This cross-sectional study analyzes changes in breast, cervical, and colorectal cancer screening before and after the COVID-19 pandemic among adults in Ontario, Canada. Did preexisting disparities in cancer screening change after the COVID-19 pandemic?In this cross-sectional study of 9 471 513 adults in Ontario, Canada, who were eligible for breast, cervical, and colorectal cancer screening before the COVID-19 pandemic, assessed at 2 time points, preexisting disparities in screening for people living in lower-income neighborhoods and for immigrants significantly widened for both breast and colorectal cancer screening.Results of this study suggest that policy makers should investigate the value of prioritizing and investing in improving access to team-based primary care for people who are immigrants and/or living with low income. Breast, cervical, and colorectal cancer–screening disparities existed prior to the COVID-19 pandemic, and it is unclear whether those have changed since the pandemic.To assess whether changes in screening from before the pandemic to after the pandemic varied for immigrants and for people with limited income.This population-based, cross-sectional study, using data from March 31, 2019, and March 31, 2022, included adults in Ontario, Canada, the country’s most populous province, with more than 14 million people, almost 30% of whom are immigrants. At both dates, the screening-eligible population for each cancer type was assessed.Neighborhood income quintile, immigrant status, and primary care model type.For each cancer screening type, the main outcome was whether the screening-eligible population was up to date on screening (a binary outcome) on March 31, 2019, and March 31, 2022. Up to date on screening was defined as having had a mammogram in the previous 2 years, a Papanicolaou test in the previous 3 years, and a fecal test in the previous 2 years or a flexible sigmoidoscopy or colonoscopy in the previous 10 years.The overall cohort on March 31, 2019, included 1 666 943 women (100%) eligible for breast screening , 3 918 225 women (100%) eligible for cervical screening , and 3 886 345 people eligible for colorectal screening . The proportion of people up to date on screening in Ontario decreased for breast, cervical, and colorectal cancers, with the largest decrease for breast screening  and the smallest decrease for colorectal screening . Preexisting disparities in screening for people living in low-income neighborhoods and for immigrants widened for breast screening and colorectal screening. For breast screening, compared with income quintile 5 (highest), the β estimate for income quintile 1 (lowest) was −1.16 ; for immigrant vs nonimmigrant, the β estimate was −1.51 . For colorectal screening, compared with income quintile 5, the β estimate for quntile 1 was −1.29 ; for immigrant vs nonimmigrant, the β estimate was −1.41 . The lowest screening rates both before and after the COVID-19 pandemic were for people who had no identifiable family physician . In addition, patients of interprofessional, team-based primary care models had significantly smaller reductions in β estimates for breast , cervical , and colorectal  postpandemic screening and higher uptake of screening in general compared with patients of other primary care models.In this cross-sectional study in Ontario that included 2 time points, widening disparities before compared with after the COVID-19 pandemic were found for breast cancer and colorectal cancer screening based on income and immigrant status, but smaller declines in disparities were found among patients of interprofessional, team-based primary care models than among their counterparts. Policy makers should investigate the value of prioritizing and investing in improving access to team-based primary care for people who are immigrants and/or with limited income. There were 41% fewer screening tests performed overall in Ontario in 2020 compared with 2019,10 and by December 2020, there was a backlog of more than 300 000 screening mammograms.11 The screening backlog was expected to be associated with delays in cancer diagnosis: between March and August 2020, there were, on average, 51% fewer high-grade cytological abnormalities detected through screening each month compared with 2019,12 and Tinmouth et al13 estimated that it would take 41 months to recover from the provincial backlog of colonoscopies, the recommended test after abnormal colorectal screening results with a fecal immunochemical test.In March 2020, the World Health Organization declared a global pandemic of COVID-19.21 For example, a 2018 report from the provincial advisor on the quality of health care found that only 55% of women living in extremely income-limited, urban neighborhoods were up to date on cervical screening compared with 66% of women in the wealthiest, urban neighborhoods and that 45.6% of people living in extremely income-limited, urban neighborhoods were overdue for colorectal cancer screening compared with only 32.5% in the wealthiest, urban neighborhoods.22 Similarly, Vahabi et al23 found that 57% of immigrants were up to date on breast cancer screening compared with 66% of nonimmigrants.The COVID-19 cancer screening backlog was occurring in the context of longstanding provincial disparities in breast, cervical, and colorectal cancer screening for people who are immigrants or with limited income, despite 3 organized provincial screening programs that provide clear, evidence-based guidelines on screening eligibility, centralized correspondence to those due for screening, and family physician audit and feedback reports for patients in their practice.24 but it is also feasible that barriers to screening were heightened in the context of COVID-19 for people experiencing disparities. In this population-based, retrospective, cross-sectional study, we aimed to assess whether changes in screening from before the pandemic to March 2022 varied by income or immigration status.It is unclear how the COVID-19 pandemic has affected preexisting screening disparities. Primary care physicians in Ontario were encouraged to consider prioritizing people who were underscreened or never screened for cancers when screening resumed,STROBE) reporting guideline.Research ethics board approval for this study was obtained by ICES  through the Privacy Impact Assessment of Sunnybrook Health Sciences Centre, Toronto, Ontario. ICES is an independent, nonprofit research institute, and its legal status under Ontario’s health information privacy law allows it to collect and analyze health care and demographic data, without consent, for health system evaluation and improvement. This study followed the Strengthening the Reporting of Observational Studies in Epidemiology . The models with additional payments include those in which payment is primarily capitation based but with a small fee-for-service component and additional payments based on quality performance, including cancer screening . Among primarily capitation-based models, those that include government-funded, interprofessional teams are known as family health teams. More than 77% of Ontario residents are formally enrolled with a family physician practicing in a patient enrollment model.28 There are also 101 community health centers in Ontario, which are nonprofit organizations that provide primary care and health-promotion services to patients and communities, with a focus on people experiencing marginalization, serving less than 2% of the population.29 These centers also are structured using interprofessional teams. Physicians working in community health centers are paid by salary.30 Family physicians who do not work in a patient enrollment model or in a community health center work under a traditional fee-for-service pay structure.Ontario is Canada’s most populous province with more than 14 million people, of whom almost 30% are immigrants.31 This data set includes patient demographic characteristics, family physician enrollment status, health care usage information, and cancer screening status. The Ontario Health Insurance Plan claims database and Discharge Abstract Database were used for billing code and diagnostic information and for exclusions. The ICES Physician Database and Corporate Provider Database were used to obtain information on family physicians. The Canadian 2016 Census of Population was used to determine neighborhood income quintile (given as Q1-Q5) based on postal code of residence. These data sets were linked using unique encoded identifiers and analyzed at ICES using SAS, version 8.3 (SAS Institute Inc).We accessed several data sets available at ICES, including the Immigration, Refugees and Citizenship Canada Permanent Residents database, which consists of detailed demographic information on Ontario’s immigrants and refugees who arrived in the country beginning in 1985. Data include their country of birth, date of arrival, and immigrant category. The Postal Code Conversion File was used to obtain Rurality Index for Ontario (RIO) classification based on postal code of residence. The Registered Persons Database was used to determine residents’ health insurance coverage. We also accessed the Primary Care Population Database, which includes every Ontario resident who is eligible for primary care with active provincial health insurance coverage and a health care contact in the previous 7 to 11 years at a given point in time.32For each cancer screening type, we assessed whether the screening-eligible population was up to date on screening (a binary outcome) at 2 time points: March 31, 2019 (prior to the COVID-19 pandemic), and March 31, 2022. Being up to date on screening was defined as having had a mammogram in the previous 2 years, a Papanicolaou test in the previous 3 years, and a fecal test in the previous 2 years or a flexible sigmoidoscopy or colonoscopy in the previous 10 years.31 The lower age thresholds were chosen to allow for individuals to be eligible for screening for the full look-back window.At each of the 2 time points, we defined the screening-eligible population for each cancer type. We started with all residents of Ontario who had at least 5 years’ history of Ontario Health Insurance Plan coverage on the index dates . Women eligible for breast screening were defined as those aged 52 to 69 years, excluding those with a history of breast cancer or mastectomy. Women eligible for cervical screening were defined as those aged 23 to 69 years, excluding those with a history of cervical cancer or hysterectomy. People eligible for colorectal screening were defined as those aged 52 to 74 years, excluding those who had a history of colorectal cancer or inflammatory bowel diseases.33 and health care use over the prior 2 years using resource utilization bands (RUBs)  from the case-mix Johns Hopkins ACG System, version 10.0 (Johns Hopkins Medicine),34 which uses outpatient billing and inpatient hospital records. For immigrants, we also determined their world region of origin based on country of birth and a previously published modification of the World Bank classification system, as screening status has been shown to vary by region of origin.36We described each study cohort based on age category, sex, neighborhood income quintile, immigrant status (immigrant or not), primary care model, rurality of residence based on postal code using the RIO classification,We used counts and frequencies to describe the 3 screening-eligible cohorts based on the aforementioned variables as of March 31, 2019. We assessed up-to-date status for each screening type at 6-month intervals, from April 1, 2012, to April 1, 2022, for the overall cohorts, as well as stratified by income quintile and immigrant status. We also compared breast, cervical, and colorectal screening at our 2 time points for the overall cohorts as well as stratified by our study variables.P < .05.For each screening type, we conducted multivariable regression analyses using generalized linear models, with the rate difference between the postpandemic and prepandemic time points as the outcome. To assess the rate difference after and before the pandemic, we aggregated individual-level data to unique combinations of each category of age group, sex , income quintile, immigration status, patient enrollment model, RIO categories, and RUBs, all of which were included in the model. The mean number of eligible people from before and after the pandemic in each cluster was used as weight to account for different size of denominators. To avoid small denominators and to prevent unstable rates in the regression models, we combined family health teams and community health centers into 1 category, as both use interprofessional teams. We combined those in RUBs 0 and 1 and 4 and 5, and we excluded people with no identifiable family physician. To reduce the outcome of unstable results associated with small clusters, we excluded clusters having fewer than 50 eligible people either before or after the pandemic in the generalized linear models. This excluded no more than 0.36% (for breast screening) of the 2019 population and no more than 0.46% (for breast screening) of the 2022 population. The threshold for statistical significance was 2-sided On March 31, 2019, there were 1 666 943 women eligible for breast screening , 3 918 225 women eligible for cervical screening , and 3 886 345 people eligible for colorectal screening  in Ontario . BetweenFrom March 31, 2019 (before the pandemic), to March 31, 2022, the proportion of people up to date for each screening type declined, by 9.4 percentage points for breast (61.1% to 51.7%), 7.3 percentage points for cervical (59.4% to 52.1%), and 3.9 percentage points for colorectal (65.9% to 62.0%) cancer screening . For breMultiple regression analysis results for those with a family physician are shown in In this population-based cross-sectional study comparing cancer screening prior to the COVID-19 pandemic vs in the postpandemic period, we found that the proportion of people up to date on screening in Ontario decreased for breast, cervical, and colorectal cancers, with the largest decrease for breast screening (9.4 percentage points) and the smallest decrease for colorectal screening (3.9 percentage points). Preexisting disparities in screening for people living in low-income neighborhoods and for immigrants widened for both breast and colorectal screening. The lowest screening rates both before and after the pandemic were for people who had no identifiable family physician, in which rates were no higher than 17.6%  and were as low as 9.6% (breast screening in 2022). We also found that patients of interprofessional models, such as family health teams and community health centers, had significantly smaller reductions in postpandemic screening and higher screening rates at each time point compared with other primary care patients.37) province-wide. Among interprofessional teams, nonphysician practitioners may play a role in identifying patients who are overdue for screening and who need screening outreach or screening education and in performing and/or ordering screening tests. However, efforts to expand access to team-based care must center on health equity .39 These efforts will require significant investment: although community health centers are designed for people experiencing marginalization, they currently serve less than 2% of the province’s population, and family health teams have traditionally been least available in geographic areas with the greatest need.40Our findings suggest that access to primary care, as well as the type of primary care model to which one has access, played a crucial role in cancer-screening recovery after the COVID-19 pandemic. These results may make a compelling argument for expanding access to interprofessional, team-based primary care as a method of increasing cancer-screening uptake . Provincial screening guidelines are different for individuals at increased risk.This population-based, cross-sectional study in Ontario, Canada, found that the COVID-19 pandemic was associated with a decrease in up-to-date status for breast, cervical, and colorectal cancer screening and that people with limited income and immigrants generally had larger decreases than their counterparts. We also found that people without family physicians had very low screening uptake both before and after the pandemic and that, among those with family physicians, access to interprofessional, team-based primary care may have been protective for screening during the pandemic. Policy makers should investigate the value of prioritizing and investing in improving access to team-based primary care for people who are immigrants and/or with limited income.


