Cervical cancer ranks as the fourth leading cause of cancer death in women worldwide. In Taiwan, although the universal health insurance system has achieved 99.9% coverage and ensured easy access to medical care, some cervical cancer patients continue to delay initiation of definitive treatment after diagnosis. This study focused on cervical cancer patients who delayed treatment for at least 4 months, and examined the characteristics, related factors, and survival in these patients.Data on patients with a new confirmed diagnosis of cervical cancer by the International Federation of Gynecology and Obstetrics (FIGO) staging system between 2005 and 2010 were obtained from the National Health Insurance Research Database and the Taiwan Cancer Registry. Logistic regression analysis was performed to analyze the association of various factors with treatment delay. The Cox proportional hazards model was used to analyze the effects of various factors on mortality risk.P < 0.05).The rate of treatment delay for cervical cancer decreased steadily from 6.46% in 2005 to 2.48% in 2010. Higher rates of treatment delay were observed among patients who were aged ≥75 years (9.91%), had severe comorbidity, had stage IV (9.50%), diagnosing hospital level at nonmedical center, or at public hospital ownership. Factors that correlated with treatment delay were age ≥75 years (odds ratio [OR] = 2.42), higher comorbidity Charlson comorbidity index (CCI) 4–6, or ≥7 , cancer stage IV (OR = 2.60), the diagnosing hospital being a regional, district hospital, or other , and at public hospital ownership. Those who delayed treatment had 2.31 times the mortality risk of those who underwent timely treatment (Delayed cervical cancer treatment in Taiwan was associated with age, comorbidity, cancer stage, diagnosing hospital level, and hospital ownership. Delaying treatment for ≥4 months substantially raised mortality risk in cervical cancer patients. In Taiwan, approximately 1567 women were diagnosed with cervical cancer and over 640 died of the disease in 2014, making cervical cancer the seventh most common cause of cancer death among Taiwanese women and a significant health concern in Taiwan. In 1995, Taiwan's Ministry of Health and Welfare began providing one Pap smear test per year to every woman aged 30 years or older, and this measure has demonstrated remarkable efficacy against cervical cancer. Among the age-standardized incidence rates for the 10 leading cancers in women from 2002 to 2011, cervical cancer (41.4%) had the highest decrease rate. The age-standardized mortality from cervical cancer fell from 11 to 4 per 100,000 population between 1995 and 2013, a 64% reduction.Cervical cancer is one of the five most common causes of cancer death in women worldwide, responsible for over 265,000 deaths in 2012, accounting for approximately 7% of cancer deaths in women. Seeking a second opinion may also cause patients to delay treatment. In Taiwan, a study by Huang et al showed lung cancer patients who had higher treatment refusal rates were associated with factors such as men, older adults, high prediagnosis Charlson comorbidity index (CCI) score, other catastrophic illnesses or injury, and advanced cancer stage. The National Health Insurance (NHI) Program covers 99.9% of the population and waives copayments for patients with cancer. The NHI Administration has also included 93% of Taiwan's health services organizations as NHI-contracted health care providers as of the end of 2014. Even given this ease of health care access and waiving of copayments for patients with cancer, a small proportion of Taiwanese patients who have been diagnosed with cervical cancer delay their treatment. The Taiwan government aims to decrease mortality from cervical cancer. In order to reduce cervical cancer mortality, we need to understand why some cervical cancer patients delayed or did not receive their cancer treatments. This study aimed to examine factors related to treatment delay in these cervical cancer patients and the effect on their survival.Among pregnant patients with cervical cancer, consideration for better fetal outcome may be a reason for choosing to delay treatment.22.1Taiwan's NHI is a compulsory single-payer health insurance program that provides comprehensive coverage for medical care. Patient data were obtained from the Ministry of Health and Welfare's National Health Insurance Research Database and cause of death database, and the Health Promotion Administration's Cancer Registry files from 2005 through 2010. Diagnoses were coded according to the International Classification of Diseases for Oncology (ICD-O) from the Taiwan Cancer Registry. Since this study used the National Health Insurance Research Database and Cancer Registry Files, which were published by Taiwan government, the patient identification information has been deleted prior to analysis, and personal privacy was protected in this study. The study has been approved by the Institutional Review Board of China Medical University Hospital (IRB No.: CMUH102-REC3-076).2.2 Patient diagnoses were coded according to ICD-O, and cervical cancer cases were distinguished by codes corresponding to the relevant primary disease site C530 through C539. Treatment delay was defined as not undergoing any conventional cancer treatment  within 4 months of a confirmed diagnosis of cervical cancer. Data from the cancer treatment registry of the Health Promotion Administration indicate that the stage or condition of cancer changes in the absence of prompt treatment, and that 4 months postdiagnosis is the time by which the 93% of cancer patients who do undergo treatment have initiated treatment. For these reasons, patients who did not undergo treatment within 4 months of diagnosis,10 were selected as study subjects for our analysis of treatment delay.A retrospective, longitudinal cohort study was carried out with patients who had a new diagnosis of cervical cancer between 2005 and 2010 and delayed treatment, followed up to the end of 2012 for survival analysis. There were 9081 patients with newly diagnosed cervical cancer which excluded stage 0 or stage missing, diagnosed after death, or other missing value in Taiwan from 2005 to 2010. For women with abnormal Pap test results, cervical cancer is diagnosed by either cervical biopsy of grossly visible lesions or colposcopically directed biopsy.2.3 , and socioeconomic status , health status (comorbidity as CCI and presence of catastrophic illness or injury other than cancer), cancer stage (stages I to IV), and characteristics of the diagnosing hospital . All cancer patients were identified in the NHI catastrophic illness or injury registry file, which contains 30 categories of patients with any severe illness or injury, such as cancer, chronic renal failure, systemic lupus erythematosus, type I diabetes, and so on, defined by the NHI. In National Cancer Registry, the clinical staging system (stages I to IV) in cervical cancer developed by the International Federation of Gynecology and Obstetrics (FIGO) which is the representative and associated with whole histologic types of cervical cancer. Deyo's adaptation of the CCI was used to assess the severity of comorbid conditions in cervical cancer patients, and the computed scores were classified into four categories: ≤3, 4–6, and ≥7. Taiwan's 359 townships and city districts were previously classified by Liu et al into 7 levels by urbanization, level 1 (most urbanized) through level 7 (least urbanized), representing highly urbanized cities, moderately urbanized cities, developing cities, average towns, aging towns, agricultural towns, and remote villages, respectively. The definition of level of urbanization has been widely used in relevant studies.,9,15,16Two outcomes were examined in this study: whether the cervical cancer patient delayed treatment past 4 months and whether the patient survived. The independent variables analyzed included demographic characteristics such as age at diagnosis, residence urbanization level2.42 test and mean age at diagnosis was determined by t-test. To adjust for the cluster effects of patients having the same diagnosing hospital, logistic regression was performed using the generalized estimating equation model to analyze the association of factors with treatment delay in cervical cancer patients. In order to control all variables, we put all relative variables in the logistic model and the Cox proportional hazard model to control other variables completely.Descriptive statistics were used to analyze, for each year from 2005 to 2010, the demographic characteristics and proportions of cervical cancer patients and of the subset who delayed treatment. Whether treatment delay was associated with patients’ demographic characteristics, socioeconomic status, health status, cancer stage, or diagnosing hospital characteristics was determined by the χTo examine the effect of treatment delay on survival in cervical cancer patients, the log-rank test was used to first determine whether patient survival, followed up through 2012, correlated with demographic characteristics, socioeconomic status, health status, cancer stage, histologic type, or diagnosing hospital characteristics. Then the Cox proportional hazard model was used to evaluate the effect of treatment delay on patient survival after adjusting for all other related variables. Finally, factors affecting the survival of treatment-delayed patients were analyzed. All statistical analyses were performed with SAS software, version 9.3 .3Between 2005 and 2010, a total of 9081 patients received a new diagnosis of cervical cancer in Taiwan, of whom 347 patients (3.82%) delayed treatment. Treatment delay declined annually during this period (Table 3.1P < 0.05). Patients aged ≥75 years delayed treatment at the highest rate of all age categories, at 9.91% (P < 0.05). Areas classified as urbanization level 6, or agricultural towns, had the lowest treatment delay rate (3.48%) of all residence area categories. While controlling for the other variables in the model, the delay rates (3.49%) in urbanization level 2, or moderately urbanized cities, of residence areas was statistically significant difference (P < 0.05).The characteristics of cervical cancer patients who delayed treatment were examined in terms of demographic characteristics, socioeconomic status, health status, cancer stage, and the diagnosing hospital characteristics Table . With reP > 0.05) after controlling for other variables. Among the insurance status categories, farmers or fishers had the leading rate of treatment delay of 4.79%, but the difference was not statistically significant (P > 0.05) after controlling for other variables.The rate of treatment delay in cervical cancer patients also varied with socioeconomic factors. With respect to income, the treatment delay rate would increase with decreasing monthly salary, but the difference among the participants with different monthly salary was not statistically significant (P < 0.05). Patients with preexisting catastrophic illnesses or injuries also had a higher rate of treatment delay after cervical cancer diagnosis (7.74%), but the difference was not statistically significant (P > 0.05) after controlling for the variables. Of the cancer stages, stage IV corresponded to the highest treatment delay rate , and stage I, the lowest . With respect to the level of the diagnosing hospital, medical centers made more cervical cancer diagnoses and had the lowest rate of treatment delay (2.43%) among the diagnosed patients than other levels of hospitals (P < 0.05). In terms of the hospital ownership, the treatment delay rate at private hospitals was lower than the treatment delay rate at public hospitals . Also, patients with CCI scores of 4 to 6, ≥7  had significantly higher odds of treatment delay than patients with CCI scores ≤3. With respect to cancer stage, the likelihood of treatment delay increased with increasingly advanced cancer stage. The odds for stage IV patients were 2.6 times  that for the stage I. The patient's monthly salary, insurance status, residence urbanization level, and other catastrophic illnesses or injuries were not significantly associated with treatment delay (P > 0.05).The risk factors for treatment delay in cervical cancer patients were analyzed by logistic regression. In order to take into consideration the cluster effects of patients having the same diagnosing hospital, a generalized estimating equation approach was used in the analysis. As shown in Table 3.3P < 0.05). The effects of other factors on survival in treatment-delayed cervical cancer patients were also analyzed, one of the factors being age at diagnosis. Relative to the reference age group, namely patients aged ≤44 years, patients aged ≥75 years were 1.63 times as likely to die . Whereas patients aged 55 to 64 years  had a lower mortality risk than the reference group. Low-income household was at 1.47 times increased mortality risk relative to the employees/employers group (P < 0.05). The presence of other catastrophic illness or injury significantly affected risk of death in treatment-delayed cervical cancer patients (P < 0.05). Comorbidity in treatment-delayed cervical cancer patients was positively correlated with mortality risk. Relative to the reference group, CCI scores of ≤3, the adjusted hazard ratio for death increased from 5.86 for CCI scores of 4 to 6 to 10.67 for scores ≥7 (P < 0.05). Mortality risk also rose with increasingly advanced cancer stage. Relative to the reference group, cancer stage I, the adjusted hazard ratios for death for stages II, III, and IV respectively increased from 1.93, 2.81 to 5.19 (P < 0.05). The effects of histologic type on survival in treatment-delayed cervical cancer patients were analyzed. Adenocarcinoma in treatment-delayed cervical cancer patients was associated with a 1.24 times increased risk of death relative to squamous cell carcinoma . The residence urbanization level, monthly salary, and hospital ownership did not significantly affect mortality risk in treatment-delayed cervical cancer patients (P > 0.05).The analysis on the effect of treatment delay on the survival of cervical cancer patients is presented in Table 3.4P < 0.05) and widened with time. The 1-year survival of treatment-delayed cervical cancer patients was 59.65%, significantly lower than that of treated patients, 90.90%. Survival decreased with increasing time to 37.92% for treatment-delayed patients and 70.75% for treated patients at 5 years  at diagnosis were more likely to delay treatment Table . Compara in 2011 found that medication cost was one of the barriers to adherence to physician-prescribed therapy among breast cancer patients. In the United States, young women with public or no insurance and with low socioeconomic status tended to have longer treatment delay times after breast cancer diagnosis. Our analysis (Table P > 0.05). The difference between our finding and those of studies conducted in other countries can be explained by that under Taiwan's NHI program, the government subsidizes the insurance premiums of the economically disadvantaged, thus lowering the barriers to medical care for low-income individuals and minimizing the effect of income on treatment seeking or delay.With respect to economic factors, Lin et al an advanced cancer stage, or worsening disease; patients’ state of health, access to pertinent information, attitude toward their disease, interaction with and encouragement from health care staff, and concern about adverse effects of treatment. In a 2012 study on breast cancer patients who refused conventional treatment and opted for alternative therapy, Citrin et al showed that negative perceptions of physician demeanor , fear of side effects, belief in the efficacy of alternative therapies (including consumption of raw fruits and vegetables and supplements) were factors in the patients’ treatment refusal. The reasons of nontreatment or interrupted treatment within 4 months among breast, colon, oral, and cervical cancer patients were studied in Tsai et al questionnaire study. The main points for treatment delay of cervical cancer patients are the fear of surgery, economic burden of household, worried about poor life quality after therapy, and no companions for treatment. According to the results of our regression analysis  can be treated with either radical surgery or radiation therapy. Stages IIB to IV (advanced stage) should be treated with chemoradiation and radiation therapy.5Under Taiwan's universal health insurance system, compulsory health insurance enrollment, along with medical outreach efforts in remote mountainous areas, has lowered barriers and enhanced access to medical care for the Taiwanese population. The results of our present analysis show that among Taiwanese patients with a new confirmed diagnosis of cervical cancer between 2005 and 2010, the rate of treatment delay of 4 months or longer exhibited a yearly decline during that period. This study can help identify cervical cancer patients at risk of delay. Higher rates of treatment delay were found in cervical cancer patients characterized by more advanced age, more severe comorbidity, more advanced cancer stage, having a nonmedical center diagnosing hospital, or diagnosing at public hospital ownership. Not undergoing treatment within 4 months was associated with a 2.31-fold increased risk of death relative to timely treatment. Cervical cancer patients with more advanced age, low-income household, other catastrophic illness or injury, higher comorbidity, more advanced cancer stage, adenocarcinoma, or diagnosing at district hospital level had a higher risk of death. Because the present analysis was performed with secondary databases as the retrospective data source, the factors involved in treatment delay in Taiwanese cervical cancer patients could not be clarified fully if this was due to doctor's delay, intentional or by mistake, patients’ decision, failure to seek treatment or inability to start treatment due to poor surgical candidacy, or inability to tolerate chemoradiation. In addition, the database lacked information on patient's marital status, education level, lifestyle, health behaviors, medical knowledge, physical and psychological status, and family care and support. A more in-depth understanding of this problem awaits further research by means of questionnaires or interviews.We are grateful for use of the National Health Insurance Research Database and the Cancer Register Files provided by Statistic Center of Department of Health and Welfare, Taiwan.


Intraductal tubulopapillary neoplasm (ITPN) depicts a distinct entity in the subgroup of premalignant epithelial tumors of the pancreas. Although the histomorphological and immunophenotypical characterization of ITPN has been described by several authors in terms of report of case series in the past, the rarity of that tumor subtype and similarity to other entities still makes identification of ITPN a challenge for radiologists and pathologists. To date, little is known about tubulopapillary carcinoma that can evolve from ITPN.In the present work, we analyze one case of ITPN associated with an invasive component and discuss the results involving the current literature. Collected patient data included medical history, clinical symptoms, laboratory tests, radiological imaging, reports of interventions and operation, and histopathological and immunohistochemical examinations. The patient initially presented with acute pancreatitis. A solid tumor obstructing the main pancreatic duct and sticking out of the papilla of Vater was detected and caught via endoscopic intervention. Histopathological examination of the specimen revealed mainly tubular growth pattern with back to back tubular glands. Immunohistochemically, the tumor was strongly positive for keratin 7 (CK7) and pankeratin AE1/AE3, and alpha 1 antichymotrypsin; negative for synaptophysin and chromogranin A, CDx2, CK20, S100, carcinoembryonic antigen (CEA), MUC 2, MUC5AC, and somatostatin; and in part positive for CA19-9. Extended pancreatoduodenectomy was performed, the final diagnosis was tubulopapillary carcinoma grown in an ITPN.The identification of an ITPN of the pancreas can be a challenging task. Endoscopic retrograde cholangiopancreaticography is an excellent tool to directly see and indirectly visualize the intraductal solid tumor and to take a biopsy for histopathological evaluation at the same time. Together with a thorough immunohistochemical workup, differential diagnoses can be ruled out quickly. To date, reports of ITPN are rare and little is known about the potential for malignant transformation and the prognosis of tubulopapillary carcinoma grown from an ITPN. Radical surgical resection following oncologic criteria is recommended; however, more data will be needed to assess an adequate treatment and follow-up standard. Intraductal tubulopapillary neoplasm was first recognized by Japanese investigators . In 2002In the current edition of the WHO tumor classification, only ITPN and intraductal pancreatic mucinous neoplasm (IPMN) are included in the category intraductal neoplasms. Other entities which may histologically mimic ITPN and may show intraductal growth such as acinar cell carcinoma and pancreatic neuroendocrine tumor are not included in this category, as they show a nonductal differentiation.ITPN is a rare tumor and accounts for less than 1% of exocrine pancreatic neoplasms. It usually appears as a solid mass obstructing the main pancreatic duct thereby causing upstream duct dilation. Due to its malignant potential, ITPN is usually treated by radical surgical resection following oncological criteria in a curative approach . PylorusWhereas histopathological and immunomorphological characteristics are well described, little is known about surgical outcome and prognosis of ITPN to date.The 73-year-old male patient initially presented in the emergency room with nausea and persistent abdominal pain in the upper right quadrant for 1 day. He reported to feel weak and exhausted for a few weeks already. A weight loss of 5 kg in 2 months was attributed to a special diet; the patient did not record loss of appetite. Night sweating was negated. The medical history did not reveal a malignant or benign disease of the gastrointestinal tract. A routinely performed screening colonoscopy 3 years ago showed normal findings.Elevation of serum amylase and lipase, liver transaminases, and bilirubin as well as the patient’s clinical presentation indicated acute pancreatitis Table .Table 1BContrast-enhanced computed tomography (CT) of the abdomen confirmed the diagnosis of pancreatitis, showing edematous peripancreatic fat, minimal amounts of ascites, accentuated peripancreatic lymph nodes, a dilated pancreatic duct in the tail and body of the pancreas, and slightly dilated intra- and extrahepatic bile ducts. There were no clear signs of a tumor or stenosing process in the pancreas and bile ducts at that time Fig. .Fig. 1CTConservative treatment and further evaluation including endosonography and endoscopic retrograde cholangiopancreaticography (ERCP) were initiated. Endosonography revealed an enlarged pancreatic head with inhomogenous parenchyma, compromising the distal bile duct, but without signs of mechanic cholestasis or cholelithiasis. The pancreatic duct was dilated to 7 mm Fig. , with a Histopathological and immunohistochemical workup  19-9, CA72-4, CA125, IgA, IgM, or