7,9-tetramethyluric acid (theacrine)”, we speculated that N-methyltransferase encoded by this gene responsible for catalyze the methylation at 9-N position in theacrine synthesis. Meanwhile, the expressions of related gene in purine alkaloid degradation pathway was probably correlated with the decreased of purine alkaloid in Kucha samples. TEA007899 and TEA023610, encoded allantoinase (ALN), and TEA020308, encoded urease (URE), were all highly expression in ND2 and ND3 with low-caffeine Kucha plant, which involved the final degradation step of “Allantoin→Allantoic acid → →NH3 + CO2”.KEGG pathway analysis indicated that purine alkaloid metabolism pathway plays significant roles in Kucha metabolism, and a number of key genes related to theacrine and caffeine biosynthesis were selected from these DEGs. Theacrine biosynthesis is catalyzed by a series of enzyme, including 31. However, until now few studies have been reported on molecular mechanism of theacrine biosynthesis in Kucha cultivars. In this study, we proposed an NMT, encoded by TEA024443, was likely the key enzyme involving in the final synthesis process of theacrine. Next, the gene of TEA024443 should be focused on verification and testing its function in theacrine biosynthesis. Further, we will construct the tea genetic population with Kucha cultivars as parents, key genes involved in theacrine metabolism were identified and analyzed by density genetic map and QTL mapping. It will make it possible to breeding new tea cultivar which is rich in theacrine yet contains low caffeine.Caffeine is seemed as a precursor of theacrine, its biosynthesis pathway is very clear. And the key genes involved in caffeine metabolism have been identified, which can provide reference for the further study of theacrineTo sum up, our study provided a new insight to illuminating theacrine molecular metabolism in Kucha plants, and it may help to the breeding improvement of Kucha plant in subsequent studies.Three tea cultivars were used as plant materials in this study, including high-theacrine cultivar ‘Niedu Kucha 3′ (ND3), medium-theacrine ‘Niedu Kucha 2′ (ND2) and low-theacrine ‘Shangyou Zhongye’ (SY) as a control. The former two are Kucha accessions from the same local population of tea landrace named ‘Niedu Kucha’, the latter one is local cultivated tea clone. All of them were collected from the same region located in the southwest part of Jiangxi province. And they are currently preserved in National Germplasm Hangzhou Tea Repository, in the Tea Research Institute of the Chinese Academy of Agricultural Sciences at Hangzhou city, Zhejiang province in China. Leaves of two Kucha plants are tasted very bitter, while that of the controlled cultivar is not bitter. Fresh healthy leaves were picked on the two terminal leaf position from ND2, ND3 and SY respectively, quickly frozen in liquid nitrogen firstly, and then stored at −80 °C for RNA extraction. And the young shoots with “two leaves and a bud” collected from three cultivars were fixed with hot air at 120 °C for 5 min and then dried to constant weight at 80 °C. These dried samples were used to detect content of purine alkaloid such as theacrine, caffeine and theobromine using HPLC method. All experiments had three biological replicates.32. A Waters High Performance Liquid Chromatography (HPLC) instrument was used to detect the compounds in this experiment. A Waters C18 column  was performed at a flow rate of 1.0 mL/min with the column temperature of 35 °C. 1% formic acid was used as mobile phase A, 100% acetonitrile as mobile phase B, 10 μL sample liquid was injected and analyzed at 280 nm, and the elution time was 45 min.Purine alkaloids such as theacrine, caffeine and theobromine were detected by HPLC methods described in a previous reported methodEASY-spin Plus Complex Plant RNA kit  was used to extract total RNA. The integrity and quantity of total RNA were estimated by NanoDrop ultraviolet spectrophotometer  and agarose gel electrophoresis. cDNA libraries established by the standard mRNA-Seq Library Prep Kit, and the library quality was assessed by using the Agilent 2100 Bioanalyzer . The cDNA libraries were sequenced by the Illumina HiSeq platform, performed by Personal Biotechnology Co., Ltd. .C. sinensis ‘Shuchazao’ genome by HISAT software with default parameters and counted by feature counts34.High-quality reads were evaluated and acquired from the raw data, which need to filter out the low-quality sequence, adaptor sequences, duplicated sequences and ambiguous (with the ratio of N > 5%). The clean reads from all the samples were mapped to http://tpia.teaplant.org/. The DEGs  among tea samples  were identified by DESeq. 2. Genes with a FDR  ≤ 0.05 and an absolute fold change value ≥ 1.0 were identified as DEGs36.Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation were downloaded from 2O, 0.6 μL primer each pair, 10 μL 2x FastFire qPCR PreMix, 0.4 μL 50x Rox Reference Dye△ and 1 μL diluted cDNA. The cycling profile was 95 °C, 60 s; 40 cycles of 95 °C, 5 s, and 60 °C, 32 s in 96-well optical reaction plates. The relative gene expression was calculated though the 2−△△Ct method37. Each sample was examined in three technical replicates.To confirm the accuracy of RNA-Seq and DEG analyses, 12 DEGs associated with purine alkaloid biosynthesis pathway were selected for qRT-PCR. The primers of specific genes for qRT-PCR were designed by Primer Premier 5, and the primer pair sequences were listed in Table Three biological replicates of sequencing data and purine alkaloid components were used for calculating the mean value and the standard deviation (SD) based the Duncan’s multiple range tests. SPSS 18.0 was used to analyze the correlations of gene expression and purine alkaloid concentrations via Pearson correlation. The volcano figure was produced with Prism 7.0 software. The double coordinate figures and heat maps were draw with R package.Supplementary Information.