After 30 min, the expression and concentration of hypothalamic CRF and AVP, the concentration of plasma adrenocorticotropic hormone (ACTH) and corticosterone (CORT), and the release of striatal DA, amygdalar GABA, and hippocampal GLU were measured. Our results indicate that the neuroendocrine changes induced by alcohol intoxication and withdrawal are mediated by CRF1, not CRF2, except for the changes in hypothalamic AVP, which are not mediated by CRF receptors.The aim of the present study was to determine the effects of alcohol intoxication and withdrawal on hypothalamic neurohormones such as corticotropin-releasing factor (CRF) and arginine vasopressin (AVP), and extrahypothalamic neurotransmitters such as striatal dopamine (DA), amygdalar gamma aminobutyric acid (GABA), and hippocampal glutamate (GLU). In addition, the participation of the two CRF receptors, CRF1 and CRF2, was investigated. For this purpose, male Wistar rats were exposed to repeated intraperitoneal (ip) administration of alcohol every 12 h, for 4 days and then for 1 day of alcohol abstinence. On the fifth or sixth day, intracerebroventricular (icv) administration of selective CRF1 antagonist antalarmin or selective CRF2 antagonist astressin Alcohol addiction has three stages including binge/intoxication, withdrawal/negative affect, and preoccupation/anticipation (craving). Each stage is characterized by specific changes in the hypothalamic neurohormones such as corticotropin-releasing factor (CRF) and arginine vasopressin (AVP), as well as extrahypothalamic neurotransmitters such as striatal dopamine (DA), amygdalar gamma aminobutyric acid (GABA), and hippocampal glutamate (GLU) ,2.The stage of binge/intoxication is associated with the activation of the hypothalamic–pituitary–adrenal (HPA) axis that is initiated by hypothalamic CRF and/or AVP . The HPAThe stage of withdrawal/negative affect is associated with the activation of the extended amygdala circuit, which is mediated by extrahypothalamic CRF and norepinephrine . The extAlcohol withdrawal syndrome consists of somatic  signs and affective  symptoms that emerge immediately after alcohol cessation ,2. The pCRF acts via two distinct CRF receptors, CRF1 and CRF2, with putatively antagonistic actions in the central nervous system (CNS) . CRF1 is2B was performed. After 30 min, the mice were decapitated without anesthesia, trunk blood was collected, and the brains were removed. From the brain, the expression and concentration of hypothalamic CRF and AVP as well as the release of striatal DA, amygdalar GABA, and hippocampal GLU were determined. From the trunk blood, the concentration of plasma ACTH and CORT were measured.The aim of our study was to determine the effects of alcohol intoxication and withdrawal on hypothalamic neurohormones and extrahypothalamic neurotransmitters in rats. Since CRF was involved in all three stages of alcohol addiction, the participation of the two CRF receptors was investigated. For this purpose, male Wistar rats were exposed to repeated intraperitoneal (ip) administration of alcohol every 12 h, for 4 days and then for 1 day of alcohol withdrawal. On the fifth day  or the sixth day , intracerebroventricular (icv) administration of the selective CRF1 antagonist antalarmin or selective CRF2 antagonist astressinN = 72). The rats were treated in accordance with the ARRIVE guidelines and the experiments were carried out in concordance with the EU Directive 2010/63/EU for animal experiments. They were housed together and kept in their home cages at a constant temperature on a standard illumination schedule with 12-h light and 12-h dark periods (lights on from 6:00). Commercial food and tap water were available ad libitum. They were also handled daily in order to minimize the effects of non-specific stress.Male Wistar rats weighing 150–250 g were used  on cobas c502 analyzer . The sensitivity of the assay was 10.1 mg/dL (0.01 g/dL). The GeneJET RNA Purification Kit, the Maxima First Strand cDNA Synthesis Kit, and the NanoDrop One device required for the determination of hypothalamic CRF and AVP expression was provided by Thermo Scientific Inc., Waltham, MA, USA. The sandwich Enzyme-linked Immunosorbent Assay (ELISA) Kits required for the determination of the hypothalamic CRF and AVP, and plasma ACTH concentrations were purchased from Phoenix Pharmaceuticals Ltd., Mannheim, Germany. The methylene chloride, sulfuric acid, and ethyl-alcohol solutions used for the determination of the plasma CORT concentration were provided by Reanal Ltd., Budapest, Hungary. The Krebs solution was provided by Reanal Ltd., Hungary. The tritium-labelled neurotransmitters including the [3H]DA, [3H]GABA, and [3H]GLU, and the Ultima Gold scintillation fluid used for the in vitro superfusion studies and liquid scintillation were ordered from Perkin-Elmer Inc., Waltham, MA, USA.The saline and alcohol solutions administered ip were provided by B. Braun Inc., Melsungen, Germany and Reanal Ltd., Budapest, Hungary, respectively. The selective CRF1 antagonist antalarmin and the selective CRF2 antagonist astressinThe rats were implanted with a stainless steel Luer cannula (10 mm long) that was aimed at the right lateral cerebral ventricle. The surgical intervention was performed under anesthesia with 35 mg/kg pentobarbital sodium . The stereotaxic coordinates for the right lateral cerebral ventricle were 0.2 mm posterior and 1.7 mm lateral to the bregma, and 3.7 mm deep from the dural surface, according to a stereotaxic atlas of the rat brain . CannulaMale Wistar rats were exposed to repeated ip administration of alcohol every 12 h, for 4 days and then for 1 day of alcohol withdrawal. The protocol of alcohol administration was based on a previous study in which 20% alcohol was administered at dose of 3 g/kg DA, [3H]GABA, or [3H]GLU incubated, then superfused for 30 min and electrically stimulated for 2 min with the means of a superfusion system provided by Experimetria Ltd., Budapest, Hungary. The superfusates were collected in Eppendorf tubes by a multichannel fraction collector (Gilson FC 203B). The total collecting time was 32 min  and the peak of the fractional release was observed at 14 min. In the corresponding figures, only these peaks were represented. Finally, the brain tissue was removed from the superfusion system and solubilized in Krebs solution using an ultrasonic homogenizer called a Branson Sonifier 250. After the addition of scintillation fluid to the samples and the remaining brain tissue, the radioactivity was measured with a liquid scintillation spectrometer  and expressed in count per minute (CPM). The fractional release was calculated as the ratio between the radioactivity of the samples and that of the remaining brain tissue.The striatal DA, amygdalar GABA, and hippocampal GLU release were measured by means of an in vitro superfusion system, which was described originally by Gaddum, and later improved by Harsing and Vizi ,20. The 2B or saline) ANOVA was performed with the estimated marginal means calculated followed by the Bonferroni post hoc test. A probability level of less than 0.05 was accepted as indicating a statistically significant difference.Data were presented as the means ± SEM. Statistical analysis of the results was performed by ANOVA, if the test prerequisites allowed, using SPSS Software v.29.0 . A two-way 2  × 3  = 1.875, p < 0.001], and a significant interaction between the two factors  were observed. On the sixth day, a significant main effect in the alcohol-treated group , a significant main effect in the antagonist-treated group , and a significant interaction between the two factors  were detected.Hypothalamic CRF mRNA expression was increased by alcohol intoxication and withdrawal, and these stimulatory effects were reduced by antalarmin, but not astressin2B . On the p < 0.001], but no significant main effect in the antagonist-treated group, yet a significant interaction between the two factors  were assessed. On the 6th day, a significant main effect in the alcohol-treated group , but no significant main effect in the antagonist-treated group and no significant interaction between the two factors were noticed.In addition, the hypothalamic CRF concentration was increased by alcohol intoxication and withdrawal, and these stimulatory effects were reversed by the selective CRF1, but not CRF2 antagonist . On the 2B  = 6.299, p < 0.001] were observed. On the sixth day, a significant main effect in the alcohol-treated groups , no significant main effect in the antagonist-treated group, but a significant interaction between the two factors  were detected.Hypothalamic AVP mRNA expression was decreased by alcohol intoxication and withdrawal, but these inhibitory effects were not significantly influenced either by antalarmin or astressin2B . On the p < 0.001], but no significant main effect in the antagonist-treated group, and no significant interaction between the two factors were assessed. On the 6th day, a significant main effect in the alcohol-treated group , but no significant main effect in the antagonist-treated group, and no significant interaction between the two factors were noticed.In contrast, hypothalamic AVP concentration was increased by alcohol intoxication and withdrawal, but these stimulatory effects were not affected by none of the selective CRF antagonists .. On the2B  = 12.523, p < 0.001], and a significant interaction between the two factors  were proved. On the 6th day, a significant main effect in the alcohol-treated group , but no significant main effect in the antagonist-treated group, and no significant interaction between the two factors were shown.The plasma ACTH level was elevated by alcohol intoxication and withdrawal, and these elevations were ameliorated by antalarmin, but not astressin2B . On the p < 0.001], a significant main effect in the antagonist-treated group , and a significant interaction between the two factors  were