STUDY QUESTION Can antioxidant treatment with N-acetylcysteine (NAC) protect ovarian follicles from ischemia-reperfusion injury in xenotransplanted human ovarian tissue? SUMMARY ANSWER Daily administration of NAC for 7–12 days post-transplantation reduced ischemia-reperfusion injury and increased follicle survival in human ovarian xenografts by upregulating the antioxidant defense system and exerting anti-inflammatory and antiapoptotic effects. WHAT IS KNOWN ALREADY Freezing of human ovarian tissue is performed with high follicular survival rates but up to 70% of follicles appear to be lost due to hypoxia and ischemia-reperfusion injury during ovarian tissue transplantation (OTT). NAC has been demonstrated to possess antioxidant and antiapoptotic properties, and studies in rodents have shown that intraperitoneal administration of NAC reduces ischemia-reperfusion injury and increases follicle survival in autotransplanted murine ovaries. STUDY DESIGN, SIZE, DURATION Pieces of frozen-thawed human ovarian tissue from 28 women aged 23–36 years were transplanted to immunodeficient mice in short- and long-term xenograft studies or cultured in vitro. Three short-term xenograft studies (1-week duration) were performed, in which saline or 150 mg/kg NAC was administered for 7 days post-transplantation (n = 12 patients per group). Two long-term xenograft studies (4 weeks of duration) were performed. In one of these studies, saline or 150 mg/kg NAC was administered for 12 days (n = 12 patients per group), while in the other study 50, 150 or 300 mg/kg NAC was administered for 7 days (n = 8 patients per group). In addition, human ovarian tissue (n = 12 pieces from three patients per group) was cultured with increasing concentrations of NAC (0, 5, 25 and 75 mM) for 4 days in vitro. PARTICIPANTS/MATERIALS, SETTING, METHODS Donated ovarian tissue was obtained from women who had undergone ovarian tissue cryopreservation for fertility preservation at the University Hospital of Copenhagen. Cortical tissue pieces (5 × 5 × 1 mm) were transplanted subcutaneously to immunodeficient mice and NAC or saline was injected intraperitoneally. Grafts were retrieved after 1 or 4 weeks and follicle density was assessed. Gene expression analysis of antioxidant defense markers (superoxide dismutase; Sod1/SOD1, heme oxygenase-1; Hmox1/HMOX1, catalase; Cat/CAT), proinflammatory cytokines (tumor necrosis factor-alpha; Tnf-α, interleukin-1-beta; Il1-β, interleukin 6; Il6), apoptotic factors (B-cell lymphoma 2; Bcl2/BCL2, Bcl-2-associated X protein; Bax/BAX) and angiogenic factors (vascular endothelial growth factor A; Vegfa/VEGFA, angiopoietin-like 4; Angptl4/ANGPTL4) was performed in 1-week-old human ovarian xenografts and in cultured human ovarian tissue. Grafts retrieved after 4 weeks were histologically processed and analyzed for vascularization by CD31 immunohistochemical staining, fibrosis by Masson’s Trichrome staining and apoptosis by immunofluorescence using cleaved caspase-3. MAIN RESULTS AND THE ROLE OF CHANCE After 1-week grafting, the relative expression of Sod1, Hmox1 and Cat was significantly higher in the group receiving 150 mg/kg NAC (NAC150-treated group) compared to controls (P = 0.04, P = 0.03, and P = 0.01, respectively), whereas the expression levels of Tnf-α, Il1-β and Il6 were reduced. The Bax/Bcl2 ratio was also significantly reduced in the NAC150-treated group (P < 0.005). In vitro, the relative gene expression of SOD1, HMOX1 and CAT increased significantly in the human ovarian tissue with increasing concentrations of NAC (P < 0.001 for all genes). However, the expression of VEGFA and ANGPTL4 as well as the BAX/BCL2 ratio decreased significantly with increasing concentrations of NAC (P < 0.02, P < 0.001 and P < 0.001, respectively). After 4-week grafting, fibrosis measured by collagen content was similar in the NAC150-treated group compared to controls (control: 56.6% ± 2.2; NAC150: 57.6% ± 1.8), whereas a statistically significant reduction in the CD31-positive vessel area was found (control: 0.69% ± 0.08; NAC150: 0.51% ± 0.07; P < 0.02). Furthermore, a reduced immunoreactivity of cleaved caspase-3 was observed in follicles of the NAC150-treated xenografts compared to controls. Follicle density (follicles/mm3, mean ± SD) was higher in the NAC150-treated group compared to the control group in the 1-week xenografts (control: 19.5 ± 26.3; NAC150: 34.2 ± 53.5) and 4-week xenografts (control: 9.3 ± 11.0; NAC150: 14.4 ± 15.0). Overall, a 2-fold increase in follicle density was observed in the NAC150-group after 1-week grafting where fold changes in follicle density were calculated in relation to grafts from the same patient. Around a 5-fold increase in follicle density was observed in the NAC150 and NAC300 groups after 4-week grafting. LARGE SCALE DATA N/A LIMITATIONS, REASONS FOR CAUTION Follicle density in the human ovarian cortex is highly heterogeneous and can vary 100-fold between cortex pieces from the same woman. A high variability in follicle density within and between treatment groups and patients was found in the current study. Thus, solid conclusions cannot be made. While intraperitoneal injections of NAC appeared to reduce ischemia-reperfusion injury in human ovarian xenografts, different administration routes should be investigated in order to optimize NAC for potential clinical use. WIDER IMPLICATIONS OF THE FINDINGS This is the first study to demonstrate the antioxidant, anti-inflammatory and antiapoptotic properties of NAC in xenotransplanted human ovarian tissue. Therefore, NAC appears to be a promising candidate for protecting ovarian follicles from ischemia-reperfusion injury. This provides the initial steps toward clinical application of NAC, which could potentially reduce the loss of ovarian follicles following OTT. STUDY FUNDING/COMPETING INTEREST(S) We are grateful to the Danish Childhood Cancer Foundation, Hørslev Foundation, Aase and Einar Danielsen’s Foundation (grant number: 10-001999), Dagmar Marshalls Foundation, Else and Mogens Wedell-Wedellsborgs Foundation, Knud and Edith Eriksens Mindefond, and Fabrikant Einar Willumsens Mindelegat for funding this study. None of the authors have any competing interests to declare.