IgE. Only IgG subclass 2 was unspecifically elevated.Eighteen days after the initial endoscopic intervention with pancreatic stenting, re-endoscopy with stent removal and follow-up endosonography were performed. Pancreatic duct diameter had normalized. A highly suspect 11-mm hypoechoic vascularized lesion with sharp limitation, contacting but not compromising the pancreatic duct, was detected in the pancreatic body. In the inhomogenously structured pancreatic head, pancreatitis seemed to have regressed, but a hypoechoic formation without a clear border was seen, suspicious of pancreatic carcinoma. The distal common bile duct was compressed in the pancreatic head with a slight prestenotic dilatation.Staging CT of the chest, abdomen, and pelvis confirmed the presence of a suspect hypodense lesion located in the head of the pancreas Fig. , and accFollowing interdisciplinary consensus in the hepato-pancreato-biliary tumor conference, oncologic extended pylorus-preserving pancreaticoduodenectomy (PPPD) was planned. After another short episode of a mild pancreatitis and thorough anaesthesiological evaluation prior to surgery, extended PPPD with pancreatogastric anastomosis was performed without complications on day 47 after initial admission to hospital. Intraoperative immediate frozen sections of the resection margins of the common bile duct and pancreas were free of tumor cells. Intraoperative sonography of the liver showed no sign of hepatic tumor spread.In the formalin-fixed surgical specimen, macroscopy revealed a narrowed pancreatic duct in a distance of 1 cm from the papilla, occluded by a drop-shaped 8-mm tumor that appeared to leave the duct wall intact for the most part. The organ parenchyma appeared partly fibrous with regular acini in the uncinate process.Histopathological examination of HE-stained sections taken form most parts of the resection specimen revealed an intraductal lumen-occluding lesion with a mainly tubular growth pattern with back to back tubular glands Figs.  and 9. IImmunohistology revealed the same findings as compared to the biopsy specimen including a strong expression of keratin 7 by all tumor cells Fig. . NeuroenDefinite histopathological diagnosis was a tubulopapillary carcinoma of the pancreatic head grown from an intraductal tubulopapillary neoplasm with a maximal tumor diameter of 28 mm.According to the Union for International Cancer Control (UICC) 8th edition TNM staging for malignant tumors, the tumor was classified as pT2, pN0 (0/25), L0, V0, Pn0, R0 (wide), which refers to a stage IB. The tumor was considered low-grade (G1) .The patient could be discharged from hospital on postoperative day (POD) 13 after an uneventful postoperative course. Six weeks after the operation, on POD 43, the patient was readmitted for 1 day due to unspecific upper abdominal pain and vertigo. Clinical examination showed a distended abdomen, but no signs of peritonitis. Abdominal sonography revealed slightly reduced bowel movements. There were no signs of cholestasis or fluid collections seen on ultrasound examination. Blood test showed normal levels of serum pancreatic and liver enzymes as well as normal inflammation parameters  without adjuvant chemotherapy.The patient recently had the second follow-up in our hepatopancreatobiliary outpatient clinic 6 months after pancreatic resection. Physical examination as well as blood tests and abdominal sonography showed normal findings, and the patient presented clinically well under pharmacological pancreatic enzyme substitution. The next follow-up appointment after three more months is planned to include an abdominal CT.Intraductal tubulopapillary neoplasm (ITPN) depicts a rare subgroup of intraductal epithelial neoplasms of the pancreas. Yamaguchi et al. were the first ones to collect and describe 10 cases of that previously undefined type of tumor [ITPN most commonly shows the morphological appearance of a solid nodular tumor arising in the main pancreatic duct and obstructing the ducts with subsequent upstream dilatation . ConsistTypical features of ITPN on histopathological examination are a tubulopapillary growth pattern, high-grade dysplasia, necrotic foci, and a lack of visible intracellular mucin, which distinguish ITPN from IPMN . In contn = 33) recently reported by Basturk et al. focused on a thorough clinicopathological characterization of those uncommon pancreatic lesions. Data from five high-volume centers of surgical pathology were collected and reviewed for that purpose. Besides the aforementioned characteristics, the authors noticed CAM5.2 labeling of all ITPNs, as a proof of epithelial tissue origin, and the absence of MUC2 and MUC5AC [Regarding immunohistochemical appearance, ITPN was reported lacking gastroenteric differentiation, typically seen in IPMN, reflected by the absence of MUC2 (and of CDX2) and MUC5AC expression. Instead, signs of pancreatic duct differentiation are revealed by expression of CK7 and/or CK19, as well as MUC1 and MUC6 , 5, 8–12d MUC5AC .Date et al. screened the literature for reports on ITPN in 2016 and retrospectively analyzed a total of 58 cases, including Yamaguchi’s first cases . The autMUC5AC has been found to be expressed commonly by pancreatic ductal adenocarcinoma (PDAC) and, less frequently, by precursor lesions such as IPMN –17. As mITPN show genetic patterns that are different from those seen in IPMN, PanIN, and pancreatic ductal adenocarcinoma, specifically an absence of KRAS and BRAF mutations , 8, 11. Intraductal tubulopapillary neoplasms have also been reported to occur in the bile ducts , 28. NakAddressing the need for early detection and discrimination of different pancreatic lesions, Tajima et al. reflected the value of endoscopic ultrasound-guided fine needle aspiration cytology (EUS-FNAC) and described their observations in one case . Three cUnspecific symptoms such as discomfort, abdominal pain, weight loss, and jaundice due to pancreatic duct obstruction may be recorded in ITPN patients . In theiIn the study reported by Basturk et al., 45% of ITPN were located in the head of the pancreas, 32% in the body/tail, and 23% diffusely involved the organ . Rooney A significant proportion of ITPN may become invasive . The invIn the study of Kölby et al., one patient died from multiple liver metastases after 7 months, two other patients had successful treatment for tumor recurrence 12 and 34 months postoperatively, and two patients required palliative care for recurrent disease with distant metastases after 18 and 24 months . The autIn the study by Basturk et al., follow-up data of 22 patients were available. Among those, the 5-year survival rate was 100% in patients without an invasive component and 71% in patients with an invasive carcinoma. Two patients died of the disease at 23 and 41 months; one patient died of unrelated causes at 49 months. After a median follow-up period of 61.5 months, 12 patients were alive with disease, while 7 patients were disease-free after a median follow-up of 19 months , 9, 12.Satisfying results have been shown after radical resection of pancreatic ITPN, compared to the more malignant course of PDAC. Following oncologic R0 resection, our patient did not show any sign of disease recurrence in the short postoperative period of 6 months. The interdisciplinary tumor board decided for initial 3-monthly follow-up in our specialized hepatopancreatobiliary surgical outpatient unit. Close clinical follow-up is recommended for early detection of disease recurrence .To date, reports of ITPN with or without invasive carcinoma are rare and case series largely make recourse to earlier publications. In order to assess the long-term outcome of ITPN and to develop appropriate therapeutic strategies, more data will be needed in the future.


Circadian and seasonal rhythms are seen in many species, modulate several aspects of human physiology, including brain functions such as mood and cognition, and influence many neurological and psychiatric illnesses. However, there are few data regarding the genome-scale molecular correlates underlying these rhythms, especially in the human brain. Here, we report widespread, site-specific and interrelated diurnal and seasonal rhythms of gene expression in the human brain, and show their relationship with parallel rhythms of epigenetic modification including histone acetylation, and DNA methylation. We also identify transcription factor-binding sites that may drive these effects. Further, we demonstrate that Alzheimer's disease pathology disrupts these rhythms. These data suggest that interrelated diurnal and seasonal epigenetic and transcriptional rhythms may be an important feature of human brain biology, and perhaps human biology more broadly, and that changes in such rhythms may be consequences of, or contributors to, diseases such as Alzheimer's disease. et al. show diurnal and seasonal rhythms of gene expression in the human brain, their relationship to histone acetylation and DNA methylation, and their disruption in Alzheimer's disease.Diurnal and seasonal rhythms modulate brain function, but we do not know the genomic basis for these rhythms. Here, Lim  Circadian and circannual rhythms are seen in many plant and animal species. Circadian rhythms modulate phenomena as diverse as bioluminescence in dinoflagellates2Notwithstanding the ubiquity of circadian and circannual rhythms—and their impact on human disease—there remain gaps in our understanding of the genetic and epigenetic mechanisms generating them and linking them to normal and abnormal tissue function, especially in the human brain. Mechanisms underlying circadian rhythms are better understood. An evolutionarily conserved transcriptional negative feedback loop lies at the core of the molecular clock in model organisms1112151618Less is known about the genetic and epigenetic mechanisms underlying seasonal rhythms, and how these molecular events influence tissue function. Seasonal rhythms of selected genes in specific brain regions have been reported in hamsters2021Using post-mortem human brain tissue obtained from two longitudinal cohort studies of ageing, we recently characterized large-scale diurnal rhythms of DNA methylation and their relation to diurnal rhythms of gene expression in the human dorsolateral prefrontal cortexUsing these data, we demonstrate interrelated diurnal and seasonal rhythms of gene expression in the dorsolateral prefrontal cortex that are linked to parallel rhythms of epigenetic modification, associated with specific transcription factor-binding sites and altered in the context of Alzheimer's disease pathology. These data suggest that seasonal and diurnal molecular rhythms may play an important role in the biology of the human dorsolateral prefrontal cortex, and their disruption may be a potential contributor to, or consequence of, Alzheimer's disease.We studied post-mortem dorsolateral prefrontal cortex samples from 757 participants in two ongoing cohort studies of older persons, the Rush Memory and Ageing Project (MAP) and the Religious Orders Study (ROS), in which participants were free of dementia at study enrolment and agreed to annual evaluations and brain donation on death. Clinical characteristics of the study participants are in To identify seasonal patterns in the expression of each gene, we considered expression levels as a function date of death relative to January 1. For diurnal patterns of gene expression, in keeping with similar studies18We found many genes, including several canonical circadian clock genes, that have robust diurnal and seasonal rhythms . Based o18F-statistic and P value . This set of genes was strongly enriched for genes previously reported as diurnally rhythmic in Brodmann's area 11 , Brodmann's area 47  and dorsolateral prefrontal cortexχ2=21.4, P=3.7 × 10−6) in other studies. The timing of clock gene expression in our data set was strongly correlated with the timing of clock gene expression in these other data sets . These results illustrate the robustness of these rhythms across brain regions and data sets. They represent an important source of transcriptional variation in the brain, and our large data set behaves similarly to what has been observed previously, enabling us to connect our results with the existing framework of brain molecular rhythms.Similar to other human brain data sets, where 8–12% of the transcriptome is diurnally rhythmic at .05 refs 1918,19, F-statistic for diurnal rhythmicity. We compared this to the median F-statistic in 10,000 null data sets generated by randomly shuffling times of death to generate an empiric P value. The transcriptome as a whole showed much greater diurnal rhythmicity than expected by chance . A similar pattern was seen in the H3K9 acetylome, with morning-acrophase H3K9Ac peaks tending to have fall seasonal acrophases, and evening-acrophase H3K9Ac peaks tending to have spring seasonal acrophases . For the DNA methylome, we plotted the timing of the nadir rather than acrophase of methylation as hypomethylation rather than hypermethylation is classically associated with transcription. However, because the acrophase always follows the nadir by π radians in a cosine curve, the analyses are statistically equivalent whether acrophase or nadir is used. The resulting patterns were different than those seen for the transcriptome and H3K9 acetylome. DNA methylation sites with fall nadirs were no more likely to have evening than morning nadirs . Similar results were seen when we limited these analyses to the most significant diurnally and seasonally rhythmic sites, or when we repeated these analyses in relation to local clock time or to the midpoint of the dark period W=4,572.6, P=0.0136, n=664 samples), and the difference in angular distribution of seasonal acrophases showed a trend toward significance . Moreover, the set of H3K9Ac peaks proximate to active TSS was relatively enriched for morning/fall acrophase peaks, while the set of peaks distant from active TSS was relatively enriched for evening/spring acrophase peaks . Similar results were seen when we limited these analyses to the most significant diurnally and seasonally rhythmic H3K9Ac peaks, or when we repeated these analyses in relation to local clock time or the midpoint of the dark period . However, there was no association between proximity to an active TSS and the timing of seasonal rhythms  or with the overall distribution of DNA methylation sites among the four temporal clusters . Similar results were seen when we repeated these analyses in relation to local clock time or the midpoint of the dark period .For DNA methylation, proximity to active TSS was associated with the timing of diurnal rhythms, as we had reported previously  with the set of H3K9Ac peaks near morning-acrophase transcripts being relatively enriched for morning-acrophase H3K9Ac peaks, and vice versa . Similarly, the angular distribution of seasonal acrophases for H3K9Ac peaks near spring versus fall-acrophase transcripts also differed , and there was a trend toward relative enrichment for spring-acrophase H3K9Ac peaks near spring-acrophase transcripts, and fall-acrophase H3K9Ac peaks near fall-peaking transcripts .Both H3K9Ac and DNA methylation are thought to influence transcription. Therefore, we examined associations between the timing of rhythms of transcript abundance and the timing of rhythms of H3K9Ac and DNA methylation at nearby sites. To examine associations between diurnal transcript and H3K9Ac rhythms, we contrasted two groups of H3K9Ac peaks—those within 2 kb of morning-acrophase transcripts and those within 2 kb of evening-acrophase transcripts. The angular distribution of diurnal acrophases for these two sets of H3K9Ac peaks differed . The angular distribution of seasonal nadirs also differed significantly between DNA methylation sites near the TSS of spring-acrophase transcripts and those proximate to the TSS of fall-acrophase transcripts . However, here there was a relative enrichment of fall-acrophase  DNA methylation sites near fall-acrophase transcripts and spring-acrophase (rather than spring-nadir) DNA methylation sites near spring-acrophase transcripts .We found similar results in the DNA methylome. The angular distribution of diurnal nadirs differed significantly between DNA methylation sites near the TSS of morning-acrophase transcripts and those near the TSS of evening-acrophase transcripts (P<0.05 by the F-test based on n=527 samples for DNA methylation and n=471 samples for H3K9Ac) epigenetic sites proximate to the most significantly rhythmic  transcripts, although statistical significance was attenuated perhaps reflecting the smaller number of sites considered  <0.05 . Effect P<0.05 for both diurnal and seasonal rhythmicity  thought to play roles in circadian and circannual rhythmicity. Meanwhile, work Data from participants in two ongoing longitudinal cohort studies of older persons were included in this study: the Rush MAP and the ROS. The ROS is a longitudinal study of ageing in Catholic brothers, nuns and priests from across the United StatesOf the 1191 samples for whom these data were available, dorsolateral prefrontal cortex RNA expression data passing quality control criteria (see below) were available from 531 samples, DNA methylation data passing quality control criteria (see below) were available from 732 samples and H3K9Ac ChIP-seq data passing quality control criteria (see below) were available from 664 samples. Data from these participants were included in the current analyses. Their characteristics are shown in This study was approved by the Institutional Review Board of Rush University Medical Centre and was conducted in accordance with the latest version of the Declaration of Helsinki. All participants provided written informed consent, and an Anatomic Gift Act for organ donation.http: //www.synapse.org; SynapseID syn3388564). We analysed only genes and transcripts on autosomal chromosomes, and excluded genes and transcripts expressed in <90% of our samples, leaving 18,709 individual GENCODE v14 genes and 42,873 individual GENCODE v14 isoforms in these analyses. All FPKM values were log-transformed before further analysis. Principal component analysis was used to assess sample quality, and only those samples with principal components 1, 2 and 3 values within 3 s.d. from their respective means were included. Data from 531 samples meeting quality control criteria as above, and with full clinical data as described below, were included in this analysis.RNA was extracted from dorsolateral prefrontal cortex blocks with the miRNeasy Mini Kit  and the RNase-free DNase Set (Qiagen). RNA concentration was quantified by Nanodrop  and an Agilent Bioanalyser was used to assess quality. Samples from which <5 μg of RNA were obtained, or samples with a Bioanalyser RNA integrity score of 5 or less, were excluded from further analysis. The strand-specific dUTP method−1. The Methylation Module v.1.8 from the Genome Studio software suite (Illumina) was used to carry out colour channel normalization and background removal, and to generate β values and detection P values for 485,513 CpG site across the human genome. These data are available through the synapse.org AMP-AD Knowledge Portal (http: //www.synapse.org SynapseID syn3157275). Probes with detection P values>0.01 in any samples, with 47/50 nucleotides matching sex chromosome sequences during sequence alignment with BLAT, in which a single-nucleotide polymorphism with a minor allele frequency ≥0.01 exists within 10 bp upstream or downstream of the CpG site, or on a sex chromosome were excluded from further analysis. This left 420,132 autosomal CpGs in the data set. Principal component analysis was used to assess sample quality, and only those samples with principal components 1, 2 and 3 values within 3 s.d. from their respective means were included. As well, samples in which at least 2 out of the 10 bisulfite conversion control probes failed to reach a value of 0.8 were excluded. Data from 732 samples meeting quality control criteria as above, and with full clinical data as described below, were included in these analyses.We assessed DNA methylation in 746 dorsolateral prefrontal cortex samples as described previously27q value cutoff of 0.001 (ref. http: //www.synapse.org SynapseID syn4896408). Five different quality filters were used to remove low-quality samples6 unique reads, (ii) non-redundant fraction≥0.3, (iii) cross-correlation≥0.03, (iv) fraction of reads in peaks≥0.05 and (v) ≥6,000 peaks. After quality control, 669 out of 712 samples remained. H3K9Ac domains were defined by calculating all genomic regions that were detected as a peak in at least 100 (15%) of our 669 samples. Regions neighboured within 100 bp were merged and very small regions of <100 bp were removed. In total, we obtained 26,384 H3K9Ac domains. A subset of 25,740 of these domains were located on autosomal chromosomes and used for subsequent analyses. To quantify H3K9Ac, we counted the number of reads falling into each H3K9Ac domain for each sample, divided by the width of each domain in kilobases and by the total number of mapped reads in each sample, and scaled this to obtain units of FPKMs. FPKM values were log-transformed before analysis. Data from 664 samples meeting the quality control criteria as above, and with full clinical data as described below, were included in these analyses.Grey matter was dissected on ice from blocks of dorsolateral prefrontal cortex, minced and crosslinked with 1% formaldehyde at room temperature for 15 min and quenched with 0.125 M glycine. After homogenizing the tissue in cell lysis buffer using the Tissue Lyser and a 5 mm stainless-steel bead, the nuclei were lysed in cell lysis buffer and chromatin was sheared by sonication. Chromatin was incubated overnight at 4 °C with the Millipore anti-H3K9Ac monoclonal antibodies  and purified with protein A sepharose beads. Finally, extracted DNA was used for Illumina library construction following usual methods and sequenced with 36 bp single-end reads on the Illumina HiSeq. Reads were aligned by the BWA algorithm against the human reference genome GRCh37 ref. 55. MACS2001 ref. 56. PooleWe computed age at death from the self-reported date of birth and the date of death. We recorded sex at the time of the baseline interview.The time of sunrise on the day of death was computed from the recorded date of death, and the latitude and longitude of the city in which each participant died.Depressive symptoms were assessed with a 10-item version of the Centre for Epidemiologic Studies Depression scale2 in sections from the frontal, temporal, parietal, hippocampal and entorhinal cortices of each participant, scaling these values using the s.d. of all participants, and then averaging across the four brain regions and three pathologies  to generate a summary score. As described previouslyAlzheimer's disease pathology was quantified as described previously58We concurrently characterized independent diurnal and seasonal patterns in the expression of each of the 18,709 genes using cosine functions, considering each transcript as a function date of death and time of death relative to sunrise on the date of death , adjusted for age at death, sex, post-mortem interval, the burden of Alzheimer's disease pathology, the presence/absence of depressive symptoms and technical covariates  as follows:t is the ZT, and d is the date of death, Ad is the amplitude of diurnal rhythmicity, As