shown. On the 6th day, a significant main effect in the alcohol-treated group , a significant main effect in the antagonist-treated group , and a significant interaction between the two factors  were proved.In parallel, the plasma CORT level was augmented by alcohol intoxication and withdrawal, and these augmentations were attenuated by the selective CRF1, but not CRF2 antagonist . On the 2B  = 98.352, p < 0.001], and a significant interaction between the two factors  were observed. On the sixth day, a significant main effect in the alcohol-treated group , a significant main effect in the antagonist-treated group , and a significant interaction between the two factors  were detected.Alcohol intoxication increased, whereas alcohol withdrawal decreased the striatal DA release, and both effects were reduced by antalarmin, but not astressin2B . On the p < 0.001], a significant main effect in the antagonist-treated group , and a significant interaction between the two factors  were assessed. On the sixth day, a significant main effect in the alcohol-treated group , a significant main effect in the antagonist-treated group , and a significant interaction between the two factors  were noticed.Similarly, alcohol intoxication increased, whereas alcohol withdrawal decreased the amygdalar GABA release and both effects were reversed by the selective CRF1, but not CRF2 antagonist . On the 2B  = 172.546, p < 0.001], and a significant interaction between the two factors  were shown. On the sixth day, a significant main effect in the alcohol-treated group , a significant main effect in the antagonist-treated group , and a significant interaction between the two factors  were proved.Consequently, the hippocampal GLU release was decreased and increased in alcohol intoxication and alcohol withdrawal, respectively, and both effects were antagonized by antalarmin, but not astressin2B . On the Our results indicate that during alcohol intoxication, the HPA axis is activated by hypothalamic CRF and is reflected by the elevation of plasma CORT and ACTH levels. Aside from the stress axis, the reward system is also activated, mainly resulting in increased striatal DA, but also increased amygdalar GABA and decreased hippocampal GLU release. During alcohol withdrawal, the HPA axis remains activated, but this time, the activation is accompanied by the decrease in the striatal DA and amygdalar GABA release, and increase in hippocampal GLU release, which is probably mediated by extrahypothalamic CRF.Regarding the hypothalamic neurohormones, previous studies have already suggested the role of CRF in the HPA-axis activation induced by alcohol based on several lines of evidence . First, Regarding the extrahypothalamic neurotransmitters, previous studies have suggested that alcohol binge/intoxication and withdrawal/negative effects are also associated with certain changes in the striatal DA, amygdalar GABA, and hippocampal GLU ,2. Acute2B [As for the role of CRF receptors in the neurohormonal changes induced by alcohol, previous in vivo experiments have already reported that alcohol induced upregulation of paraventricular CRF1 expression , and tha2B . This is2B ,44,45. IIn conclusion, our results indicate that the neuroendocrine changes induced by alcohol intoxication and withdrawal are mediated by CRF1, not CRF2, except for the changes in hypothalamic AVP, which are not mediated by CRF receptors. Therefore, our study demonstrates that 4-day ip administration of alcohol followed by 1-day abstinence in rats is a valid model for alcohol intoxication and withdrawal, characterized by specific changes in hypothalamic neurohormones and extrahypothalamic neurotransmitters, which can be used for therapeutical purposes.


Leishmania braziliensis can either respond well or poorly to the treatment or heal spontaneously; It seems to be dependent on the parasite and/or host factors, but the mechanisms are not fully understood. We evaluated the in situ immune response in eighty-two active lesions from fifty-eight patients prior to treatment classified as early spontaneous regression (SRL-n = 14); treatment responders (GRL-n = 20); and non-responders . Immunohistochemistry was used to identify cell/functional markers which were correlated with the clinical characteristics. PRL showed significant differences in lesion number/size, clinical evolution, and positive parasitological examinations when compared with the other groups. SRL presented a more efficient immune response than GRL and PRL, with higher IFN-γ/NOS2 and a lower percentage of macrophages, neutrophils, NK, B cells, and Ki-67+ cells. Compared to SRL, PRL had fewer CD4+ Tcells and more CD163+ macrophages. PRL1 had more CD68+ macrophages and Ki-67+ cells but less IFN-γ than GRL. PRL present a less efficient immune profile, which could explain the poor treatment response, while SRL had a more balanced immune response profile for lesion healing. Altogether, these evaluations suggest a differentiated profile of the organization of the inflammatory process for lesions of different tegumentary leishmaniasis evolution.Localized cutaneous leishmaniasis caused by  Leishmania (Viannia) braziliensis and the most common clinical presentation is termed localized cutaneous leishmaniasis (LCL) [Leishmaniasis is an infectious disease mainly distributed in tropical and sub-tropical regions with varied clinical presentation and the potential to cause deformities and death. The main clinical presentations are visceral (VL) and tegumentary leishmaniasis (TL); the latter of which can be subdivided into cutaneous (CL) and mucosal (ML) leishmaniasis. In the last two decades, more than 1,000,000 cases of TL were reported to the Pan American Health Organization (PAHO), with a yearly average greater than 50,000 cases. In Brazil, TL is mainly caused by is (LCL) ,3,4,5. Lis (LCL) . Some hyis (LCL) ,8,9.Of the cases that demand specific treatment, some patients do not reach lesion healing after one complete treatment cycle, which is characterized as therapeutic relapse. The phenomenon of non-response to primary treatment has been increasingly reported in the literature and is a great concern because the therapeutic arsenal for leishmaniasis is still quite restricted ,13,14,15Leishmania species of the Viannia subgenus also triggers macrophage activation. Macrophages are highly plastic cells, showing a spectrum of phenotypes depending on the stimulus received from the environment. Macrophages have enormous plasticity and can perform diverse functions depending upon their phenotypic state, but for didactic purposes, they have been classified as Classically activated (M1) macrophages or alternatively activated (M2) macrophages [The literature shows that spontaneous healing or the development of severe lesions in TL is influenced by the immune response and characteristics of the organized inflammation in the lesion sites ,22,23,24rophages . M1 favorophages ,33,34,35rophages . Extracerophages ,37,38,39A few studies have assessed and compared the immune response in cases of CL with early spontaneous healing and those that respond poorly to treatment ,42,43,44The purpose of this study was to evaluate and compare some aspects of the in situ immune response in the active cutaneous lesions of patients presenting early spontaneous regression of TL (without treatment) and responders or non-responders to specific treatment with meglumine antimoniate (prior to treatment and at therapeutic relapse). These findings were then correlated with clinical and parasitological parameters in order to identify possible related patterns.Leishmania. The study was approved by the institutional Research Ethics Committees of the Instituto Oswaldo Cruz and Instituto Nacional de Infectologia Evandro Chagas (CEP-IOC: CAEE 88890518.6.0000.5248 and CEP-INI: CAEE 88890518.6.3001.5262). The variables of interest for the characterization of the study group were sex, age, size and number of lesions, lesion location and its characteristics, and response to the treatment. When possible, the Leishmania species, isolated by the culture of the tissue obtained during the biopsy, was characterized using multilocus enzyme electrophoresis (MLEE), PCR of HSP-70, restriction fragment length polymorphism (RFLP) with HaeIII and BstUI enzymes, and/or genetic sequencing [Eighty-two active lesions from fifty-eight patients with the localized cutaneous form of TL, as confirmed by parasite detection , were selected and classified into three groups: (A) lesions that evolved early spontaneous regression during the diagnosis procedure and consequently no treatment was required ; (B) “responders to meglumine antimoniate treatment” with no relapse for at least 1 year of longitudinal monitoring ; and (C) “non-responders to meglumine antimoniate treatment” . The specific treatment comprised the use of meglumine antimoniate administered intramuscularly at 5 mg Sb5+/kg/day for 30 days as previously described ,53,54. Pquencing .Leishmania sp. . The sections were incubated with biotinylated secondary antibody , stained using the Histostain®-Plus enzyme labeling kit containing streptavidin peroxidase  and then the AEC staining kit (Zymed Laboratories Inc.), with intermediate washing steps in PBS. Following this sequence, the sections were stained with Mayer’s hematoxylin (Dako), then a coverslip was added with Faramount Mounting Medium (Dako). Negative controls without primary antibodies were carried out. Slides were analyzed on an optical microscope and the percentage of labeled cells was determined by counting 500 cells or 10 fields (1000× magnification). For NOS2 specifically, label intensity was determined by the number of positive sites per 20× field: discrete (1 positive site), moderate (2 positive sites), intense (3 positive sites), and very intense (4 a 5 positive sites), as previously described [The immunohistochemistry technique was carried out on 3 to 4-μm sections of cryopreserved