IMPORTANCEBecause the incidence of pediatric-onset inflammatory bowel disease (IBD) is increasing, knowledge of the long-term risk of cancer in this patient population is required. OBJECTIVE To evaluate the relative rate of cancer among patients with pediatric-onset IBD. DATA SOURCES A comprehensive systematic search was performed of MEDLINE and Embase from the date of database inception to October 31, 2021. STUDY SELECTION All unselected, population-based cohort studies of pediatric-onset IBD assessing the risk of cancer were included. Tertiary center referrals and insurance database studies were excluded. All articles were assessed by 2 independent reviewers. DATA EXTRACTION AND SYNTHESISThe study followed the Preferred Reporting Items for Systematic Reviews and Meta-analyses reporting guideline for data extraction and used the Newcastle-Ottawa Scale for assessment of the risk of bias and the quality of included articles. MAIN OUTCOMES AND MEASURESA random-effects model meta-analysis was conducted of included studies using the inverse-variance method to assess the relative rate of cancer overall and by IBD subtype (Crohn disease or ulcerative colitis), sex, and thiopurine exposure among patients with pediatric-onset IBD. Pooled relative rates (pRRs) along with 95% CIs were calculated for combined studies. RESULTSOf 4628 articles screened, 5 population-based studies from North America and Europe were eligible for inclusion. These studies comprised 19 812 individuals with pediatric-onset IBD followed up for 283 540 person-years in which 715 cases of cancer were identified. Meta-analysis of pRR estimates showed a 2.4-fold increased rate of cancer among patients with pediatric-onset IBD (pRR, 2.46; 95% CI, 2.06-2.93), seen among patients with Crohn disease (pRR, 2.03; 95% CI, 1.67-2.46) and those with ulcerative colitis (pRR, 2.61; 95% CI, 2.00-3.40). This increased rate is primarily due to an increased rate of liver (

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Aim Patients with Crohn's disease (CD) often suffer from perianal fistulizing disease. Their risk of anorectal cancer remains uncertain. We aimed to examine the long‐term risk of anorectal cancer in a population‐based cohort of CD patients with anorectal fistula. Method Our study population covered all individuals (n = 7 987 520) aged 15+ years living in Denmark from 1978 to 2018. We identified all patients with CD and anorectal fistula in the Danish National Patient Register (NPR) and 50 matched noninflammatory bowel disease (IBD) individuals from the general population. Using Cox regression analyses, we examined the risk of anorectal cancer in CD fistula patients versus non‐IBD individuals. All patients with CD were identified using codes from the International Classification of Diseases and their data extracted from the NPR. The main outcome measure was cases of anorectal cancer. Results A total of 2786 CD patients with anorectal fistula and 139 300 non‐IBD individuals were followed for 1 553 917 person‐years. During follow‐up, anorectal cancer was observed in 19 CD patients (0.68%) and 340 non‐IBD individuals (0.24%), corresponding to a 2.9‐fold increased hazard ratio (HR) of anorectal cancer in CD fistula patients (95% CI 1.80–4.53), with a particularly high risk of anal cancer (HR 15.13, 95% CI 6.88–33.31) and a mean time from CD fistula diagnosis to anorectal cancer of 6.7 (SD 6.5) years. The risk was slightly higher in women than men and had no apparent relation to treatment with tumour necrosis factor‐α inhibitors. Sensitivity analyses using CD nonfistula patients for comparison revealed similar results. Individual data on smoking and infection with human papilloma virus were not available. Conclusion Patients with CD and anorectal fistula have a three‐fold increased risk of anorectal cancer compared with the general population. The number needed to surveil to detect one case of anorectal cancer in this patient population was 2160 patients per year in patients with long‐standing fistula (>6 years).