is the amplitude of seasonal rhythmicity, φd is the time of the acrophase of diurnal rhythmicity and φs is the date of the acrophase of seasonal rhythmicity. For these analyses, the diurnal period was fixed at 24 h and the seasonal period was fixed at 365 days. This is a limitation of any study design where each individual contributes only one data point to each 24-h or 365-day sampling period. All times of death were converted to radians  for analysis and then converted back to hours for the purposes of visual representation. Dates of death were similarly converted to radians . In secondary analyses, we repeated all analyses using two alternative reference times: local clock time (with ZT0=midnight), to which the timing of artificial light exposure is linked, and the midpoint between sunset and sunrise, which is invariant across the seasons. For computational efficiency, we fit equivalent linearized models of the formwhere Ad, φd, As and φs from and, To quantify the contribution of diurnal rhythmicity to the model fit in F-statisticand determined the Fd. In similar way, we quantified the contribution of seasonal rhythmicity to the model fit in F-statistic.The greater the contribution of diurnal rhythmicity to the overall model fit, the greater the value of We then repeated the above analyses for each of the 42,873 individual isoforms, 25,740 autosomal H3K9Ac peaks and 420,132 autosomal DNA methylation sites.P value and setting a threshold of P<0.05. We compared this list of putatively diurnally rhythmic genes to three other published data sets . To compute an empiric P value, we compared this to the median Fd across all transcripts in each of 10,000 null data sets (Fd_null) generated by randomly shuffling the times of death, and determined the proportion of null data sets for which Fd_null>Fd_observed. We then did the same for seasonal rhythmicity, using null data sets generated by randomly shuffling dates rather than times of death. Then, we repeated these analyses for the 25,740 autosomal H3K9Ac peaks and 420,132 autosomal DNA methylation sites.Next, we quantified the degree of diurnal rhythmicity across all 18,709 genes or 42,873 transcripts by computing the median χ2-statistic for the corresponding 2 × 2 contingency table using the observed data (χ2obs), comparing this to the corresponding χ2-statistics computed by repeating these procedures on 10,000 empiric null data sets generated by shuffling both the times and dates of death (χ2null), and calculating an empiric P value by determining the proportion of 10,000 null data sets in which χ2null>χ2obs. We then repeated this analysis for the H3K9Ac and DNA methylation data, except that for the DNA methylation data, we considered the time of the nadir rather than acrophase of methylation, as hypomethylation rather than hypermethylation is classically associated with transcription. Finally, as a sensitivity analysis, we repeated these analyses considering only those transcripts/peaks/sites that were both seasonally rhythmic and diurnally rhythmic at P<0.05.We next examined for patterns in the timing of diurnal and seasonal rhythms of RNA expression. By visual inspection, and in keeping with prior work19W-statistic (Wd_observed) and compared this to the corresponding W-statistic for each of 10,000 null data sets generated by randomly shuffling times of death (Wd_null), and calculated an empiric P value by determining the proportion of 10,000 null data sets where Wd_null>Wd_observed. We then did the same for the angular distribution of seasonal acrophases, using null data sets generated by shuffling dates of death. Next, we examined whether the classification of individual peaks into diurnal/seasonal clusters differed between the two groups of H3K9Ac peaks by calculating the χ2-statistic, χ2observed, for the corresponding 4 × 2 contingency table , comparing this to the corresponding χ2-statistics generated by repeating the above procedure on 10,000 null data sets generated by shuffling times and dates of death, and calculating an empiric P value by determining the proportion of 10,000 null data sets in which χ2null>χ2observed. We then repeated the above procedure for DNA methylation sites. Finally, as a sensitivity analysis, we repeated these analyses considering only those transcripts/peaks/sites that were both seasonally and diurnally rhythmic at P<0.05.We previously showed that for rhythms of DNA methylation, the time of the diurnal acrophase varies depending on distance from the nearest TSSBoth H3K9Ac and DNA methylation are thought to influence transcription. Therefore, we examined associations between the timing of rhythms of transcript abundance and the timing of rhythms of H3K9Ac and DNA methylation at nearby sites. We first examined diurnal rhythms of H3K9Ac, using the 471 samples where both sets of data were available.W-statistic as above, computing an empiric P value by comparing this to the equivalent W-statistic calculated on 10,000 null data sets generated by randomly shuffling times of death. We then determined whether the two groups differed in the proportion of H3K9Ac peaks classified as morning or evening by calculating the corresponding χ2 statistic, and computing an empiric P value by comparison with the equivalent χ2-statistic calculated from 10,000 null data sets generated by shuffling times of death. After considering diurnal rhythms of H3K9Ac as above, we then repeated this analysis considering seasonal rhythms. Next, we repeated these analyses for DNA methylation sites using the 527 samples with both RNA-seq and DNA methylation data. Finally, as a sensitivity analysis, we repeated these analyses considering only those transcripts/peaks/sites that were seasonally or diurnally rhythmic at P<0.05.We considered two groups of H3K9Ac peaks: those within 2 kb of the TSS of transcripts empirically classified as morning acrophase, and those within 2 kb of the TSS of transcripts empirically classified as evening acrophase, and compared the angular distributions of their diurnal acrophases using the Mardia–Watson–Wheeler The local transcription factor environment has an important influence on the circadian timing of transcription in model systems3233We then used logistic regression models of the formP values and analysis-wide FDRs by comparing the effect estimates above to those generated from 10,000 null data sets generated by randomly shuffling times or dates of death. We identified a set of candidate transcription factors potentially involved in regulating diurnal and/or seasonal rhythmicity if their binding sites had an FDR<0.05 for one of diurnal or seasonal rhythmicity for any of RNA, H3K9Ac or DNA methylation. As a sensitivity analysis, we repeated this procedure considering only transcripts, H3K9Ac peaks or DNA methylation sites with P<0.05 for diurnal or seasonal rhythmicity, and calculated Spearman's correlation coefficients relating the transcription factor effect estimates estimated on the basis of all transcripts/H3K9Ac peaks/DNA methylation sites, and those estimated on the basis of only those transcripts/H3K9Ac peaks/DNA methylation sites with P<0.05 for diurnal or seasonal rhythmicity.to examine the independent association of the local presence of binding sites for each of the 161 ENCODE transcription factors with the odds of having a spring versus fall or evening versus morning transcript, H3K9Ac peak or DNA methylation site. We estimated uncorrected Next, to examine for shared regulatory effects of these transcription factor-binding sites on diurnal and seasonal rhythmicity, we plotted their estimated coefficients for diurnal versus seasonal rhythmicity for each of transcript expression, H3K9Ac and DNA methylation, and calculated Spearman's correlation coefficients. Next, to examine for shared regulatory effects of these transcription factor-binding sites on transcript and epigenetic diurnal rhythms, we calculated Spearman's correlation coefficients relating to their effects on diurnal transcript versus epigenetic rhythms (H3K9Ac and DNA methylation). We then repeated this for seasonal rhythms.Brain disorders such as Alzheimer's diseasey1 and y2 are age at death and male sex.where Here, the relative amplitudes of diurnal and seasonal rhythmicity in individuals with Alzheimer's disease are given byand the relative amplitudes of diurnal and seasonal rhythmicity in individuals without Alzheimer's disease are given byMoreover, the acrophases of diurnal and seasonal rhythmicity in individuals with Alzheimer's disease are given byand the acrophases of diurnal and seasonal rhythmicity in individuals without Alzheimer's disease are given byP values. We then repeated this procedure for seasonal acrophases. To quantify the effect of Alzheimer's disease on the amplitude of transcript rhythms, we calculated the median difference in relative amplitude between individuals with and without Alzheimer's disease, first considering all transcripts, and then considering separately transcripts in each of the four temporal clusters. This procedure was repeated in 10,000 null data sets generated by shuffling times of death to generate empiric P values. We then repeated this procedure for the relative amplitude of seasonal rhythms. As a sensitivity analysis, we then repeated the above considering only those transcripts with individual P<0.05 for both diurnal and seasonal rhythmicity. Finally, we repeated the above analyses for the H3K9Ac peaks, and DNA methylation sites.To depict graphically the association between a pathological diagnosis of Alzheimer's disease and the timing of transcript rhythms, we classified each transcript into diurnal and seasonal classes as above, based on the model predicted acrophase for samples without Alzheimer's disease, and separately plotted the model-predicted diurnal and seasonal acrophase times for those with Alzheimer's disease. To more formally quantify the association between a pathological diagnosis of Alzheimer's disease and the timing of transcript rhythms, we calculated the median difference in predicted acrophase timing between individuals with and without Alzheimer's disease, first considering all transcripts and then considering separately transcripts in each of the four temporal classes. This procedure was repeated in 10,000 null data sets generated by shuffling times of death to generate empiric https://www.synapse.org/#!Synapse:syn3388564). H3K9Ac ChIP-seq data that support the findings of this study have been deposited in the synapse.org AMP-AD knowledge portal with the accession code syn4896408 (https://www.synapse.org/#!Synapse:syn4896408). DNA methylation data that support the findings of this study have been deposited in the synapse.org AMP-AD knowledge portal with the accession code syn3157275 (https://www.synapse.org/#!Synapse:syn3157275).RNA-seq data that support the findings of this study have been deposited in the synapse.org AMP-AD knowledge portal with the accession code syn3388564 .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures.2(FPKM). Acrophases in units of radians.Parameters of model fit seasonal and diurnal cosine curves for 18,709 GENCODE v14 genes. Mean level and amplitudes expressed in units of log2(FPKM). Acrophases in units of radians.Parameters of model fit seasonal and diurnal cosine curves for 42,873 GENCODE v14 isoforms. Mean level and amplitudes expressed in units of logParameters of model fit seasonal and diurnal cosine curves for 25,740 H3K9Ac peaks. Mean level and amplitudes expressed in units of ln(FPKM). Acrophases in units of radiansParameters of model fit seasonal and diurnal cosine curves for 420,132 DNA methylation sites. Mean level and amplitudes expressed refer to beta values of methylation. Acrophases in units of radians.