tissue from the lesions affixed to silanized slides as previously described ,43. Afteescribed . The picTo enable double labeling, immunofluorescence was performed following the same steps as conventional immunohistochemistry regarding the application of the primary antibodies. Subsequently, sections were stained with specific fluorophore-conjugated secondary antibodies, Alexa Fluor 488 (Invitrogen), R-phycoerythrin , and DyLightTM 633 . Fluoromount-G Mounting Medium with DAPI (Invitrogen) was used to mount the slide and coverslip. Images were captured on a Zeiss Axio lmager M1 microscope with the AxioVision software .p values ≤ 0.05 were considered significant.A specific database was constructed for this study using the R 4.2.1 software  and GraphPad Prism 8.0 . The Shapiro–Wilk test was used to verify the normality of the data. Analysis of immunohistochemistry results of the active primary lesions was compared two-by-two using the Mann–Whitney non-parametric test, while the Wilcoxon test was used for samples paired in the same patient (PRL). The Kruskal–Wallis test was used for the analysis and characterization of patient data and comparison of three or more groups using Dunn’s Test correction. The chi-square exact test was used to analyze NOS2 expression. Data were presented in distribution as the mean ± SEM (minimum error) or as the median. When applicable, p = 0.032) and greatest lesion size (p = 0.05), which varied from 5 to 90 mm in diameter , as well as the lesion at therapeutic failure (PRL2) showed significant differences in all cell and functional markers, except for CD8, mast cell tryptase, and CD206. p < 0.0001; CD4+ and CD8+ T lymphocytes presented homogenous distribution throughout the dermis in all groups. PRL had fewer CD4+ T cells than SRL specifically, while GRL was found to have significantly more CD4+ cells compared with the lesions of the other groups (p < 0.0005), mainly due to more CD22+ cells in PRL when compared with SRL, as SRL presented the lowest concentration of this cell marker . SRL presented the lowest amount of CD68+ staining and the highest IFN-γ+ staining   and CD206 (mannose receptor). Both markers were homogenously distributed throughout the tissue of all lesions and no statistically significant differences were observed in the groups regarding CD206 (= 0.005) .Double staining of the biopsy sections was performed to observe co-localization of macrophage markers (CD68 or CD163) with the functional markers, NOS2 and arginase, which are indicative of M1 and M2 macrophages, respectively. Although quantification could not be made, M1 and M2 macrophages could be detected in all groups, as shown in p = 0.009; p = 0.05, Neutrophils were present in all lesions regardless of the group evaluated or the clinical characteristics of evolution time and number of lesions. Heterogeneous distribution was observed throughout the tissue. Although the mean values of these cells appeared to be similar across the groups , there wMast cells were homogenously distributed throughout the dermis of all lesions. In general, the staining for mast cells, via mast cell tryptase, was present in small concentrations (around 5 to 8%) in the lesions, with no statistical differences between the groups .p < 0.0001, NK cells, identified by the CD56 marker, were also homogeneously distributed throughout the tissue of the lesions. A significant difference was identified when the groups were compared altogether (p < 0.0001) . The other groups showed similar values, about three times higher than that of the SRL group. Statistically significant differences were observed when SRL was independently compared to the other groups   . Interesp = 0.08).Regarding the presence of parasites detected by immunohistochemistry , we obseIn the present study, biopsies of the active lesions from LCL patients were collected prior to meglumine antimoniate treatment. These patients either healed spontaneously during the diagnosis process , or were considered good (GRL) or bad (PRL) responders to the specific treatment. Evaluation of cell and functional markers, prior to the treatment, within the lesions of these groups, indicated differences in modulation of the skin immune response. In general, patients presented clinically similar lesions, but PRL patients showed a greater number of lesions and it was easier to find parasites using different diagnostic exams. The majority of PRL patients presented three or more positive tests, while in most patients with spontaneous resolution, only one exam indicated positivity, this was usually by PCR detection. In LCL, the relationship a between higher number of lesions and the difficulty in lesion healing has already been reported in the medical literature ,56. WhenWhen the primary lesions of the patients were compared, patients with early spontaneous resolution and those with good treatment response had the most balanced immune responses with a type 1 profile, i.e., a higher proportion of CD4+ T cells and higher IFN-γ expression, than in the lesions of patients who experienced a therapeutic relapse. It is also noteworthy that when we compared SRL with GRL, we could see that in spontaneous healing lesions, there were lower concentration markers of inflammatory activity, such as the receptor marker for IL-2R (CD25), the Ki-67 proliferative cell marker, as well as markers for neutrophils, macrophages, NK cells, and CD8 T cells, suggesting that the inflammatory process was more controlled. This might also be another indirect explanation as to why these lesions seem to have a smaller number of parasites, as many of these cells are involved in cytotoxicity processes that are associated with the presence of parasites and involved in parasite elimination, as already described in murine models. However, they can lead to tissue destruction when exacerbated, worsening the clinical aspect of the lesions and the response to treatment ,58,59.Leishmania spp. has already been demonstrated in both in vitro and in vivo infection models [When the groups needing specific treatment were compared, those that responded well presented higher proportions of CD4+ and IFN-γ+ cells than either the initial or reactivated lesions of the non-responders (PRL1 and PRL2). In CL, CD4+ T cells and IFN-γ lead to the activation of macrophages toward the destruction of parasites . The expn models ,66, and n models . Patientn models .Leishmania spp. [With regards to neutrophils, another phagocytic cell type, our results showed similar infiltration of these cells in the initial lesions of the treatment responders and non-responders, wherein both groups had higher proportions of this cell type compared to lesions of early spontaneous resolution. However, the NET formation was more evident in patients with good evolution towards healing (GRL and SRL). The NET formation is identified as a mechanism of parasite control in different infectious diseases, including with extracellular nia spp. ,37,39,69nia spp. . Furthernia spp.  demonstrLeishmania spp., is reported to be crucial for infection control [The importance of a type 1 immune response, characterized by cytotoxicity, with optimal concentrations of IFN-γ and lymphocytes, and macrophages activated towards the elimination of  control ,73,74,75 control . Thus, cBesides a type 1 response, phenomena aimed at regulating the in situ immune response are extremely important for the balance between parasite elimination and tissue preservation. The inflammatory process progresses with both pro-inflammatory and regulatory activities. T reg cells can modulate the innate and adaptive immune responses and have the ability to control excessive or misdirected effects of the immune response. This modulation involves different mechanisms such as the suppression of T cell proliferation and cytokine production, the secretion of immunosuppressive cytokines, and the induction of T cell apoptosis in several diseases including infectious ones . In rece+ cells that varied from 10 to 16% of the cells in the lesions, with a predominance in the lesions of responders to the specific treatment. This was more evident when these data were compared to lesions of therapeutic relapse in non-responder patients (PRL2). T reg cells participate in the resolution of dermal lesions and thus could be used as markers for immunotherapeutic strategies in leishmaniasis caused by species of the Vianna subgenus [L. braziliensis consistently present a lower proportion of FoxP3+ cells than lesions caused by other species of the Viannia subgenus, suggesting that the parasite species may influence the stimulation of immune regulation [Our results demonstrated a percentage of FoxP3subgenus . Asymptosubgenus . On the gulation .L. braziliensis is considered the most exclusive species [L. braziliensis, including the two patients from Amazonas and the patient from São Paulo.It has been demonstrated that the species of the parasite can influence the progress of infection . However species ,79. Six L. major, Trichuris muris, Nyppostrongylus brasiliensis, and Toxoplasma gondii was also due to its regulatory action [IL-33 has been associated with a type 2 immune response  and withy action . The auty action . In humay action . The lity action . In this2 activity). Altogether, these evaluations , the inflammatory activity seems insufficient to control the parasite, at least in the initial stages of the disease. The cases of patients who required treatment but presented a good therapeutic response (GRL) would be located in the middle of those two extremes, with evidence of a type 1 immune response and control of parasite load. A better understanding of the dynamics of the inflammatory process associated with the phenomena of LCL lesions towards healing may support the development of new treatments, including those based on immunotherapy, as well as the development of tools for vaccine design leading to better control of the infection.