The emergence of multidrug-resistant (MDR) and extended-spectrum beta-lactamase (ESBL)-producing uropathogens has complicated the treatment of urinary tract infections (UTI). Paediatric UTI is a common illness, which if not treated properly, may lead to acute and long-term complications, such as renal abscess, septicaemia, and renal scarring. This study aimed to determine the prevalence of MDR and ESBL-producing uropathogens among children.During the study period (April 2017–April 2018), midstream urine samples were collected following aseptic procedures from children < 16 years in Siddhi Memorial Hospital. Standard culture and biochemical tests were performed to identify uropathogens and antimicrobial susceptibility test was done by modified Kirby-Bauer disc diffusion method following Clinical and Laboratory Standard Institute (CLSI) guidelines. ESBL-producing uropathogens were screened by ceftazidime (30 μg) and cefotaxime (30 μg) discs, and confirmed by the combination disc tests: ceftazidime + clavulanic acid (30/10 μg) or cefotaxime + clavulanic acid (30/10 μg) as recommended by CLSI.Escherichia coli  and Klebsiella pneumoniae  were common among the uropathogens. Among them, 80.3% were resistant to amoxycillin and 51.2% were resistant to cotrimoxazole. Most of them were susceptible to amikacin, nitrofurantoin, and ofloxacin. MDR was detected in 34.5% (n = 70/203) and ESBL producers in 24.6% (n = 50/203) of them. The proportion of MDR isolates was higher in children < 5 years  than children ≥ 5 years  (P = 0.03).We processed 5545 non-repeated urine samples from the children with symptoms of UTI. A significant growth of uropathogens was observed in 203 samples (3.7%). The median age of the children was 24 months (interquartile range (IQR), 12–53 months). Nitrofurantoin, ofloxacin, and amikacin can be used for the empirical treatment for UTI in children in Bhaktapur, Nepal. MDR and ESBL-producing uropathogens are prevalent; this warrants a continuous surveillance of antimicrobial resistance. Escherichia coli and Klebsiella pneumoniae, cause UTIs. Effective antimicrobial treatment of these infections is necessary; if untreated, they may complicate to renal abscess, septicaemia, renal scarring, and renal failures which can be life threatening in children [Urinary tract infection (UTI) is a common cause of illness in children. Gastrointestinal bacteria, such as children , 2. Not children .The growing prevalence of antimicrobial resistance among uropathogens is an enormous concern. The increase in proportion of multidrug-resistance (MDR) and ESBL-producing uropathogens in children has not only limited the treatment by rendering the first-line antibiotics ineffective but also complicated the treatment by increasing the hospital stay. The problem of antimicrobial resistance has become worse in resource-limited countries where antimicrobials are available over-the-counter –6. SurveIn this study, we aimed to determine the prevalent uropathogens, their antimicrobial susceptibility, and the prevalence of MDR and ESBL producers in children attending a paediatric hospital in Nepal with symptoms of UTI.We carried out this cross-sectional study from April 14, 2017 to April 13, 2018, in the Department of Microbiology, Siddhi Memorial Hospital (SMH) in Bhaktapur, Nepal. SMH is a 50-bedded hospital that provides tertiary care to children. We included children less than 16 years of age in this study.5colony-forming units/mL) of a single organism. To isolate and identify the organisms, we performed standard microbiological methods including colony morphology, Gram staining, catalase, oxidase, and in-house set of biochemical tests [We collected urine specimens following aseptic procedures, such as midstream urine, catheterisation, and suprapubic aspiration from children with clinical symptoms suggestive of a possible UTI. We transported the urine samples to the laboratory immediately and processed by the semi-quantitative streaking method. We inoculated 10 μL of urine onto a cysteine lactose electrolyte deficient (CLED) agar plate using a sterile calibrated wired loop. Inoculated plates were incubated at 37 °C in aerobic conditions for 18 h. We diagnosed