GPX4 Expression After Kono-S vs Conventional Anastomosis

Postoperative recurrence of Crohn's disease frequently occurs at or near the ileocolic anastomosis.1 Increasing evidence suggests that oxidative stress plays a central role in anastomotic healing, fibrosis and mucosal recurrence. Surgical injury triggers neutrophil recruitment and reactive oxygen species (ROS) production, which can initiate lipid peroxidation within intestinal epithelial membranes.3 Lipid peroxidation products such as malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) activate inflammatory and fibrotic signalling pathways including mitogen-activated protein kinase (MAPK), nuclear factor-κB (NF-κB) and transforming growth factor- β (TGF-β).2,4 GPX4 is a key antioxidant enzyme that detoxifies lipid peroxides and prevents ferroptosis, a regulated form of oxidative cell death.5 Reduced GPX4 activity has been described in inflammatory bowel disease and may contribute to epithelial injury and disease progression.5 Recent results have demonstrated that a reduced GPX4 expression at time of surgery predicts disease recurrence after ileocolonic resection independent of established clinical risk factors (e.g., smoking, penetrating disease, sex and age). Despite the recognised importance of oxidative stress in intestinal inflammation, its role at the surgical anastomosis in Crohn's disease has not been directly studied.

The Kono-S anastomosis is a functional end-to-end, antimesenteric configuration designed to reduce luminal distortion and faecal stasis while excluding mesenteric inflammatory influence. Early studies suggested reduced endoscopic recurrence compared with conventional anastomoses, although long-term clinical results remain mixed.6 The biological mechanisms underlying any benefit remain poorly understood.

The investigators hypothesise that the Kono-S anastomotic configuration may reduce local oxidative stress at the anastomotic site by limiting reactive oxygen species exposure and subsequent lipid peroxidation, as reflected by lower levels of markers such as malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) compared with conventional techniques. This more favourable redox environment may support preservation of epithelial antioxidant defence mechanisms, including maintenance of glutathione peroxidase-4 (GPX4) expression and activity, thereby reducing susceptibility of intestinal epithelial cells to ferroptosis. In turn, reduced lipid peroxidation may attenuate activation of key pro-inflammatory and pro-fibrotic signalling pathways, including MAPK, NF-κB and TGF-β, with potential downstream effects on mucosal inflammation and fibrotic remodelling.4 Overall, these mechanisms may contribute to improved anastomotic healing and a lower risk of early endoscopic recurrence.

Inclusion criteria

• Adults (≥18 years) with confirmed Crohn's disease who underwent ileocolic or small bowel resection with a primary anastomosis.

• Documented anastomotic configuration (Kono-S or conventional).
• Availability of archived pathology material from the resection specimen containing the anastomotic site.
• At least one additional tissue timepoint available: preoperative endoscopic biopsy or follow up endoscopic biopsy within 24 months.
• Available postoperative endoscopic follow up data (Rutgeerts score) where applicable.

Exclusion criteria

• • Indeterminate colitis or non-Crohn's pathology on histology.

• Lack of retrievable tissue or poor tissue quality (e.g., extensive necrosis or autolysis) precluding IHC.
• Unclear anastomotic type.
• Absence of epithelial component in the available tissue (e.g., only serosa or fibrofatty tissue).
• Major confounders that could affect GPX4 expression, such as severe ischaemia, uncontrolled sepsis, or immediate reoperation at the anastomotic site.
• Patients with permanent stomas and no anastomotic follow up where interpretation would be ambiguous.