In this review, the authors will discuss the highlights of early onset colon cancer (EOCRC), an increasing but still unclear phenomenon. Indeed, the risk factors are largely similar to classic colon cancer (CRC), but it is still unclear what causes an increased risk of CRC at an early age. In addition, the issue of screening, which allows early diagnosis of the disease, is also important; the younger segment of the population is not included in such programs except if they belong to the highest-risk groups. If the phenomenon of EOCRC becomes more prominent, screening programs will have to be reviewed but with consideration of the consequences for health economics. In this review, we will focus on the epidemiological, prognostic, clinico-pathological, molecular, and therapeutic features of EOCRC; in particular, we will try to highlight the characteristics of the metastatic stage and the differences with CRC in older segments of the population.Despite a recent overall decrease in colorectal cancer (CRC) incidence and mortality, there has been a significant rise in CRC diagnoses in young adults. Early onset colorectal cancer (EOCRC) is defined as CRC diagnosed before the age of 50. Possible predisposing conditions include not only genetic syndromes but also other risk factors, such as microbiome alteration, antibiotic exposure, obesity, diabetes mellitus, and inflammatory bowel disease. EOCRC tends to be diagnosed later than in the older counterpart because of a lack of awareness and the fact that screening for CRC usually starts at the age of 50. Furthermore, CRC in young adults seems to be related to unique molecular features and more aggressive clinical behavior. This paper aims to provide an in-depth review of this poorly understood subject, with a comprehensive review of the state of the art and considerations for future perspectives. Early onset colorectal cancer (EOCRC) is defined as CRC diagnosed in individuals younger than 50, which is generally considered the ideal age to start screening programs in the average-risk population. Although the overall incidence of colorectal cancer (CRC) is declining, the number of new diagnoses in patients younger than 50 is alarmingly increasing ,2. ModifEOCRC seems to have different features than CRC in older patients. EOCRC generally develops with more aggressive features, is diagnosed at a more advanced stage ,5,6,7, aOf note, the advanced stage at diagnosis might be related to the fact that screening campaigns do not involve the population under the age of 50, except among individuals with a family history of CRC or those affected by chronic IBD ,10. The Given the greater proportion of young patients that present with advanced disease at the time of diagnosis, young adults with Stage IV EOCRC represent a small but increasing and relevant population  that neeCRC is the third most common malignancy and cause of cancer death worldwide in both genders, mostly over the age of 50 . Since tConsidering the current data and despite general trends toward population aging, a retrospective cohort study foresees by 2030 an increase in colon cancer diagnosis of 90% in the 20–34 age cohort and 27.7% in the 35–49 age cohort, with an even higher rise for rectal cancer diagnosis of 124% and 46% for the two subgroups, respectively ,22.As displayed in In the United States (US), age-specific CRC risk has returned to the levels recorded in those born around 1890 . Here, aA similar trend was also observed in European countries, where from 2004 to 2016, CRC IRs increased by 7.9% annually in the age group of 20–29 years, 4.9% among those aged 30–39 years, and 1.6% in the 40–49 cohort [With regard to non-Western populations, in recent years, the incidence of EOCRC has also been increasing in Arabic countries, which could be attributed to improved diagnostic strategies and changes in lifestyle and dietary habits, which have become more similar to those of Western countries . An IR iWhen talking about geographic differences, an important factor to consider is the presence of a private healthcare system, as EOCRC seems more prevalent among patients with no insurance coverage or ready access to care (16.5% vs. 4.7%), often of nonwhite ethnicity (29.5% vs. 17.6%) [Considering that most EOCRC diagnoses are made in people with an average screening risk, and almost half of these patients are aged between 45 and 49 (SEER-Stat Database), since 2018, the American Cancer Society has indicated 45 as the optimal age to initiate CRC screening .Screening in individuals younger than 50 is universally recommended for people with an elevated risk of CRC because of chronic IBD, familial syndromes, or with a family history of CRC in a first-degree relative (FDR) . In thesAs shown in EOCRC is mainly a sporadic disease (80%): only about 1 out of 5 CRC patients diagnosed under the age of 50 carry a germline mutation, and almost half of them show no family history related to the corresponding hereditary cancer syndrome . HoweverLS is the most common cause of hereditary CRC and accounts for 2–3% of all CRC cases ,38. It iAPSs include familial adenomatous polyposis (FAP), polymerase proofreading-associated polyposis (PAPP), NTHL1-associated polyposis (NAP), and MUTYH-associated polyposis (MAP). FAP is caused by germline mutations in the APC tumor suppressor gene, and it has been associated with 1% of all CRC cases. In the process leading from adenoma to tumor, there is an initial mutation of the APC gene, which normally inhibits cell proliferation, resulting in the instability of other genes, such as KRAS, SMAD4, and P53 . TherefoCarriers of the APC mutation typically develop classic colonic polyposis with 100 to 1000 adenomas and show close to a 100% lifetime risk for CRC. The presence of attenuated phenotypes (20–100 polyps) is acknowledged ,42. The Li–Fraumeni syndrome, associated with germline TP53 mutations, causes less than 1% of EOCRC. Several other genes have been explored ; however, their association with EOCRC has been demonstrated in only 2% of cases .CF has recently been listed as a familial CRC syndrome. The loss of CFTR alters a variety of cellular processes and might lead to carcinogenesis. By the age of 40, 50% of CF patients develop adenomas, among which 25% are aggressive advanced adenomas or adenocarcinomas. This led to the development of new CF CRC screening recommendations, and the initiation of endoscopic screening has been lowered to the age of 40 in CF patients and to the age of 30 in organ transplant recipients ,44.The rising incidence of EOCRC has involved patients born after 1960, which suggests population-level behavioral changes or earlier points to exposure . We hereCompared with individuals without metabolic syndrome, those with 1, 2, or ≥3 metabolic comorbidities have a 9%, 12%, and 31% higher risk of EOCRC, respectively. These positive associations are true for colon but not rectal cancer .Obesity is surely one of the main modifiable risk factors associated with CRC. A prospective study involving US female nurses aged 25 to 42 revealed that the relative risk (RR) for incident EOCRC for each 5-unit increment in BMI in women was 1.2, considering weight gain since early adulthood . SeveralType II diabetes mellitus (DM) is also associated with an increased risk of CRC, with a RR of 1.3. DM individuals might develop CRC 5 years earlier than the average population due to prolonged exposure to hyperinsulinemia, which is directly implicated in oxidative stress and in tumor proliferation promotion ,22,51.Interestingly, even though metabolic syndrome mostly affects people over 50, CRC incidence in this age group is stable or declining. This might be due to the fact that older individuals are more often on maintenance medications , which may have a chemopreventive role. For example, it has been reported that low-dose metformin was associated with a decrease in metachronous adenomas or polyps after a polypectomy ,53.High consumption of red and/or processed meat and sugary beverages is associated with an increased risk of CRC, whereas vegetables, fruits, fiber, and calcium are related to a lower CRC risk . There iSome meta-analyses ,56 amongAlcohol consumption and smoking are associated with a significant increased risk of CRC development  and EOCRC incidence (RR 1.71) .Long-term use of NSAIDs and aspirin have displayed a protective role against the development of EOCRC . The AmeIBD promotes carcinogenesis in CRC as it leads to an inflammatory environment, with increased free radical species, circulating adipokines, and upregulated antiapoptotic and proliferative pathways ,58. Of nBacteroides fragilis, Fusobacterium nucleatum, Streptococcus bovis, and Escherichia coli) can promote CRC through the alteration of colonic integrity, bile acid composition, and the production of toxins and metabolic products of short chain fatty acids (SCFAs), which can influence systemic inflammatory responses [Intestinal dysbiosis can be caused by various factors, such as high BMI, dietary habits, and chronic inflammation. Specific intestinal bacterial species  and distal colon  than in the rectum . Conversely, physical activity reduces the incidence of proximal  and distal colon cancer  but does not significantly affect rectal cancer. Smoking, on the other hand, appears to increase the risk of distal colon and rectal cancer . Alcohol consumption, diabetes, and the use of NSAIDs generally increase the risk of CRC but do not appear to correlate with a specific anatomical site [While in many other cancer types, young people and adolescents have been shown to have a worse prognosis ,68,69, tHawk et al. collected data from 63.894 patients with mCRC throughout a period of 35 years and showed an improvement in OS for young patients compared to older patients  . Other aIn the literature, the discrepancies in mEOCRC outcomes can be influenced by the inclusion or exclusion of hereditary cancer syndromes in data collections. Indeed, Lynch syndrome-associated CRC typically shows increased survival rates rather than sporadic cases due to the possibility of responding to immunotherapy ,78,79.It is widely reported that young adults tend to receive more aggressive treatments in terms of both surgery and chemotherapy in comparison to older patients with the same disease stage ,81. For Regarding mortality rates, Siegl et al. recently published updated CRC statistics from the American Cancer Society. The results show that in the last few years, death rates have an opposite trend based on age, similar to the incidence trend. Among adults older than 50 years old, mortality has had a steady decline of 0.5% annually since 2005 thanks to improved treatments. On the other hand, young adults have presented a constantly increasing death rate of approximately 1% per year since 2004 .Several studies have shown that EOCRC has peculiar features compared to LOCRC, regarding its clinical presentation, anatomical sites, and histological classification.p= 0.03) [EOCRC is typically diagnosed in more advanced pathologic stages (Stages III or IV) ,85. A stp= 0.03) . Moreovep= 0.03) . The lacp= 0.03) . p < 0.05) [The main symptoms observed are rectal bleeding, abdominal pain, and changes in bowel habits. It is reported that hematochezia can be developed from 6 months to 1–2 years before diagnosis ,87,88. C < 0.05) . The fir < 0.05) . Further < 0.05) . In orde < 0.05) ,90.Many studies have shown a different location of CRC based on age. The majority of mEOCRC is localized in the left colon , with a percentage of 41% compared to 34% of mLOCRC ,86.An increased predisposition to synchronous and metachronous tumors or finding polyps afterward is also reported .p< 0.001) [Another explanation of this late diagnosis involves tumor biology. EOCRC tends to have histopathological features associated with rapid carcinogenesis and a tendency to metastasize precociously. Young patients’ cancer is usually poorly differentiated and high grade, and it shows perineural, lymphovascular, and venous invasion ,92. EOCR< 0.001) .In conclusion, as shown in Recent studies hint at a substantial molecular difference between EOCRC and LOCRC ,95,96. H(1)Chromosomal instability (CIN), which is the classic pathway and causes mutations of multiple oncogenes, such as KRAS, and deletions of suppressor genes, such as APC and p53;(2)Microsatellite instability (MSI), which is usually sporadic, but there are also hereditary causes. MSI-CRC can be caused by hereditary germline mutations occurring in the MMR genes  and tumor somatic hypermethylation of