Differential expression of ALOX15 and GPX4 in the testicular tissue of men with obstructive and non-obstructive azoospermia: a cross-sectional study.

Background: Ferroptosis is an iron-dependent form of non-apoptotic cell death. Oxidative stress (OS), accumulation of ferrous ions, and lipid peroxidation products are key features that induce ferroptosis. This study examined the levels of arachidonate 15-lipoxygenase (ALOX15), which promotes lipid peroxidation, and glutathione peroxidase 4 (GPX4), a vital antioxidant enzyme, in testicular tissue from patients with obstructive azoospermia (OA) and across non-obstructive azoospermia (NOA) subgroups categorized into mild, moderate, and severe hypospermatogenesis (HS). Methods: Testicular tissue from 72 azoospermic men undergoing testicular sperm extraction (TESE) for fertility treatment was collected and histologically evaluated. Patients with successful sperm retrieval (SSR+) were included in further immunohistochemistry analysis. The expression of ALOX15 and GPX4 was assessed by immunofluorescence microscopy. The primary outcome measures were the number of ALOX15-positive germ cells per 1 mm Results: The overall sperm retrieval rate was 62.5% (45/72). Patients with SSR+ were divided into the OA group (N=14) and the NOA group (N=31), showing mild (N=11), moderate (N=12), and severe (N=8) HS. The expression of both ALOX15 (P=0.004) and GPX4 (P=0.004) was significantly higher in the OA group than in the NOA group. The expression of ALOX15 was significantly lower in moderate and severe HS compared to OA and mild HS. GPX4 expression levels were similar between patients with OA and those with mild and moderate HS. In contrast, significantly lower GPX4 expression was detected in patients with severe HS compared to both OA and mild HS. Additionally, patients diagnosed with comorbidities unrelated to the reproductive system exhibited higher GPX4 expression (P=0.04) than those without. Conclusions: ALOX15 and GPX4 enzyme levels decreased gradually with increasing severity of spermatogenic impairment. Similar GPX4 expression levels between OA and NOA with mild and moderate HS suggest relative preservation of GPX4-positive germ cells in these groups. In contrast, reduced expression observed in severe HS may reflect the diminished presence of advanced germ cells that physiologically express GPX4. Therefore, our findings demonstrate an association between enzyme expression and spermatogenic severity, but do not establish a direct causal link to OS activity.