a UTI when we observed a significant growth  following the guidelines of the Clinical and Laboratory Standards Institute (CLSI), Wayne, USA . We testWe screened all Gram-negative bacilli for ESBL production. We tested the susceptibilities to third-generation cephalosporins by using ceftazidime (30 μg) and cefotaxime (30 μg) discs. As recommended by CLSI, we considered the isolates as potential ESBL producers if the zone of inhibition was ≤ 22 mm for ceftazidime or ≤ 27 mm for cefotaxime. The potential ESBL producers were then examined for confirmation by the combination disc test as the CLSI guideline. In this test, we tested the isolates against a ceftazidime (30 μg) disc and a ceftazidime + clavulanic acid (30/10 μg) combination disc, and we compared the inhibition zones around these discs. We considered that the test was positive for the confirmation of ESBL producers if the inhibition zone diameter was ≥ 5 mm larger with the combination disc than the ceftazidime disc. Similarly, we also performed the confirmation test with a cefotaxime (30 μg) disc and a cefotaxime + clavulanic acid (30/10 μg) combination disc in all potential isolates; the results were interpreted as positive if the inhibition zone diameter was ≥ 5 mm larger with the combination disc than the cefotaxime disc. Having positive results in any one of these two confirmatory tests, we confirmed the isolates as ESBL producers.We analysed the data in Stata 14 . The chi-squared test was used to compare the proportions of isolates between males and females, and between younger than 5 years and equal or older than 5 years.n = 153, 75.3%) of isolates were from the children less than 5 years. E. coli was the most common isolated uropathogen  followed by K. pneumoniae   in male children. There were no significant differences in the proportions of bacterial isolates between males and females, and between children less than 5 years and the older-aged groups  and there were 101 male and 102 female children. Three-quarters  of the uropathogens. The proportions of MDR in male and female children were similar  (P = 0.8). We observed a higher proportion of MDR in children younger than 5 years  than older aged group  (P = 0.03)  of the uropathogens. The proportions of ESBL producers were not different between male  and female children  (P = 0.3), and between children under 5 years  and older-aged children  (P = 0.9). MDR was detected in 34.2% of E. coli and 36.7% of K. pneumoniae isolates. ESBL producers were detected in 27.2% of E. coli and 23.3% of K pneumoniae isolates  countries than the resistance of E. coli—53.4% to ampicillin, 8.2% to co-amoxiclav, and 2.1% to ciprofloxacin—in OECD countries [The prevalence of antimicrobial resistance to first-line antimicrobials for UTIs in children has increased in resource-limited countries. A systematic review on 58 observation studies shows the pooled prevalence of resistance in E. coli and 36.7% of K. pneumoniae were MDR. Our findings were similar to those of Shrestha et al. who reported that 34% isolates were MDR in Dharan, a city in the eastern part of Nepal, in 2018 [E. coli [Increasing resistance of uropathogens to three or more groups of antimicrobials (MDR) has become a severe threat to the health care system . Gram-ne in 2018 . However[E. coli , 18. The[E. coli , 20. We Infections with ESBL-producing organisms in children are associated with longer hospital stays, frequent complications, and increased mortality . ESBL-prThis study has limitations. This study was conducted in one hospital for a period of 1 year, so the findings may not be generalisable. We could not evaluate the risk factors and outcomes of MDR and ESBL producers. The genotypes of ESBL producers could not be determined.We found a high prevalence of MDR and ESBL-producing uropathogens in children in Bhaktapur, Nepal. The prevalent uropathogens were susceptible to nitrofurantoin, ofloxacin, and amikacin, some of which may be used for the empirical treatment of UTIs in children. Improved antimicrobial stewardship in hospitals and a restricted use of over-the-counter antimicrobials maybe some of the ways to decrease the prevalence of antimicrobial resistance in the community.