MLH1. Nearly 10% of CRCs are caused by MLH1 promoter hypermethylation and are often in association with the BRAF V600E pathogenetic variant in sporadic CRC ;(3)CpG island methylator phenotype (CIMP) pathways. In some cases, CRC may be caused by the hypermethylation of promoter CpG island sites, resulting in the silencing of suppressor genes. Sometimes, there is MLH1 gene involvement and, thus, microsatellite instability ,100,101.The three main mechanisms underlying the occurrence of CRC are as follows ,20,96: events, or a correlation with genomic clusters .The literature data on the mutational incidences of genes are controversial, perhaps because older studies do not clearly distinguish between MSS and MSI tumors; in particular, there are many discrepancies for KRAS and BRAF V600E mutations . Some stMoreover, there appears to be no difference between young and old patients regarding mutations on codons 12 and 13 of KRAS. The percentage of tumors with p53 overexpression seems to be lower in EOCRC than in LOCRC , but accp = 0.32), NRAS , BRAF , PIK3CA , and BRAF V600E . Of note, mutations in the Wnt pathway were most common in LOCRC patients compared with EOCRC patients , whereas the P53 pathway alteration involved 85.2% of EOCRC patients and 81.2% of LOCRC patients (p = 0.499). Finally, no significant differences were detected between EOCRC and LOCRC patients in the mutation frequencies of the PI3K, MAPK, or TGF-b pathways [Few studies in the literature focused on metastatic disease. Ting Xu et al. analyzed mutational differences in various genes between young and elderly metastatic patients, including KRAS  patients, it is also present in 10% to 20% of sporadic CRC patients ,109. Germline mutations in the DNA MMR genes hMLH1 and hMSH2 and, to a lesser extent, hMSH6 and hPMS2 are associated with an increased lifetime risk of CRC and extracolonic cancers . AlthougMSI-H tumors more frequently involve the right colon, are mucinous, have normal p53 expression, have a higher frequency of metachronous and synchronous tumors, and have a better prognosis than MSS . No gendMSS tumors have less involvement of the right colon and fewer synchronous and/or metachronous tumors . MoreoveUp to 63% of EOCRC with MSS is euploid (CIN-negative) ,113,114.The data on the frequency of MSI in the young and old are contradictory in the literature. Some studies argue that most EOCRC is MSS. MSI-h does not appear to be the main carcinogenic pathway ,91, and Differences between the young and old are also present when considering MSI-H tumors; in fact, BRAF is mutated in 5% of EOCRC MSI-H cases and in 48% of LOCRC cases . The BRAp = 0.004) and in MSH6 . These data suggest that younger CRC patients may be more likely to benefit from immunotherapy with checkpoint inhibition [Lynch syndrome-related genes are more frequently mutated in the young than in the old, mostly with differences in MSH2  with metastatic colon cancer and dMMR have poorer survival than pMMR patients . Moreover, Ting Xu et al. evaluated 31 patients with mEOCRC, among which 16 were MSS and the remaining 15 were MSI-H. Sixteen patients had mutations in one of the MMR genes . In addition, germline mutations were found in MUTYH (12% of patients), RAD50 (9%), TP53 (3%), and FANCL (3%). No patients showed mutations in APC or POLD1/POLE . MACSs are more frequent in young people, are often already metastatic at diagnosis, and have a worse prognosis than CIN and MSI ,20. HoweConversely to the traditional anatomical distinction, the colon cannot simply be divided into the right and left but must be considered a continuum of molecular alterations that fade into each other upon progression in the colic tract . There iIn EOCRC, MSI is predominantly in the right colon compared to the left colon (30% vs. 17%) . In the Right colon tumors have a higher rate of BRAF mutations than left ones . FurtherSurely science will have to strive to understand in detail what differences are present in the right and left colon between the young and the old, so that oncologists can be better guided in their choice of treatments.p = 0.027) and underwent more surgeries  than their older counterparts. Furthermore, the younger cohort demonstrated better survival in first-line treatment  [p = 0.325), even though they had more advanced stages at diagnosis  and worse cancer-specific survival  [To date, an early age of onset does not influence the treatment algorithm for CRC in the major clinical guidelines, and EOCRC patients have the same therapeutic indications as their older counterparts. Nevertheless, in clinical practice, the age at diagnosis might influence physicians’ decision-making. Considering that younger patients tend to have better performance status and fewer comorbidities, the evidence shows that EOCRC is likely to receive more aggressive treatment, with conflicting data on the potential prognostic advantages ,121,122.< 0.001) . In anot< 0.001) .The only potentially curative option for mCRC is radical surgery, which should always be performed when feasible. Liver metastasis resection should be considered when sufficient remnant parenchyma is preserved (≥30% liver). Loco-regional ablative treatments, such as radiotherapy, thermal ablation, and chemoembolization, might be associated with surgery. However, up to 55–80% of patients will experience relapse following metastatic resection . CytoredIn order to optimize the treatment choice, testing for KRAS, NRAS exon 2, 3 and 4, BRAF mutations, and MMR status should be carried out in every new metastatic CRC diagnosis . RAS mutp = 0.0002) and survival  compared with FOLFIRI  at the price of a worse toxicity profile [p = 0.043) [Palliative fluoropyrimidine-based backbone chemotherapy, combined with anti-VEGFR bevacizumab, represents the standard first-line treatment for metastatic, proficient MMR, RAS, or BRAF-mutated CRC. Patients deemed unfit for combination treatments might receive single-agent fluoropyrimidine with or without bevacizumab . Patient profile . BRAFV60 profile ). Nevert profile . Based o= 0.043) . Pooled = 0.043) . TherefoThe MMR status aids clinicians to identify patients who might benefit from first-line immune checkpoint inhibition ,139 and Patients with good PS and no significant comorbidities who progress on a first-line therapy should be offered a second-line treatment. The management is driven by the previous regimen administered: the switch from a first-line oxaliplatin-based regimen to a second-line irinotecan-based regimen and vice versa represents the standard option. Antiangiogenic and anti-EGFR antibodies might be added to backbone chemotherapy, according to the presence or absence of RAS mutations . Furtherp < 0.001) [Patients fit for beyond second-line treatment might receive oral agents, such as regorafenib and trifluridine–tipiracil (TAS-102), which demonstrated increased survival compared to BSC in phase III trials . The pha< 0.001)  and a ma< 0.001) . Patient< 0.001)  and laro< 0.001) .Much work is underway to identify other potentially targetable driver mutations for EOCRC in order to design personalized therapeutic approaches and minimize chemotherapy-related side effects. A comprehensive analysis of 18,218 specimens collected from younger (<40 years old) and older (≥50 years old) CRC patients demonstrated that overall genomic alterations were similar in most of the studied genes. Younger patients had frequent TP53 and CTNNB1 alterations, whereas APC, BRAF, FAM123B, KRAS, and PIK3CA mutations were observed mostly in older patients. Interestingly, TMB in the MSI-H cohort increased with age . GeneticTo conclude, prognostic features, predictive biomarkers, and specific treatment algorithms for EOCRC are yet to be determined. To date, there is no evidence that the intensification of therapies confers survival benefits; therefore, a heavier toxicity profile and long-term implications should be considered in treatment planning for young mCRC patients.The frequency of colon cancer is increasing in the population under the age of 50 years old compared with a decrease in cases in older people. The reasons behind this changing phenomenon are still unknown. Certainly, there is a multifactorial component, but scientific research efforts will have to be focused on understanding the pathogenesis of these cancers to counteract their spread.As the younger population is not covered by screening programs, EOCRC is often diagnosed at a more advanced stage. Furthermore, mEOCRC shows different features compared with mLOCRC, being biologically more aggressive and less differentiated. From a clinical and therapeutic point of view, mEOCRC does not differ from its counterpart, although young people often tend to undergo more aggressive treatments due to their better performance status. From the molecular perspective, however, studies do not always concur, even though, in general, mEOCRC seems to have more KRAS, Wnt, and p53 mutations, and a higher rate of dMMR.In conclusion, much effort still needs to be made to understand this emerging issue, starting from the identification of predisposing factors and the development of new preventive strategies. Given the alarmingly rising incidence of EOCRC, it might be useful to consider modifying the screening programs and including younger patients with no familial predisposition, even if this would have a significative impact on the healthcare economy. It might be possible to think of charging younger people for the testing of occult blood in stools, guaranteeing tax relief in any case, and performing only colonoscopies at the full expense of the healthcare system.


For every 1 g/day DHA intake, an increase of 0.38% in DHA content and 0.78% in total omega-3 content was observed in DHM . DHM saturated fatty acids were positively associated with erythrocyte dimethyl acetals, plasma stearic acid, trans fatty acids intake, and breastfeeding duration and negatively associated with erythrocyte margaroleic acid . DHM cholecalciferol was associated with plasma cholecalciferol levels and dairy intake . Other weaker associations were found for free thiamin, free riboflavin, pyridoxal, dehydroascorbic acid, and the lipid profile in DHM. In conclusion, the diet and nutritional status of donors influence the fatty acid profile and micronutrient content of DHM.The influence of the diet and nutritional status of milk donors on the nutritional composition of donor human milk (DHM) is unknown. The present study aimed to determine the nutritional profile of DHM and the associations between donors’ dietary intake and nutritional status and the micronutrient and lipid composition in DHM. For this purpose, 113 donors completed a food frequency questionnaire, provided a five-day weighed dietary record, and collected milk for five consecutive days. Nutrient determinations in donors’ erythrocytes, plasma, urine, and milk were performed. Multiple linear regressions were conducted for the evaluation of the associations. We highlight the following results: DHM docosahexaenoic acid (DHA) was positively associated with donors’ plasma DHA content and donors’ DHA intake (R Donor human milk (DHM) is considered the best feeding option for preterm infants when their mother’s own milk is not available. Its use is increasing in neonatal units, primarily for infants at high risk of developing necrotizing enterocolitis or feeding intolerance, such as those weighing <1500 g and/or those at less than 32 weeks of gestational age (GA), infants with congenital heart disease, or those with severe intestinal disorders ,5. TheseAccording to studies carried out in different geographical areas of the world, there is evidence that the content of fatty acids (FAs) and some micronutrients in human milk (HM) depends on the maternal deposits of each nutrient at the end of gestation and maternal intake during the lactation period ,7,8,9. HIn this context, micronutrients can be classified in a simplified way into two groups with respect to their secretion patterns in milk in relation to maternal intake and status and the response to supplementation. In general, water-soluble vitamins (except folate), fat-soluble vitamins, iodine, and selenium are considered to belong to a group of micronutrients for which secretion into HM is dependent on maternal intake or status, and maternal supplementation may increase their concentrations in milk ,9,10,11.Nevertheless, the results of the different surveys have not always been concordant , and theMoreover, HM is a very complex and dynamic fluid, and its nutritional composition