P-069 The expression patterns of ALOX15 and GPX4 in the seminiferous epithelium of men with non-obstructive azoospermia (NOA) due to primary testicular failure
Authors: Romac L , Sapunar D , Romac P , Schwenner-Radovniković J , Maretić M 
Published: July 2024

Abstract

Study question
Does the expression of ALOX15 and GPX4 differ in NOA men with a history of cryptozoospermia from NOA who never had sperm in their ejaculate?

Summary answer
There was no significant difference between these 2 groups of patients.

What is known already
Oxidative stress (OS) has been investigated as a prime suspect for many unexplained pathologies, including primary testicular failure (PTF). The fact that male germ cells are rich in polyunsaturated fatty acids (PUFAs) but have a limited amount of cytoplasmic scavenging enzymes, is the reason behind their extreme vulnerability to OS. The accumulation of lipid peroxidation markers leads to the inactivation of glutathione peroxidase 4 (GPX4), the lipid repair enzyme, and the exacerbation of arachidonate 15-lipoxygenase (ALOX15), a lipoxygenase enzyme that promotes lipid degradation. This study investigates the activity of these antagonizing enzymes in the testicular tissue of NOA patients.

Study design, size, duration
This observational study was conducted in the period from May 2020, following the Ethics Committee of the University of Split School of Medicine approval, to October 2023. The testicular tissue from 57 patients diagnosed with NOA (according to WHO criteria) and referred to their first microTESE as a part of fertility treatment was collected and processed.

Participants/materials, setting, methods
Samples were prepared for histological evaluation and immunodetection (fixation; embedding in paraffin; H&E staining; and staining with anti-ALOX15 and anti-GPX4 antibodies). Two groups of NOA patients were observed: Crypto-NOA and NOA group. The main outcome measure was the number of ALOX15 positive germ cells per 1 mm² of the seminiferous tubule surface (N/ mm²) and the percentage (%) of GPX4 positive surface per surface of the tubule measured by the ImageJ program.

Main results and the role of chance
Spermatozoa were retrieved in 37 of 57 (64,91 %) men; samples of 4 patients had to be excluded due to poor fixation. There were 18 patients in the Crypto-NOA group, and 10 in the NOA group, while 5 patients were diagnosed with obstructive azoospermia (OA) and were used as controls.
For comparison of the baseline data of Crypto-NOA and NOA patients, we used the Mann-Whitney test, and with statistical significance set at the level of P &lt; 0.05, we found that there were significantly more patients with cryptorchism in the NOA group.
As a tool for histological evaluation, we used the diagnostic code designed by McLachlan et al. and found similar spermatogenic phenotypes between Crypto NOA and NOA patients. The most represented phenotype was hypospermatogenesis with a moderate reduction in late spermatid number mixed with normal tubules (38,8 % in Crypto-NOA; 40% in NOA), following the phenotype where late spermatids are present in some tubules but mixed with tubules showing no progression past the spermatocyte stage.
Using the Mann-Whitney test, we did not find a significant difference in the expression of ALOX15 and GPX4 in the seminiferous epithelium of Crypto-NOA and NOA patients.

Limitations, reasons for caution
The testicular tissue of NOA patients is characterized by focal or patchy spermatogenesis which led to the exclusion of several samples due to the fixation of tissue with a homogenous pattern of Sertoli cell-only or germ-cell arrest.

Wider implications of the findings
This study will provide additional information regarding the cause of impaired spermatogenesis and help to determine if the patient is a potential candidate for antioxidant therapy. Our next step is to associate the expression patterns of ALOX15 and GPX4 with the success rates of in vitro fertilization procedure outcomes.

Trial registration number
not applicable