Ningmitai (NMT) capsule has been widely prescribed for the treatment of chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS), but the mechanism remains unclear. This study aims to evaluate the therapeutic effects of the NMT capsule in the experimental autoimmune prostatitis (EAP) rat models and explore its possible mechanisms. β and TNF-α. The MDA level was detected to reflect the level of oxidative stress. The bilateral dorsal root ganglia of T3/L1 to S4 were dissected to measure the substance P expression.  A total of fifty male Sprague Dawley rats were used in this study. Prostate extract was obtained for the induction of EAP rat models. The EAP rats were randomly divided into the model group, NMT low-dose group (0.45 g/kg/d), NMT medium-dose group (0.90 g/kg/d), and NMT high-dose group (1.80 g/kg/d), with six rats per group. Three NMT treatment groups were administered with the NMT capsule by gavage for 30 days. HE staining was used for histopathological analyses of prostate tissues. Western blotting was used to measure the expression of proinflammatory factors IL-1P < 0.05), especially in the NMT medium-dose group and NMT high-dose group. Moreover, the expression of IL-1β and the level of MDA were significantly decreased (P < 0.05). In addition, NMT treatment could significantly alleviate substance P expression in dorsal root ganglia.  EAP rat models were successfully constructed, in which extensive infiltration of inflammatory cells was found. Treatment of NMT capsule for 30 days and the infiltration of inflammatory cells were significantly mitigated (P in EAP rats. This provides a theoretical basis for the clinical application of NMT capsule for CP/CPPS treatment. Our findings demonstrate that the NMT capsule can alleviate inflammatory response and oxidative stress and reduce the production of substance  Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is defined as chronic pelvic pain not caused by other identifiable pathology, according to the classification of prostatitis developed by the National Institutes of Health (NIH) in 1994 . Due to α-blockers have been widely used for the management of CP/CPPS [α-Blockers are used to improve urinary symptoms of CP/CPPS patients. However, several RCTs research studies showed heterogeneous outcomes of monotherapy with α-blockers [α-blockers may not be recommended as a first-line monotherapy [Although antibiotics and  CP/CPPS , the effblockers –7. Theseotherapy .β is higher in CP/CPPS patients when compared with controls [Autoimmune inflammation is one of the main causes of CP/CPPS , 10. A scontrols . On the controls . Hence, Herba Agrimoniae), Touhualiao (Herba Polygoni Capitati), Bai Mao Gen (Rhizoma Imperatae), Dafengteng (Radix Cocculi Trilobi), Sankezhen (Berberidis radix), Furongye (Folium Hibisci Mutabilis), and Lianqiao (Fructus Forsythiae suspensae) [Ningmitai (NMT) capsule has been widely prescribed for the treatment of CP/CPPS in China. A meta-analysis  of thirtspensae) . Some ofspensae) , 16. TheFifty healthy male Sprague Dawley (SD) rats (12 weeks old) were used in this study, all of which were purchased from the Laboratory Animal Center of Sun Yat-sen University . The animals were kept in the standard, pathogen-free environment on a 12-h light/12-h dark cycle with free access to laboratory chow and water. The animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Sun Yat-sen University.EAP rat model could simulate the inflammatory characteristics of typical CP/CPPS. It is considered as a suitable model for human CP/CPPS . To estaThirty SD rats were randomly divided into five groups with six rats per group: normal group (group N), model group (group Mo), NMT low-dose group (group L), NMT medium-dose group (group M), and NMT high-dose group (group H). NMT capsule was provided by Shanghai Xintian Pharmacy Company. Rats in group Mo and three NMT treatment groups had been induced into EAP as described above. Among NMT treatment groups, the therapeutic dose of NMT capsule in group L was 0.45 g/kg by gavage (0.10 g crude drug/mL), that in group M was 0.90 g/kg, and that in group H was 1.80 g/kg each day. Group N and group Mo were administered an equal volume of saline by gavage. All groups were given gavage once a day for 30 days. Then the body weight was measured. The rats were killed with an overdose injection of pentobarbital (>100 mg/kg), and their prostates were weighed up.The prostatic index  was calcPI = weight of prostrate (mg)/body weight (g).Prostate tissues were deparaffinized in xylene and dehydrated in alcohol. Then, the tissues were stained in Harris hematoxylin solution for 8 min before bluing in 0.2% ammonia water or saturated lithium carbonate solution for 1 min. After rinsing in 95% alcohol, tissues were counterstained in the eosin-phloxine solution for 2 min. Finally, the tissues were mounted with a xylene-based mounting medium. Images were captured using a Leica confocal microscope. Then analyzed the histopathological features of the prostate gland. To evaluate the aggressiveness of inflammation, three fields of view were randomly selected from each slice, and the total numbers of inflammatory cells were counted.The bilateral dorsal root ganglia of T3/L1 to S4 were dissected for IHC staining to evaluate the substance P (SP) expression. It was performed using a Dako Envision System  following the manufacturer's protocol. Sections were blocked using serum-free protein block buffer  for 30 min, after which they were incubated with the antibodies of SP, SP-DE4-21 .