depends not only on maternal or infant factors but also on its chronobiology  ,19,20,21Furthermore, DHM requires its own studies as it is not comparable to breast milk in some respects . FirstlyTherefore, because of the great importance of nutrition in the development of very preterm or critically ill newborns, it is essential to have information on the nutritional composition of DHM and the factors that might impact it, such as diet, in order to improve our recommendations to donors. To the best of our knowledge, the only study in which a dietary assessment of milk donors was performed was a randomized, controlled trial about the effect of donors’ supplementation with docosahexaenoic acid (DHA) on the DHA content in DHM conducted in the United States .3, iodine, calcium, phosphorous, and selenium) of raw DHM and their associations with donors’ dietary intake and nutrient status.Hence, the aim of this study was to determine, in a large population of donors from a milk bank in Madrid (Spain), the macronutrient composition, lipid profile , and micronutrient composition DThis study was part of a larger research project, a cross-sectional observational study conducted at the Aladina MGU Regional Human Milk Bank (RHMB) at the “12 de Octubre” University Hospital in Madrid, Spain. Two previous articles based on this research project have recently been published ,36.The main aim of the present work was to study the correlations between the diet, nutritional status, and nutritional milk composition  of milk donors. For this purpose, we recruited donors by opportunity at the RHMB between August 2017 and February 2020. The inclusion criterion was to be an active donor in our RHMB  without communication barriers. In our RHMB, the requirements to be a milk donor are that potential donors must have a healthy lifestyle, not be suffering from illnesses or taking any medications that are incompatible with the donation, be successfully breastfeeding their own child, and want to donate their surplus milk voluntarily and altruistically. Normally, milk donors are accepted from 3 weeks postpartum to ensure that breastfeeding is well established. There are no exclusion criteria regarding the age of the donor or the gestational age of her child, nor is there an upper limit on the length of breastfeeding. Women who wish to donate milk after the death of their baby, vegetarians or vegans with normal plasma cobalamin values committed to taking the recommended vitamin B12 supplements, and women with well-controlled hypothyroidism are accepted as donors. The statistical calculation for the sample size was undertaken in G*Power 3.1 ; for an The study was carried out in agreement with Declaration of Helsinki and was approved by the Clinical Research Ethics Committee of the Hospital Universitario “12 de Octubre” (protocol code 15/269). All participants gave written informed consent. The full study protocol and laboratory studies have been previously described in detail in the abovementioned studies ,36.The study protocol is shown in In summary, the study of participating donors comprised a health and sociodemographic survey; somatometric measurements; blood and urine determinations to study their nutritional status; a dietary study including a food frequency questionnaire (FFQ) and a five-day weighed dietary record, taking into account the intake of pharmacological nutrient supplements; and a detailed study of the nutritional composition of their milk. At the first visit to the RHMB (day 0), fasting urine and blood samples were collected. Plasma and red blood cells were obtained from the blood. All biological samples were frozen at −80 °C until analysis of the biochemical indicators of nutrition. In addition, milk donors provided their informed consent, underwent a somatometric study , filled in the health and sociodemographic survey, and completed a food consumption frequency questionnaire. Donors were trained in how to complete the five-day weighed dietary record and collect and keep frozen milk samples at home. They also were supplied with the required material.Within the following 15 days after the visit to the RHMB, the five-day dietary recording and the collection of the five-day milk samples were carried out almost concomitantly for 6 days in a row, with milk collection starting 1 day after the start of the dietary recording and ending 1 day after the recording of the diet had been completed. We ensured that the DHM we analyzed met the same conditions and underwent the same processes as the raw milk donated to our RHMB. Therefore, in order to replicate the donors’ milk expression routine, participants were not asked to express milk at any particular time, the only requirement being complete emptying of one or both breasts, and the milk was collected in the same sterile transparent bottles that the donors use under normal donation conditions. From days 2 to 5, donors collected a 25 mL milk sample from each expression (at least one per day) and froze it at −20 °C. On day 6, they collected and froze one complete milk extraction. Donors were asked to deliver their dietary records and their frozen milk samples to the RHMB during the first 15 days following the end of the study. Donors transported the milk samples in isothermal containers with cold packs, and the milk remained frozen at the RHMB at −20 °C until thawing for aliquoting.In the RHMB, different milk samples from the same day were mixed and aliquoted, but the milk from different days was not mixed, as the composition of the milk from each day was analyzed individually. Therefore, the milk samples that were analyzed went through a process of extraction, freezing at the donors’ homes, transport to the RHMB, and thawing to collect the aliquots, similar to the process that DHM usually undergoes at our RHMB. The milk samples were subsequently frozen until analysis in the respective laboratories at −80 ºC instead of −20 °C, as the freezing time was much longer than that of the DHM at the RHMB. Before analysis in the laboratory, the milk was thawed again. Thus, the studied milk underwent a total of two freeze–thaw cycles, just like the DHM in our RHMB, the only difference being that it was not pasteurized. ® software  to calculate daily nutrient intakes [The milk samples collected from days 2 to 5 were used for the study of vitamins and minerals, and the complete milk collection sample from day 6 was used for the integral characterization of the lipid fraction. The data on the intake of food, beverages, and pharmacological nutrient supplements obtained from the five-day dietary record were analyzed using DIAL intakes . Nutrien intakes ,42,43,44 intakes  for lactLipid analyses were conducted by the Food Lipid Biomarkers and Health Group at the Institute of Food Science Research . Fatty acid methyl esters were analyzed by GC-MS in erythrocytes, plasma, and milk. After fat extraction from human milk samples from 20 randomly selected donors, the separation and quantification of lipid classes were performed using an HPLC evaporative light scattering detector (ELSD), and the determination of TAG molecular species was undertaken using GC-FID.3, iodine, calcium, phosphorous, and selenium were determined in milk. The erythrocyte glutathione reductase activity coefficient (EGRAC), riboflavin, nicotinamide, pantothenic acid, pyridoxamine, and hemoglobin were determined in erythrocytes. The EGRAC, which assesses erythrocyte glutathione reductase activity in terms of an excess of flavine adenine dinucleotide compared to baseline activity, is a functional property used to determine the nutritional riboflavin status. Thiamin, riboflavin, nicotinamide, pantothenic acid, pyridoxine, pyridoxamine, folic acid, cobalamin, holotranscobalamin, homocysteine, ascorbic acid, retinol, 25(OH)D3, 1,25(OH)2D3, α-tocopherol, γ-tocopherol, triacylglycerols, total cholesterol, HDL-cholesterol, and LDL-cholesterol were determined in plasma. Creatinine, methylmalonic acid, iodine, sodium, calcium, and phosphorous were determined in urine. Vitamin and mineral analyses were conducted by the NUTREN-Nutrigenomics Group of the Department of Experimental Medicine at the University of Lleida, Spain. Micronutrient determinations were carried out using chromatographic analyses, mass spectrometry, and immunoassays. Free thiamin, free riboflavin, nicotinamide, pyridoxal, pantothenic acid, folic acid, cobalamin, ascorbic acid, dehydroascorbic acid, retinol, α-tocopherol, γ-tocopherol, cholecalciferol, 25(OH)DDeterminations of macronutrients  in DHM were carried out at the RHMB using Fourier-transform mid-infrared (FT-MID) spectroscopy in a milk analyzer .A full description of the analytic techniques employed for each of the nutrients is available in a previous paper , as mentStatistical analysis was performed with the STATA 14 program.For descriptive analysis, qualitative variables were presented as absolute and relative frequencies and quantitative variables as medians and the interquartile range or as means and the standard deviation depending on their distribution, which was established by performing the Shapiro–Wilk normality test. p < 0.05) with milk content in the bivariate analysis were entered into the models. Once the independent variables and their probable interactions were introduced in each of the models, the variables with p > 0.05 were removed, and confounding variables were evaluated until the most parsimonious model was obtained. Each model was diagnosed, and some outliers were removed to improve the fit. For each model, regression diagnosis was performed with the residuals; the assumptions of linearity with each independent variable were assessed with scatter plots and, in addition, homoscedasticity of variance was assessed with the Breusch–Pagan test and normal distribution with p–p plots. The independence of observations was also assessed with vif and omitted variable bias was tested with ovtest. The following diagnostic plots were used for the influential points: residual versus fitted values (rvfplot), augmented component-plus-residual plots (acpr plots), leverage against normalized squared residuals (lvr2plot), and Cooks’ distance. In the models’ tables, the influencing points identified and removed for each model after individual analyses were recorded.For the multivariate analysis of associations, multiple linear regressions (MLRs) were performed using each of the lipids, macro- and micronutrients in DHM as dependent variables, except for iodine, which had been previously studied . For vit+4 weeks of gestational age and the other with a full-term infant). One of the omnivore donors left the study early; thus, complete data were obtained from 113 donor women.A total of 114 milk donors were recruited: 112 were omnivores  and 2 were ovo-lactovegetarians . BMI at the time of the study was classified as normal for 71% of the sample (n = 6), fish (n = 3), meat (n = 3), eggs (n = 2), nuts (n = 1), and others (n = 3). In total, 87 of the 109 donors who specified the type of salt consumed (79.8%) reported taking iodized salt.The median age of donors was 35.6 years and they had a median body mass index (BMI) of 22.9 kg/me sample . In term+4 weeks of gestational age) and one lost one of her twins at 27+0 weeks of gestational age during the first week of life. Another donor had one fetal death at 22+6 weeks of gestational age 2 months previously  at the time of the study. The median gestational age at birth was 39st pumps .Regarding nutrient supplementation, 112 of 113 donors (99.1%) reported consuming pharmacological nutritional supplements during pregnancy and 102 of 113 donors (90.3%) during lactation. Most participants took supplements of folic acid, vitamin B12, and iodine during pregnancy. The number of donors who took supplements of each nutrient, both during gestation and lactation, and the median daily dose that they consumed are available in The results of the five-day dietary record, the FFQ, and the analysis of nutrients in the erythrocytes, plasma, and urine of the donors for the assessment of their nutritional status are displayed as Data on the nutritional composition of the DHM are presented in Models in which significant associations were found are described below.Levels of FAs in DHM are expressed as percentages of fat .trans fatty acids (TFAs) consumed in the dietary record, and breastfeeding time. For each 1% increase in DMA in erythrocytes, adjusted SFA levels in DHM increased by 2.7%. In addition, for each 1% increase in plasma C18:0 levels, the adjusted