Delayed hematological recovery, graft failure, and acute graft-versus-host disease (GVHD) still remain major problems in cord blood transplantation (CBT). Mesenchymal stem cells (MSCs) are known to support bone marrow stroma and promote hematopoiesis. Additionally, MSCs possess immunomodulatory properties and are used clinically for the treatment of acute GVHD. Therefore, the use of MSCs to enhance engraftment and prevent GVHD after allogeneic hematopoietic cell transplantation has been explored. Recent clinical trials have shown the feasibility and safety of intravenous cotransplantation of MSCs with cord blood cells in pediatric patients, but not in adult patients, who are at greater risk of graft failure. As for the route of administration of MSCs, direct intrabone marrow injection of MSCs is thought to enhance the engraftment of cord blood cells more than intravenous injection. Based on these background findings, this clinical trial was designed to develop a new strategy to enhance engraftment and prevent GVHD after CBT.6 cells/kg of patient body weight. On the day of CBT, MSCs are injected into the intrabone marrow of the patient 4 hours before the infusion of a single cord blood unit. The conditioning regimen varies according to patient age and disease. GVHD prophylaxis consists of a combination of tacrolimus and methotrexate. The primary endpoint of this study is infusional toxicity of MSCs within 14 days after transplantation.This is a single-center, phase I, clinical study to evaluate the safety of CBT combined with intrabone marrow injection of ex vivo expanded MSCs from bone marrow of a third-party donor. Adult patients with hematological disorders are eligible for this study. The target sample size is 5, and the registration period is 3 years. The target dose of MSCs infused is 0.5 × 10 Besides these approaches, cotransplantation of cord blood and mesenchymal stem cells (MSCs) has recently been reported.–14Cord blood transplantation (CBT) has been increasingly used as a curative treatment for various hematological disorders. However, delayed hematological recovery and a higher rate of graft failure after CBT lead to an increased risk of transplant-related mortality in the early period after transplant. In bone marrow, MSCs differentiate into bone-marrow stroma cells, osteocytes, osteoblasts, and endothelial cells. All of these cells form the bone marrow microenvironment, known as the hematopoietic stem cell niche, and support hematopoiesis.,17 Besides this hematopoietic support capacity, MSCs can modulate immune responses by producing several cytokines and growth factors, and this immunomodulatory property of MSCs has already been applied to the treatment of graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (HCT).,19 In addition, MSCs are able to evade allogeneic rejection because of low expression levels of HLA molecules and no expression of costimulatory molecules, such as CD80, CD86, and CD40.,21 Furthermore, MSCs can be easily expanded ex vivo and stored by cryopreservation. Therefore, ex vivo expanded and cryopreserved MSCs derived from a third-party donor can be used for patients without considering HLA matching. Because of these properties, MSCs have been explored for application in enhancing engraftment and preventing GVHD after allogeneic HCT.MSCs are a heterogeneous subset of stromal stem cells and can be isolated from many tissues, such as bone marrow, adipose tissue, cord blood, and placenta. MSCs have the capacity for self-renewal and can differentiate into mesodermal lineage cells.–14 However, cotransplantation of MSCs and cord blood cells has not been evaluated in adult patients, who are at greater risk of graft failure because of a lower cord blood cell dose per patient body weight. As for the route of MSC administration, several animal model experiments have shown that MSCs infused intravenously were trapped in lung,,23 and direct intrabone marrow injection of MSCs could enhance the engraftment of transplanted cord blood cells more than intravenous injection. Additionally, intrabone marrow injection of MSCs has been reported to be safe in previous clinical studies.,26The feasibility and safety of intravenous cotransplantation of MSCs with cord blood cells in pediatric patients have been reported by 4 clinical trials.Based on these background findings, to develop a new strategy not only to enhance engraftment but also to prevent GVHD after CBT, a first phase I clinical trial was designed to evaluate the safety of CBT combined with intrabone marrow injection of ex vivo expanded MSCs for adult patients with hematological disorders.22.1This study is a single arm, nonrandomized, open-label, single-center, phase I trial to evaluate the safety of CBT combined with intrabone marrow injection of MSCs generated from bone marrow of a third-party donor. The target sample size is 5, and the registration period is 3 years. The compliance of this study with the Act on the Safety of Regenerative Medicine was confirmed by the Ministry of Health, Labour and Welfare of Japan . This study was registered with the University Hospital Medical Information Network Clinical Trials Registry . Written informed consent will be obtained from all patients and donors before registration, in accordance with the Declaration of Helsinki. Patients and donors will be registered in this study after independent review by the data center in the Department of Hematology and Oncology, Nagoya University Graduate School of Medicine. Independent monitoring will be planned according to the Japanese clinical trial guideline at least annually.2.2The inclusion and exclusion criteria of the patients are listed in Table 2.36 cells/kg of patient weight. The volume of bone marrow aspirate is determined according to patient weight; if the patient weight is <35, 35 to 50, or ≥50 kg, the volume of bone marrow aspirate is 10, 15, or 20 mL, respectively. Mononuclear cells are isolated by centrifugation of bone marrow using Ficoll-Paque PREMIUM . The separated mononuclear cells are seeded in T-25 cell culture flasks at 1.0 to 2.0 × 107 cells/flask in D-MEM medium  containing 5% human platelet lysate and 2 IU/mL heparin  (referred to as culture medium) and cultured at 37°C in a humidified incubator containing 5% CO2. After culturing for 3 or 4 days, nonadherent cells are removed, and the adherent cells are further cultured. Cells are harvested at subconfluent using TrypLE Select . Cells at passage 1 are seeded in the same number of T-75 culture flasks from T-25. Cells at passage 2 are seeded in the same number of T-225 culture flasks from T-75. Cells at passage 3 or 4 are seeded in the threefold number of T-225 flasks. MSCs are harvested after passage 3 or 4 according to the cell count, sampled, and cryopreserved at −150°C in CP-1  until the day of CBT.The inclusion and exclusion criteria of the MSC donors are listed in Table Human platelet lysates are prepared from single-donor platelet concentrate provided by the Japan Red Cross Blood Center by the Application for the use of blood donated in Japan based on the “Guidelines on the use of donated blood in R&D, etc.”. Platelet concentrate is frozen at −30°C and thawed twice and then stored at −30°C. The frozen platelet concentrate is thawed at 4°C and centrifuged to obtain supernatant as platelet lysate.6 cells/kg of patient weight, absence of contamination by pathogens , and immune phenotype characterized by the expression of CD73, CD90, and CD105 surface molecules (≥90%) and the absence of CD14, CD19, CD34, CD45, and HLA-DR expressions (≤10%).Criteria for release of MSCs for clinical use are as follows: viability ≥70%, viable cell count ≥0.2 × 102.4The conditioning regimen is not defined in this study. In vivo purging of T cells using treatments such as antithymocyte globulin is prohibited. GVHD prophylaxis consists of the combination of tacrolimus and short-term methotrexate. Granulocyte colony-stimulating factor is administered from 7 days after transplantation until neutrophil engraftment.2.5On the day of CBT, MSCs are thawed, washed, and resuspended in 2 to 10 mL of a saline solution. The premedication with hydrocortisone 100 mg and chlorpheniramine 10 mg is administered approximately 30 minutes before injection of MSCs. After local anesthesia, a standard bone marrow aspiration needle is inserted into the iliac bone on one side. To assess that the needle is securely inserted into the bone marrow cavity, aspiration of <0.5 mL bone marrow is done. Then, approximately 5 mL of MSC suspension are injected slowly. This procedure is repeated on the iliac bone on the contralateral side. Four hours after MSC injection, cord blood is infused intravenously with the standard procedure.2.6The primary endpoint of this study is infusional toxicity of MSCs within 14 days after transplantation. Infusional toxicity is defined as adverse events that could not be explained by other complications, such as regimen-related toxicity or infection, that generally occur after transplantation. Secondary endpoints include the rate of engraftment, the time to hematopoietic recovery, the incidences and severities of acute and chronic GVHD, the incidences of regimen-related toxicities and infection, and the probabilities of nonrelapse mortality (NRM) at 100 days, relapse, disease-free survival (DFS), and overall survival (OS) at 1 year after transplantation.2.79/L for 3 consecutive days. The time to neutrophil engraftment is defined as the first day of achieving an absolute neutrophil count greater than 0.5 × 109/L for 3 consecutive days. The time to platelet and reticulocyte recoveries is defined as the first days of achieving a platelet count more than 20 × 109/L, and a reticulocyte count more than 1% for 3 consecutive days without transfusions. Primary graft failure is defined as the lack of neutrophil engraftment in patients surviving at least 60 days, and secondary graft failure is defined as neutrophil engraftment followed by a decline in neutrophil count to below 0.5 × 109/L for 3 consecutive days. Acute GVHD is diagnosed and graded according to the consensus criteria. Chronic GVHD is evaluated according to the traditional Seattle criteria and the NIH criteria for diagnosis and severity of chronic GVHD. Relapse is defined as recurrence of disease after transplantation. NRM is defined as death without disease relapse. The probabilities of relapse, NRM, acute GVHD, and chronic GVHD are estimated on the basis of cumulative incidence curves to accommodate the following competing events: death without relapse for relapse, relapse for NRM, and death without GVHD and relapse for GVHD. The probability of DFS is calculated from the day of transplantation until death from any cause, relapse, or last follow-up according to the Kaplan–Meier method. The probability of OS is calculated from the day of transplantation until death from any cause or last follow-up according to the Kaplan–Meier method.Engraftment is defined as neutrophil recovery greater than 0.5 × 103This clinical phase I trial has been designed to evaluate the safety of CBT combined with intrabone marrow injection of ex vivo expanded MSCs. We consider that coinfusion of MSCs, which are known to promote hematopoiesis and modulate immune reaction, will both enhance the engraftment of cord blood cells and prevent the occurrence of GVHD. This strategy might be applicable not only to CBT, but also bone marrow or peripheral-blood stem-cell transplantation, leading to benefits such as suppression of severe GVHD in HLA-mismatched settings and reduction of the burden imposed on hematopoietic stem cell donors by decreasing the required stem cell number. We hope that this study will provide a basis for further clinical trials of this strategy for the improvement of the outcome of hematopoietic cell transplantation.Conceptualization: Tatsunori Goto, Makoto Murata.Funding acquisition: Makoto Murata.Methodology: Tatsunori Goto, Makoto Murata, Seitaro Terakura, Tetsuya Nishida, Yoshiya Adachi, Yoko Ushijima, Kazuyuki Shimada, Yuichi Ishikawa, Fumihiko Hayakawa, Nobuhiko Nishio, Satoshi Nishiwaki, Akihiro Hirakawa, Katsuyoshi Kato, Yoshiyuki Takahashi, Hitoshi Kiyoi.Writing — original draft: Tatsunori Goto, Makoto Murata.Writing — review and editing: Tatsunori Goto, Makoto Murata, Seitaro Terakura, Tetsuya Nishida, Yoshiya Adachi, Yoko Ushijima, Kazuyuki Shimada, Yuichi Ishikawa, Fumihiko Hayakawa, Nobuhiko Nishio, Satoshi Nishiwaki, Akihiro Hirakawa, Katsuyoshi Kato, Yoshiyuki Takahashi, Hitoshi Kiyoi.