β-actin , IL-1β , and TNF-α . Then, the membranes were incubated with secondary antibodies for 1 hour at room temperature. Finally, immunodetection was performed using Chemi-Doc Imaging System .The prostate protein levels were analyzed by Western blotting. In brief, total protein was extracted by RIPA Lysis Buffer and separated by polyacrylamide gels, and then, was transferred onto PVDF membranes. After blocked with 5% BSA solution, the membranes were incubated with primary antibodies, including GAPDH , Superoxide dismutase (SOD) and malondialdehyde (MDA) were detected according to the corresponding assay kit. The SOD activity was detected by the Superoxide Dismutase assay kit (WST-1 method) . The MDA expression was detected by the Malondialdehyde assay kit (TBA method) . The prostate tissue was weighed, and a 10% tissue homogenate was made in a glass homogenizer for total protein quantification using Pierce BCA Protein Assay Kit (Thermo 23225).P < 0.05 were considered to be statistically significant.The statistical analysis was performed with SPSS for Windows . Continuous variables were expressed as the mean ± standard deviation. Multiple groups were compared using one-way analysis of variance (ANOVA). Values of In our study, three doses of NMT capsule were administered to EAP rat models to investigate the effects of NMT capsule on CP/CPPS. The effects of NMT treatment were summarized in It was suggested in the Introduction that NMT capsule may treat CP/CPPS through the anti-inflammatory effect. To investigate the histopathological change in prostate tissues after the treatment, HE staining was used. As shown in β and TNF-α [β in the model group was significantly increased in comparison with the normal group. With the treatment of NMT capsule for 30 days, it has a significant reduction of IL-1β expression compared with the group Mo. However, although the oral administration dose of NMT varied from 0.45 to 1.80 g/kg, there is no significant difference between the treatment groups. But the expression of TNF-α was not significantly different between these five groups . After NMT treatment, the MDA level was significantly down-regulated (P < 0.05).The previous study also shows that oxidative stress has a significant correlation with CP/CPPS. The SOD activity and MDA level were most commonly used to evaluate the oxidative stress state. In our study, the vitality of SOD was not significantly different between the normal group, model group, and treatment group. Whereas the level of MDA was significantly higher in the model group , and inducible nitric oxide synthase (iNOS) [To date, oxidative stress has been regarded as an important factor in the process of chronic prostatitis , 30. In e (iNOS) . These se (iNOS) . MDA, ane (iNOS) , 34. We Currently, the pelvic and suprapubic perineal pain associated with CP/CPPS is believed to be caused by dorsal root ganglia axon reflex, in which the release of SP mediates this persistent pain status . ChemicaHerba Agrimoniae), Touhualiao (Herba Polygoni Capitati), Bai Mao Gen (Rhizoma Imperatae), Dafengteng (Radix Cocculi Trilobi), Sankezhen (Berberidis radix), Furongye (Folium Hibisci Mutabilis), and Lianqiao (Fructus Forsythiae suspensae) [Herba Agrimoniae) could suppress lipopolysaccharide-induced nitric oxide production in BV2 microglial cells. Furthermore, it also suppressed the lipopolysaccharide-induced production of IL-1β, TNF-α, and IL-6 in a dose-dependent manner. Liao et al. [Herba Polygoni Capitati) contains substances of certain structural types and with antibacterial and anti-inflammatory activities. These studies suggest that these compounds may play an anti-inflammatory and antioxidative role in the treatment of CP/CPPS. In this study, we failed to identify which component of the NMT capsule playing the major role, since the NMT capsule is traditional Chinese medicine preparation containing a variety of complex ingredients. Furthermore, investigations are needed to explore which component plays a key role.NMT capsule is a pure Chinese medicine preparation consisted with Xian He Cao (spensae) . Recent spensae)  showed to et al.  found thIn summary, our findings demonstrate that the NMT capsule can alleviate inflammatory response and oxidative stress and reduce the production of substance P in an autoimmune prostatitis rat model. This provides a theoretical basis for the clinical application of NMT capsule for CP/CPPS treatment.