SFA content in DHM increased by 2%, and for each 0.1% of kcals. consumed from TFA in the dietary record, the adjusted percentage of SFAs in DHM increased by almost 0.6%. On the other hand, for each 0.1% increase in levels of erythrocyte C17:1, adjusted SFA levels in DHM decreased by approximately 1.4%. Finally, for each month of breastfeeding time, the adjusted percentage of SFA in DHM increased by 0.2%.The percentage of total saturated fatty acids (SFAs) in DHM was associated with the donors’ erythrocyte deposits of dimethyl acetals (DMAs) and C17:1 (margaroleic acid), plasma C18:0 (stearic acid) levels, The amount of monounsaturated fatty acids (MUFAs) in DHM was positively associated with donors’ plasma MUFA levels and erythrocyte C17:1 levels. Adjusted MUFA levels in DHM increased by approximately 0.5% per 1% increase in plasma MUFAs and by almost 1.4% for each 0.1% increase in the erythrocyte C17:1 level.PUFA levels in DHM were weakly associated with donors’ daily average PUFA intake according to the five-day dietary record. For each 5 g of PUFAs in the daily diet, PUFAs in DHM increased by nearly 0.7%.A positive association was found between linoleic acid levels in DHM and the linoleic acid levels in donors’ erythrocytes and average intake of meat, fish, and eggs in the five-day dietary record. For every 1% increase in the percentage of linoleic acid in erythrocytes, adjusted levels in DHM increased by 0.6%. Moreover, for each daily serving of meat, fish, or eggs consumed, the adjusted percentage of linoleic acid in DHM increased by 0.7%. For linolenic acid (C18:3-n3), no significant associations were found between donor clinical and somatometric characteristics, donor plasma/erythrocyte levels or breastfeeding characteristics and DHM content.DHA levels in DHM were associated with DHA donors’ plasma levels and the average daily DHA intake according to the five-day dietary record. For every 1% increase in DHA in donors’ plasma, the percentage of DHA in DHM increased by approximately 0.2%, and for every 1 g of daily DHA intake, the adjusted percentage of DHA in DHM increased by almost 0.4%. The association between total omega-3 in DHM and donors’ DHA intake was positive and significant. For every 1 g of DHA per day in the five-day dietary record, the percentage of omega-3 in DHM increased by 0.8%.Associations were found between free thiamin levels in DHM and donors’ thiamin plasma levels, daily dairy intake in the five-day dietary record, and breastfeeding time.For each 0.1 mcg/L of plasma thiamin, adjusted free thiamin levels in DHM increased by 1.1 mcg/L. On the other hand, for each dairy serving/day from the dietary record, adjusted free thiamin levels in DHM decreased by 2.9 mcg/L, and for each month of lactation, thiamin levels in DHM decreased by 0.5 mcg/L.Regarding riboflavin, a positive association was found between DHM-free riboflavin and the daily intake of riboflavin in the five-day dietary record, as well as receiving vitamin B2 supplementation during lactation. For 1 mg/day of riboflavin intake, the adjusted levels of free riboflavin in DHM increased by 21 mcg/L, and if donors received riboflavin supplementation during lactation, the adjusted levels in DHM increased by approximately 40 mcg/L.In relation to pyridoxal levels in DHM, associations were observed with respect to vitamin B6 supplementation during pregnancy and breastfeeding time. If donors received vitamin B6 supplements during pregnancy, adjusted pyridoxal levels in DHM increased by 11.6 mcg/L, and for each month of breastfeeding, adjusted pyridoxal levels in DHM decreased by 0.8 mcg/L.No significant associations were found for nicotinamide, pantothenic acid, folic acid, or cobalamin levels in DHM.There were associations between dehydroascorbic acid in DHM and donors’ plasma ascorbic acid levels, average daily fruit intake in the dietary record, and breastfeeding time. For each average daily serving of fruit from the five-day dietary record, adjusted dehydroascorbic acid levels in DHM increased by 0.3 mg/dL. Breastfeeding time and plasma ascorbic acid levels were negatively associated with dehydroascorbic acid levels in DHM, but the adjusted values were very low.There was a positive association between cholecalciferol levels in DHM, donors’ cholecalciferol plasma levels, and daily dairy intake. For each 10 pg/mL of plasma cholecalciferol, adjusted cholecalciferol levels in DHM increased by 173 pg/mL. According to the five-day dietary record, for each average daily serving of milk and dairy products, adjusted cholecalciferol levels in DHM increased by 521 pg/mL, equivalent to almost 21 UI/L.3 in DHM and donors’ 1,25(OH)2D3 plasma levels, vitamin D supplementation, and breastfeeding time. For each 10 pg/mL of 1,25(OH)2D3 in donors’ plasma, 25(OH)D3 in DHM increased by 1.6 pg/mL; in addition, if donors received vitamin D supplementation during lactation, 25(OH)D3 in DHM increased by 35.7 pg/mL, and for each month of breastfeeding, 25(OH)D3 in DHM increased by 2.2 pg/mL.There was a positive association between 25(OH)DThe association found between iodine levels in DHM and iodine supplementation was described in a previous publication . No signNo associations were found between carbohydrate and protein content in DHM and donors’ clinical and somatometric characteristics, nutritional biochemical determinations, or dietary intake.This cross-sectional study investigated the associations between macronutrients, the fatty acid profile, lipid classes, the relative composition of phospholipids, molecular species of triacyclglycerols, and 14 micronutrients  in raw DHM and the diet, as well as the nutritional status of 113 milk donors from the Regional Human Milk Bank in Madrid, Spain. To the best of our knowledge, this is the first study in which these associations have been evaluated in donor human milk. For this purpose, donors completed an FFQ, provided a five-day weighed dietary record, and collected milk for 5 consecutive days. In addition, somatometric measurements and nutrient determinations in donors’ erythrocytes, plasma, urine, and milk were performed. One of the most important findings of the present study was the correlation between donors’ DHA intake and DHA plasma levels and the DHA content of the raw DHM. This result is important because, in a recent study , the DHAWe also found a positive correlation between maternal PUFA intake and the PUFA content in DHM. This positive correlation was found in a previous work that studied the PUFA content in breast milk of non-donor lactating mothers during the first month of lactation . It is remarkable that other correlations found in this study regarding lipid content have not been identified before. The DHM linoleic acid content increased with the erythrocyte linoleic acid content, as well as, interestingly, with combined intake of meat, fish, and eggs. The proportion of DHM MUFAs increased with plasma MUFA levels and erythrocyte margaroleic acid levels. The proportion of DHM SFAs increased with increased erythrocyte DMA content, plasma stearic acid, TFA intake, and breastfeeding time, but decreased with increased erythrocyte margaroleic acid content. In terms of micronutrients, various associations were found between the donors’ diet/nutritional status and the DHM composition. Regarding B-group vitamins, we found associations with the content of free thiamin, free riboflavin, and pyridoxal in milk. We were not able to demonstrate positive associations between the milk content of other B-group vitamins and maternal intake or erythrocyte/plasma levels, although all of them (except folate) are considered to be dependent on diet and maternal stores . Free thiamin in DHM was positively correlated with plasma thiamin levels and negatively associated with breastfeeding time and the daily intake of dairy. Free riboflavin in DHM was positively associated with riboflavin intake and vitamin B2 supplementation during lactation. Pyridoxal in DHM was positively associated with vitamin B6 supplementation during pregnancy and negatively associated with breastfeeding time. In the literature, the concentrations of these three vitamins in human milk are strongly associated with maternal intake ,8,9,101.Donors’ fruit consumption was associated with dehydroascorbic acid content in DHM. One of the most consistent findings of the two systematic reviews conducted on the effect of maternal diet on the nutritional composition of breast milk ,9 was th3 levels) and the daily intake of dairy. Furthermore, the content of 25(OH)D3 in DHM increased with the use of vitamin D supplementation during breastfeeding. These findings are in line with observations from previous studies [For fat-soluble vitamins, associations were found only with vitamin D. The vitamin D content of the DHM was similar to the usual values for vitamin D in breast milk, widely known to be low for the needs of the infant . It was  studies ,9. Neith studies . The α-t studies , althoug studies ,13. Finally, we found no association between the carbohydrates, proteins, lipid classes, phospholipids, molecular species of triacylglycerols, calcium, phosphorus, and selenium content in the DHM and the independent variables studied. The selenium content in DHM was not associated with dietary intake in the present study, although it is known that the diet influences selenium HM content . The calIn summary, we studied the factors that might influence the nutritional content of DHM, and we highlight the following as strengths: the detailed study of the supplementation habits of donors since gestation, the five-day weighed dietary record, and the determination of nutrients in donors’ erythrocytes, plasma, and urine. In addition, the dietary study was carried out concomitantly with the collection of milk over five consecutive days. Milk from different donors or different days was not pooled to allow us to determine associations between the content of each nutrient in DHM and donors’ intake, status, and characteristics. However, our study has some limitations. It is important to point out that the studied DHM comprised raw milk, and although the impact of pasteurization on the nutrient content in DHM has been previously studied ,33, we dHowever, we consider that the results obtained in this study are relevant since we have shown that there are associations between the nutritional composition in raw DHM and the diet, as well as the nutrient deposits of donors—despite the heterogeneity among the donors regarding the duration of pregnancy and breastfeeding time—and the procedures to which raw DHM is subjected, such as extraction, freezing, mixing, and storage in transparent glass containers. trans fatty acids intake, and breastfeeding duration and negatively associated with erythrocyte margaroleic acid. DHM cholecalciferol was associated with plasma cholecalciferol levels and dairy intake. Other weaker associations were found for free thiamine, free riboflavin, pyridoxal, dehydroascorbic acid, 25(OH)D3, total MUFAs, total PUFAs, and linoleic acid in DHM. We found no associations between the carbohydrates, proteins, lipid classes, phospholipids, and molecular species of triacyclglycerols in DHM and the clinical characteristics of the donors or their somatometric study, nutritional biochemical determinations, or dietary intake. In conclusion, the diet and nutritional status of donors influence the fatty acid profile and some of the micronutrients in the raw DHM. One of the most important results was the positive correlation found between the DHA content in the DHM and both the DHA intake and the plasma DHA levels of the donors. In our study, for every 1 g/day of DHA intake, an increase of 0.38% in DHA content and 0.78% in total omega-3 content was observed in DHM. DHM saturated fatty acids were positively associated with erythrocyte dimethyl acetals, plasma stearic acid, Investigation of the composition of DHM and the factors that influence it is essential. Infants receiving DHM are in a state of nutritional emergency, and it is therefore desirable that the milk they receive is in accordance with their needs. Studies such as the present one may help to improve dietetic recommendations for milk donors to ensure a more suitable composition for DHM. However, much remains to be learned and it is possible that the development of personalized medicine will allow us to better understand the variability in the composition of DHM and the different needs of preterm infants. Answers to open questions are likely to be found in this new field.