The primary outcome was gestation-specific sex difference in the occurrence of mortality, combined morbidities, and individual morbidity. A total of 2228 VLBWI were enrolled . Mortality was not different between sexes. The risk of bronchopulmonary dysplasia and combined morbidities was significantly higher in males ≤25 weeks of gestation . Males had a significantly higher incidence of periventricular leukomalacia at 23 and 29 weeks of gestation. The risk of severe retinopathy of prematurity was higher in females >25 weeks of gestation. Although both sexes have similar risk for mortality, male sex remains an independent risk for major morbidities, especially at ≤25 weeks of gestation. The risk of each outcome for males has a specific pattern with increasing gestational age.This study aims to determine whether male sex has adverse effect on mortality and morbidities in very low birth weight infants (VLBWI) <30 weeks of gestation and to ascertain this sex effect, stratified by gestational age, adjusting for perinatal risk factors. This is a population-based study from Korean Neonatal Network for VLBWI born at 23 Infants were divided according to birth weight into very low birth weight infants  and extremely low birth weight infants 2. Approximately 15 million infants are born preterm each year, representing a preterm birth rate of 11.1%3. In Korea4, preterm birth accounts for approximately 8% of live birth, similar to East Asia (7.4%) and Western countries (8.6%)3. Complications of preterm birth are the single largest direct cause of neonatal deaths, responsible for 35% of the world’s 3.1 million deaths a year5, with VLBWI being a main cause of neonatal death and major complications6. To date, a number of guidelines for clinicians and parents on neonatal outcomes at various gestational ages have been developed9. Many factors are known to affect the mortality and morbidities of preterm infants. Among them, higher birth weight, higher gestational age, and the use of antenatal steroid have been consistently linked to better prognosis10. However, information on gestation-specific sex effect on outcomes is very limited12. Sex difference in clinical study is important because hormonal, physiological, and developmental differences between males and females could lead to sex-specific outcomes13. Previous studies have shown that the male sex is associated with an increased risk of mortality in preterm infants16. Male infants are also associated with worse respiratory outcomes, such as respiratory distress syndrome and bronchopulmonary dysplasia (BPD), as well as intraventricular hemorrhage (IVH) and retinopathy of prematurity (ROP)20. Later surfactant production and higher density of androgen receptors in male fetus contribute to increased alveolar resistance and the modulation of bronchiole budding in the early fetal lung23, and these effects may continue and worsen the respiratory outcome in males after birth. Recently, a few studies have not found any difference in sex and mortality, due to progress in neonatal intensive care24. Although gestational age is the most powerful factor for neonatal outcomes, only a few studies showed the sex effects, adjusted by gestational age12. Furthermore, as far as we know, there were no sex studies that considered various perinatal factors including antenatal steroid use, maternal diabetes, chorioamnionitis, intrauterine growth restriction (IUGR), and multiple births in preterm infants.Preterm birth is defined by the World Health Organization (WHO) as all births before 37 completed weeks of gestation26, we focused on VLBWIs born at <30 weeks of gestation. We aimed to ascertain sex effect, stratified by gestational age in this population, adjusting for perinatal risk factors that may affect neonatal outcomes. Furthermore, because previous studies have reported that preterm infants born at ≤25 weeks of gestation showed especially higher mortality and morbidities28, we performed a subgroup analysis according to gestational age group, from 23 to 25 weeks of gestation and from 26 to 29 weeks of gestation.The present study is a retrospective observational study based on the Korean Neonatal Network (KNN), which is a nationwide database on VLBWI across South Korea. The aim of the present study was to investigate whether male sex has disadvantage on mortality and short-term morbidities in preterm infants. Because mortality and major morbidities frequently occur at gestational age ≤29 weeks, with lower risk from 30 weeks onwardA total of 2228 VLBWIs  with gestational age of <30 weeks were registered to the KNN during the study period. Severe congenital anomaly was noted in 65 infants . In male infants, there were 6 congenital heart diseases, 4 genitourinary tract defects, 2 central nervous system anomalies, 12 digestive organ anomalies, 1 pulmonary abnormality, 2 chromosomal anomalies, and 5 other anomalies. In female infants, there were 12 congenital heart diseases, 1 genitourinary tract defect, 2 central nervous system anomalies, 10 digestive organ anomalies, 1 pulmonary abnormality, 1 chromosomal anomaly, and 5 other anomalies. The number of male and female infants in the two gestational age groups and at individual gestational age is shown in Fig. P = 0.026), but in the subgroup analysis, this significance disappeared. Overall, male infants had higher birth weight (P < 0.001), height (P < 0.001), and head circumference (P = 0.001) at birth and at discharge . In the subgroup analysis, the 23–25 weeks of gestation group showed that male infants had higher weight and head circumference at birth  and at discharge . In the 26–29 weeks of gestation group, male infants had higher birth weight, height, and head circumference at birth  and at discharge  .The prevalence of maternal diabetes, histologic chorioamnionitis, multiple births, and the use of antenatal steroid were not different between sexes Table . The motP = 0.011). Males also showed a higher occurrence of BPD (P = 0.012), IVH grade ≥3 (P = 0.049), and cystic periventricular leukomalacia (PVL) (P = 0.014) (Table P = 0.038). This OR tended to decrease and then lost significance from 26 weeks of gestation. BPD showed a similar pattern; the OR of males on BPD was significant at 24 and 25 weeks of gestation , and this significance disappeared from 26 weeks of gestation. Males had a significantly higher incidence of PVL at 23 and 29 weeks of gestation . In contrast, males had a lower incidence of ROP at 26 and 27 weeks of gestation . There was no significant sex difference in IVH grade ≥3 and necrotizing enterocolitis (NEC) at any gestation age  and BPD . In the 26–29 weeks of gestation group, female infants had a higher incidence of ROP , and male infants had a higher incidence of PVL   born in Canada and found that the prevalence of BPD for infants born between 24 and 26 weeks were higher in males. Although their study is different from ours in study population and statistics, their finding is generally consistent with the present study. Ito et al.38 stated that male sex was a disadvantage for BPD in preterm infants at ≥26 weeks of gestation by using a 10-year database from the neonatal research network in Japan. As the medical situations and the study period were different in this database, the results seen were different from those in our study. In contrast to Binet et al.10 and Ito et al.38, the present study adjusted the perinatal factors that might affect neonatal outcomes and demonstrated that male sex is an independent risk factor of major morbidities, especially in infants at ≤25 weeks of gestation. Interestingly, in contrast to previous studies40, the present study found that severe ROP occurred more often in female infants after 26 weeks of gestation, and this remains after adjusting for perinatal risk factors including IUGR, maternal hypertension, use of antenatal steroid, and so on. At present, the explanation for the better outcome of ROP in male infants at this gestational period is unclear. Insulin-like growth factor 1 (IGF-1) is critical to normal vascular development, and the duration of low IGF-1 concentrations is strongly correlated with severity of ROP42. Additional investigations for IGF-1 and sex according to gestations are warranted.To date, many studies have confirmed a survival advantage for female infants compared with male infants44.The strength of our study is that it is based on a large, geographical cohort, including approximately 70% of VLBWI in South Korea; therefore, the present study has greater relevance than a single-centered study. Although the present study was a retrospective study, data collection was done prospectively using the same strict guidelines of the KNN. However, we could not evaluate the long-term neurological outcome. Previous studies have demonstrated a correlation between short-term morbidities and long-term neurological outcomesMale VLBWIs born at <30 weeks of gestation have similar incidence of mortality compared with female infants, which remains after adjusting for perinatal risk factors that may affect neonatal outcomes. However, male infants showed increased risk for combined morbidity, BPD, severe IVH, and PVL. Each outcome for males has a specific risk pattern with increasing gestational age. Subgroup analysis showed that the risk for combined morbidities and BPD is higher in males ≤25 weeks of gestation. In contrast, severe ROP occurred more often in females >25 weeks of gestation. Our results showed that sex difference, stratified by gestational age, is a major factor for neonatal outcomes of preterm infants. A guideline on neonatal outcomes that includes male disadvantage, especially for BPD and combined morbidities in infants ≤25 weeks of gestation, will help clinicians’ understanding and parents’ education.+0 and 29+6 weeks of gestation between January 2013 and December 2014 and admitted to a neonatal intensive care unit (NICU) participating in the KNN6. Infants with severe congenital anomaly were excluded. The KNN is a nationwide database on VLBWI from 69 hospitals across South Korea. VLBWIs in these hospitals accounted for approximately 70% of VLBWIs in South Korea. Data were collected at each participating center and entered into a data program, and the study variables were defined according to the KNN manual. The present study was approved by the KNN data management committee.This is a population-based study of VLBWIs born 23The KNN registry was approved by the institutional review board (IRB) at each participating hospital, and informed consent was obtained from the parents at enrollment by the NICUs participating in the KNN. All methods were carried out in accordance with the IRB-approved protocol and in compliance with relevant guidelines and regulations.The primary outcome was gestation-specific sex difference in the occurrence of mortality and the prevalence of combined morbidities among five outcomes and individual morbidity.+0 to 29+6 weeks of gestation. Furthermore, we divided the enrolled infants into two gestational age groups: the 23+0–25+6 weeks of gestation group and the 26+0–29+6 weeks of gestation group.To identify gestation-specific sex outcomes, outcomes were tested at individual gestational age from 23The infants’ data were collected from the KNN database Fig. . Sex, ge2. BPD was defined as a requirement for supplemental oxygen at 36 weeks of postmenstrual age45. IVH was defined using Papile’s criteria by cranial ultrasonography46. Because imaging was conducted using the individual policy of each hospital, the worst grade among several cranial ultrasonography results during hospitalization was adopted. PVL included only cystic PVL revealed by cranial ultrasonography or magnetic resonance imaging which was performed during hospitalization. NEC was defined according to Bell’s criteria (stage 2 or higher)47. ROP was defined according to the international classification of ROP48, and the maximum stage of ROP was adopted. To investigate the prevalence of combined morbidities, the present study developed the variable ‘combined morbidities’, which was defined as having one and more morbidities among the five morbidities. To develop the variable of combined morbidities, the value of each morbidity  was added, and the ‘0’ value in the sum was use to designate no combined morbidities. IUGR was defined as birth weight less than the 10th percentile for the gestational age based on a sex-specific growth chart33.VLBWI was defined infants born <1,500 g according to the WHOχ2 test, and t test was used for continuous variables. Adjusted sex difference in mortality and morbidities was tested by multiple logistic regression. Multiple logistic regression was performed at individual gestational age and then for the two gestational age groups. Criteria for entry and removal were P < 0.05 and P > 0.10, respectively. The fit of models was checked with the Hosmer-Lemeshow goodness-of-fit test49. A P value < 0.05 with two-tailed comparisons was considered significant.Statistical analyses were performed using SPSS. Data are expressed as number (%) with OR and 95% CI or mean (standard deviation). Univariate analyses of categorical variables were performed by The datasets analyzed during the current study are not publicly available due to the policy of Research of Korea Centers for Disease Control and Prevention but are available from the corresponding author on reasonable request.