To evaluate factors affecting oocyte/embryo quality in PolyCystic Ovary Syndrome (PCOS) patients undergoing Assisted Reproductive Technology (ART) cycles.This case-control retrospective study was performed on PCOS patients referred to the infertility department of Imam Khomeini Hospital in Ahvaz from October 2017 to September 2019. Demographic and reproductive characterizations including age, gender, abortion history and infertility type (primary and secondary infertility) were extracted from patient’s records. TSH, AMH, LH, FSH, prolactin, lipid profile and blood glucose was measured. Biochemistry pregnancy was checked by determination of serum βHCG level and then, clinical pregnancy was confirmed by observing of pregnancy sac and fetal heart rate using Transvaginal USS.p = 0.24) were considered as case and control groups respectively. Retrieved oocyte numbers were significantly higher in PCOS patients (p = 0.024), but there was no significant difference in number of oocyte subtypes  between two groups. The embryo numbers and its subtypes did not differ significantly in both groups. The clinical pregnancy rate was insignificantly lower in PCOS patients (p = 0.066) and there was a significant correlation between retrieved oocyte numbers with age and AMH level  respectively. Cholesterol level had shown a positive significant correlation with number of MI oocytes  and MII oocytes significantly affected by age  and AMH level . Using Receiver operation characteristic (ROC) curve analysis, the cut-off value of total number of oocytes was > 10.5 with area under curve of 0.619±0.054(sensitivity 55.56% and specificity 69.66%)One-hundred thirty-five patients include 45 PCOS and 90 Non-PCOS patients with mean age of 31.93 ± 5.04 and 30.8 ± 5.38 (The results of this study showed that although the number of oocytes in PCOS patients was significantly higher than non-PCOS patients, the quality of oocytes was not statistically different. The number and quality of embryos were not significantly different in both groups. Our results indicated a significant relationship between the level of AMH and the number of retrieved oocytes and embryos. We found there is a significant correlation between cholesterol level and number of MI oocytes. According to Rotterdam criteria, a woman has at least two of these three characteristics: clinical and / or biochemical hyperandrogenism, ovarian dysfunction, and Polycystic ovary Morphology (PCOM) to be diagnosed as PCOS . Almost follicle . This exfollicle . It shoufollicle . The oocfollicle . These qfollicle . Since fThis case-control retrospective study was performed on PCOS patients referred to the infertility department of Imam Khomeini Hospital in Ahvaz from October 2017 to September 2019. Inclusion criteria were patients with PCOS (case group), non-PCOS patients with tubal factor or male factor (control group). Exclusion criteria included patients with Follicle Stimulating Hormone (FSH) more than 12 IU/mL, ovarian surgery history, ovarian tumor, systemic diseases, endometriosis and age more than 38 years. In this study using “Power and Sample Size” software and based on paper written by Banchhita et al. [mm were seen, COH was started (Antagonist cycle). Depending on the patient’s condition, a dose of 150 or 225 units of Gonal-F  or Cinnal-F was administrated. When the follicle size reached 13 or 14mmcetr, Cetrorelix  0.25 mg subcutaneous was started. Gonadotrophin and Cetrorelix continued until two follicles with 17mm in size and several 15mm sized follicles were seen. Oocyte retrieval was performed 34 to 36 hours after injection of 10,000 units U-HCG (Pregnyl-Chorionic Gonadotrophin) or GnRH agonist (0.5 cc Superfact-Buserline acetate) and the number and grade of oocytes were recorded which included: Metaphase II (MII), Metaphase I (MI), and Germinal Vesicle (GV). MII oocytes have excellent quality and the main goal of Controlled ovarian Hyperstimulation (COH) is to achieve maximum number of MII oocytes. MI-type oocytes are lower in maturity than MII-type and are not a favorable treatment option. GV oocytes have the lowest quality and are not a good choice for transferring. Twenty-four hours after fertilization, the embryos morphology was evaluated to check 2-pronuclear (2PN) morphology. Then if no ovarian hyperstimulation syndrome (OHSS) occurred and endometrium more than 8 mm thickness was seen, 1 or 2 embryo was transferred.Demographic and reproductive characterizations including age, gender, abortion history and infertility type (primary and secondary infertility) were extracted from the patient’s records. Thyroid-stimulating hormone (TSH), Anti-Müllerian hormone (AMH), Luteinizing hormone (LH), FSH and prolactin hormone level were measured by enzyme-linked immunosorbent assay (ELISA) test. Biochemical factors including, Fasting Blood Sugar (FBS), Glucose Tolerance Test (GTT) (75gr), Triglyceride, cholesterol and Vitamin D level were assessed. Patients in both groups underwent ART antagonist treatment cycle. On the 3rd day of menstruation, Transvaginal USS was done. If endometrium thickness was thin or no follicles greater than 10Two weeks after transfer, biochemistry pregnancy was checked by determination of serum βHCG level. Then, clinical pregnancy was confirmed by observing of pregnancy sac and fetal heart rate using Transvaginal USS.The mean value of variables compared using independent t-student or Mann–Whitney U test and proportions were compared using the chi-square test. Correlations between the variables were determined by Pearson correlation coefficient test. All statistical analysis was performed using SPSS version 22 and P-value of less than 0.05 considered significant.p = 0.24) years were considered as case and control group respectively. Although most PCOS patients had secondary infertility (31(68.9%)), primary infertility was more common in non-PCOS patients (50(55. 6%)).The mean values of infertility duration in PCOS and Non- PCOS patients were 7.4 and 6.3 years, respectively(p = 0.11). Although the mean level of FSH, LH and prolactin were similar, The AMH level was significantly higher in PCOS patients (p < 0.0001). FBS, Cholesterol and Triglyceride were non-significantly higher in PCOS patients (Table p = 0.024). However, oocyte subtypes  did not shown any significant differences between two groups.135 patients include 45 PCOS patients with mean age of 31.93 ± 5.04 and 90 Non-PCOS with mean age of 30.8 ± 5.38 (p=0.066)  and AMH level , respectively. While the MII oocytes significantly affected by age  and AMH level .The embryo numbers and its subtypes did not differ significantly in both groups. The clinical pregnancy rate was insignificantly lower in PCOS patients (6) Table . There wWe calculated the cut-off values of total number of retrieved oocytes, MI, MII, GV using ROC curves to predict in vitro fertilization (IVF) outcomes. Cut-off value for total oocyte number was >10.5 with area under curve of 0.619±0.054(sensitivity 55.56% and specificity 69.66%)   Table .Table 3Spearman correlation test showed negative correlation between total number of embryo and age but the total number of embryo, embryo grade C and B had positive correlation with AMH. There was no significant correlation between embryos grade A and D with the other variables Table .Table 4Our results have shown the number of retrieved oocytes was significantly higher in PCOS patients rather than non-PCOS. However, we could not find any statistically significant differences in number of oocyte subtypes , number of embryo subtypes  and pregnancy rate between two groups. It was seen that; the higher rate of retrieved oocyte numbers didn’t guarantee the higher clinical pregnancy rate in PCOS group. What is important in increasing the pregnancy rate is the quality of retrieved oocytes and embryos, not the higher number of them. Despite the higher number of retrieved oocytes, the number of high-quality oocytes and embryos didn’t differ significantly in two groups. In according to our results, Ludwig et al.  and PlacThis study showed that the number of oocytes in PCOS patients was significantly higher than non-PCOS patients but; the number and quality of embryos were not significantly different in both groups. We also found there is a significant correlation between cholesterol level and number of MI oocytes. So, it seems that lowering blood cholesterol levels can increase the success rate of the ART cycle however, more studies are needed to confirm this finding.