Capreolus capreolus) in a large sample set of 1312 females (305 yearlings and 1007 adults), hunted throughout Slovenia, central Europe, in the period 2013–2015. Body mass positively affected probability of ovulation and potential litter size (number of corpora lutea), although its effect was more pronounced in yearlings than in adults. Between age groups, we found clear differences in responses of both reproductive parameters to body mass which influences primarily reproductive performance of younger, and in particular, lighter individuals: at the same body mass yearlings would at average have smaller litters than adults, and at lower body mass also young to middle-aged adults would have smaller litters than old ones. In addition, while yearlings have to reach a critical threshold body mass to attain reproductive maturity, adult females are fertile (produce ova) even at low body mass. However, at higher body mass also younger individuals shift their efforts into the reproduction, and after reaching an age-specific threshold the body mass does not have any further effects on the reproductive output of roe deer females. Increased reproductive capacity at more advanced age, combined with declining body mass suggests that old does allocate more of their resources in reproduction than in body condition.We analysed effects of females’ body mass and age on reproductive capacity of European roe deer ( Capreolus capreolus) has one of the largest distribution ranges among wild ungulates and is widespread and abundant almost all over the European continent [The European roe deer . Apart ontinent –8) and dontinent , individontinent , 12–16).Roe deer are the only artiodactyls to display embryonic diapause  which enBody mass affects a number of different components of roe deer reproductive potential: (i) In the first instance body mass is crucial for females to reach reproductive maturity . To reacThe effect of body mass on reproductive potential of roe deer females has been studied in several populations across Europe  [Dama dama) [Mothers might invest more in reproduction as they age , and oldelaphus) , 35 and ma dama) . In roe ma dama) , smallerma dama)  or highema dama) , 39. To ma dama) , this stma dama) , 33) norAll individuals of roe deer used in the study were hunted during the regular hunting activity prescribed by the state of Slovenia within the yearly hunting management plans. We used only tissues of already dead individuals therefore no animal was shot or killed by any other means for the purposes of the research.2) is located in Central Europe  at the junction of four large European geographical units, i.e. the Alps, the Pannonian Basin, the Dinaric Alps, and the Mediterranean region. The climate is diverse, but roughly with a continental climate in the northeast, a severe alpine climate in the high mountain regions, and sub-Mediterranean climate in the coastal region.Reproductive organs of roe deer females were collected in 76 hunting grounds, distributed in 14 hunting management districts continuously throughout Slovenia, covering the whole range of environmental conditions and population characteristics of the species’ distribution in the country. Slovenia . In total, 1896 samples were collected, and 1312 of them were suitable for analysis considering the scope of this paper . Immediately after the cull and dissection, hunters placed uteri into plastic bags and stored them frozen until collection; the lower jaw was also removed and the left half of the mandible retained for age assessment. For each specimen, sampling date, location, eviscerated carcass mass  and age group  were recorded immediately after the hunting episode. Afterwards, samples were defrosted and analysed in the laboratory of the institute ERICo Velenje and at the Slovenian Forestry Institute. To determine the fertility and potential litter size of each female, the presence and number of corpora lutea (CL) were determined by dissection of ovaries. Fertile females were those that had CL in their ovaries, meaning that they were able to reproduce as they ovulated. The number of CL per female was considered as a potential litter size of each individual (see [Reproductive organs (uteri with ovaries) of female roe deer were sampled in the period 2013–2015, within regular hunting operations ..st SepteFor all animals, age was assessed jointly by the first and the last author of the manuscript by macroscopic inspection of tooth development and tooth-wear in mandible samples accompanying each specimen , 44. Duest September to 31st December. As body mass of both yearlings and adults linearly increased with consecutive day in the year, we performed correction of this variable using a General Regression Model , (ii) mean number of CL in fertile individuals , and (iii) synthesis of both of the above parameters–mean number of CL, including also non-fertile individuals . All statistical analyses were performed using the first two parameters , while the third parameter  was used in all graphical presentations.To explore the reproductive potential of roe deer females at the population level, as in several previous studies , we useTo visualize differences in potential reproductive output among females of different ages, we calculated the relative frequencies of the number of CL per doe  for each of the age classes and plotted them in Prior to statistical analyses, we grouped all individuals into three age categories in order to minimize possible errors in the age assessment, taking into account age-specific changes in both body mass and potential reproductive output : yearlinvs. adults × fertile vs. non-fertile; (ii) age class of adults × fertile vs. non-fertile; (iii) yearlings vs. adults × potential litter size; (iv) age class of adults × potential litter size.The effects of age and body mass on reproductive potential were firstly analysed by the chi-square test for homogeneity . We analA similar procedure was used to analyse the effects of body mass on reproductive potential. For this analysis, we grouped all individuals into five body mass classes as follows: <12 kg (n = 106); 12–13.9 kg (n = 263); 14–15.9 kg (n = 395); 16–17.9 kg (n = 351); and >18 kg (n = 197). We analysed the following combinations of variables: (v) body mass × fertility for all individuals; (vi) body mass × fertility for adults; (vii) body mass × potential litter size for all individuals; (viii) body mass × potential litter size for adults. To meet the criteria of minimal theoretical frequency, required by the chi-square test for homogeneity, we pooled the first two body mass classes into one  prior to analysis (vi), and pooled all females with the number of CL = 3 (n = 48), 4 (n = 5) and 5 (n = 1) into one group (CL = 3 or more) prior to analyses (vii) and (viii).vs. non-fertile; binomial error) and potential litter size  as dependent variables and explored differences in the effects of both yearlings vs. adults, and between both age categories of adults. In all GLM analyses in addition to age (fixed factor), the independent variables were body mass (covariate) and the interaction of body mass × age; we also explored the effects of the year of sampling  as a fixed factor. We built all possible models and used the Akaike information criteria (AIC) to select the best and other still informative models with ΔAIC < 2. We displayed parameter estimates and other base statistics only for the best model, while for the other models we showed only the model structure and ΔAIC , but homogeneity tests are not convenient to explore these interactions/effects. Therefore, we also analysed our data with generalized linear models (GLM). As in the homogeneity tests, we used fertility , averaging 94.1% in yearlings, 98.7% in young to middle-aged adults, and 98.0% in older (8+) females, respectively. Among 1312 analysed females, only 30 individuals (2.3%), i.e. 18 yearlings (5.9%) and 12 adults (1.4%), did not ovulate in the year of sampling. A significant increase in fertility was found between yearlings and adults ; Table 2Fertile females carried 1 (19.9%), 2 (75.8%), 3 (3.8%), 4  or 5 CL  . The potBody mass affected fertility and potential litter size. With increasing body mass, there is a clear increase in reproductive potential of roe deer females, which was confirmed both by the test for homogeneity  and the The mean eviscerated body mass of roe deer females depended on the age of the individual; it increased from 13.8 ± 0.3 kg in yearlings to 16.2 ± 0.3 kg in middle-aged females, where it reached its peak. After this age it started to decrease, and females from the oldest age class (elderly does) on average weighed 14.5 ± 0.7 kg. The largest increase in body mass was observed between yearlings and 2-year-old does , confirming that yearlings had still not reached their full body size. While the decrease in body mass was observed in the last two age classes, in the case of reproduction females tend to reach the peak at an older age .Body mass and age had a synergistic effect on the reproductive parameters of roe deer. In yearlings, 50% of animals with eviscerated body mass <10 kg were infertile, and some infertile individuals were also in body mass categories up to 15.9 kg. In contrast, all (6) yearlings with eviscerated body mass >18.0 kg had two CL. There were some very rare individuals which failed to ovulate among adults, primarily in the body mass category up to 11.9 kg. In yearlings and adults, the proportion of females with two or more CL sharply increased with increasing body mass; however, when taking into account the effects of body mass (body mass is constant), there was a higher proportion of adults with two or more CL than yearlings. In adults, 54 does (5.4%) had more than two CL, showing strong body mass dependence: none of them had body mass <12.0 kg and 11% of does with eviscerated body mass >18.0 kg had three or more CL .vs. adults) and the effects of body mass per se, the interaction of body mass × age category also affected both fertility and potential litter size . Although we analysed reproductive capacity of roe deer females before implantation and ovulation per se is relatively low-cost process [In our study, 50% of yearlings with eviscerated body mass <10.0 kg failed to ovulate while all yearlings heavier than 16.0 kg were fertile . Yearlin process , 33, theAlces alces) in which ovulation rate was lower and more variable among primiparous females than in older cows, and at the same body mass prime-aged cows had higher probability to ovulate than yearlings [Rangifer tarandus) [Odocoileus virginianus) [Sus scrofa) [Ovis aries) [The same age-dependent effect of body mass on fertility was observed in moose  , white-tinianus) , wild bo scrofa) –65, and s aries) .Body mass affects ovulation in roe deer females and even stronger the number of offspring that they can produce, which was clearly indicated also in our study. Our data on the effects of maternal body mass on both potential litter size and potential reproductive output fit well with existing findings, i.e. that young, primiparous females have on average smaller litters than adult does (e.g. ), and thOur data indicate that there is an age-dependent shift in reproductive potential and probably also in allocation of resources from body growth into reproduction. While young females  less than middle-aged females . AlthougConsidering the pronounced impact of body mass on reproductive potential of roe deer as well as a high rate of reproduction in early life it may be expected that reproductive potential of roe deer females would decrease toward the end of their life-span. However, our data showed only a slightly decreasing trend of potential reproductive output of very old females which was observed several years after body mass culmination . Actual So far, a study from France (Chizé) showed a marked decrease in fertility among roe deer females, older than 12 years . DecreasWe analysed reproductive capacity of roe deer females in the early stage of pregnancy by counting CL. Since the ovulation is a relatively low-cost process , 33, CL Apart from body mass and age, population density and some environmental factors may also affect reproductive performance of roe deer females . For exS1 Dataset(XLSX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.S1 TableThe explanatory variables are year, age class, day in a year (covariate) and interaction year × age class; model was built using the best subset approach and Mallow’s Cp selection criterion; last level of each categorical variable served as contrast (estimate = 0) for the remaining levels of that variable; study period 2013–2015, n = 1312.(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.