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

Introduction

Azoospermia is diagnosed when two or more semen analyses, followed by centrifugation, show a complete absence of spermatozoa in the ejaculate (1). It affects approximately 15% of infertile men and can be classified into obstructive azoospermia (OA) and non-obstructive azoospermia (NOA) (2). OA, which occurs in 40% of azoospermia cases, is caused by physical blockage anywhere between the rete testis and the ejaculatory ducts, while exocrine and endocrine functions, as well as normal spermatogenesis, remain preserved (2-4). A higher proportion of patients are diagnosed with NOA, the most severe type of male infertility, which is a result of testicular failure caused by congenital or acquired conditions (5). The most common causes are genetic causes, which include Klinefelter syndrome, Y chromosome microdeletions, single-gene mutations, as well as non-genetic causes such as cryptorchidism and varicocele; however, the majority of cases remain idiopathic (6).

Oxidative stress (OS) plays a significant role in the pathophysiology of male infertility (7). Spermatozoa are highly vulnerable to OS because of their high polyunsaturated fatty acids (PUFAs) content in the membrane (8) and the limited presence of cytoplasmic scavenging enzymes (9). In the male reproductive tract, the glutathione peroxidase-glutathione reductase system is a primary mechanism for removing reactive oxygen species (ROS) and repairing oxidative damage (10). Meanwhile, lipoxygenases (ALOXs) are non-heme, iron-dependent dioxygenases that promote lipid peroxidation of PUFAs (11). Maintaining a balanced expression of arachidonate 15-lipoxygenase (ALOX15) and glutathione peroxidase 4 (GPX4) is crucial for achieving intracellular redox balance (12,13). When the defense mechanisms of normal sperm cells fail, an imbalance develops between ROS production and total antioxidant capacity (TAC). Elevated ROS levels initiate a series of events that lead to plasma membrane lipid peroxidation.

The accumulation of lipid peroxidation products is considered a key execution step in ferroptosis, an evolutionarily conserved form of programmed cell death (14), characterised by the catalytic action of ferrous ions and the loss of antioxidant capacity of the lipid repair enzyme GPX4 (15). Ferroptosis has been implicated in pathological cell death linked to degenerative diseases (16-18) and carcinogenesis (19-21). New evidence suggests that ferroptosis plays a key role in the pathophysiology of the testis (22). Bromfield et al. were the first to explore the mechanisms of ferroptosis in the male germline lineage. Using developing germ cells from the mouse as the experimental model, they confirmed caspase-independent cell death following exposure to OS conditions and reciprocal upregulation of ALOX15 and downregulation of GPX4 protein expression (23). Recent studies confirm the role of ferroptosis in testicular injury (24-26).

Nevertheless, research on ferroptosis in human testicular tissue is still limited; most of the studies are based on animal models or involve the use of mature human spermatozoa obtained through ejaculation (27-29).

This study aims to investigate whether expression levels of ALOX15 and GPX4 differ between patients with OA and NOA patients with successful sperm retrieval (SSR+), and across NOA subgroups categorized as mild, moderate, and severe hypospermatogenesis (HS). We present this article in accordance with the STROBE reporting checklist (available at https://tau.amegroups.com/article/view/10.21037/tau-2025-1-1005/rc).

Methods

Study population

This was a single-center cross-sectional study. The patients were referred for their first testicular sperm extraction (TESE) as part of fertility treatment at the Polyclinic CITO, Women’s Health Clinic in Split, Croatia. Testicular tissue samples were obtained from 72 adult male patients who were medically and legally capable of providing informed consent and were diagnosed with azoospermia, confirmed by semen analyses of at least two samples over six months, including verification of centrifuge precipitates (1). To distinguish OA from NOA, all participants underwent a comprehensive physical examination, ultrasound imaging, semen analysis [according to World Health Organization (WHO) criteria] (1), karyotype analysis, Y-chromosome microdeletions analysis, and hormonal analysis (30,31). The inclusion criteria for the OA group were clinical diagnosis of obstruction, histologically normal spermatogenesis, normal hormonal profile, and normal-sized testes. For both OA and NOA groups, testis size was measured using ultrasound imaging. The NOA group, comprised of patients with severe spermatogenic deficiency as a consequence of primary testicular failure (PTF). It included patients with normal or low testosterone, elevated FSH, small testicular size, and no identifiable obstructive causes on examination and imaging. Patients with a history of chemotherapy or radiotherapy were excluded to avoid introducing treatment-induced gonadotoxic injury as a distinct pathophysiological entity, thereby ensuring a more biologically homogeneous cohort with intrinsic PTF. Patients with hypogonadism or without spermatozoa in the ejaculate due to specific therapies were also omitted from the study. As endocrine tests do not always distinguish with certainty normal (OA) from profoundly impaired spermatogenesis (NOA), testicular biopsy was a key investigation for defining the status of spermatogenesis (32). Patients who met the inclusion criteria for OA were referred to conventional TESE (cTESE), and for retrieving sperm from men with NOA, microdissection sperm extraction (microTESE) was performed. Classification of spermatogenesis in histological biopsy was described according to McLachlan et al. (32). Patients with SSR+ were divided into two groups. The OA group (N=14) comprised men with an obstructive etiology and preserved spermatogenesis: congenital bilateral absence of the vas deferens (CBAVD) (N=2), aspermia (N=2), epididymal cysts (N=1), epididymitis (N=3), prostatovesiculitis (N=1), ejaculatory duct obstruction (EDO) (N=2), hydrocele (N=1), no clear site of obstruction (N=1), and vasectomy (N=1). The NOA group (N=31) was further categorized by severity of spermatogenic impairment into mild (N=11), moderate (N=12), and severe (N=8) HS. The quantitative degree of HS was defined in the same manner as previously determined by Hessel et al. (33). Mild HS was defined as >60% of seminiferous tubules containing complete spermatogenesis, moderate HS as 30-60% of seminiferous tubules containing complete spermatogenesis, and severe HS as <30% of seminiferous tubules containing complete spermatogenesis. Twenty-seven men with unsuccessful sperm retrieval (SSR−) were diagnosed with: Sertoli-cell only phenotype (SCO) (N=16), germ cell arrest at the primary spermatocyte stage (N=4), germ cell arrest at spermatogonia stage (N=4), and complete hyalinization of seminiferous tubules (N=3) and were excluded from the study because documented patterns of ALOX15 and GPX4 in the human testis (https://www.proteinatlas.org/) show that ALOX15 protein expression is absent in Sertoli cells, spermatogonia, and spermatocytes and is limited to haploid germ cells, while GPX4 shows negative to very weak positivity in Sertoli cells, and strong positivity is expressed in both haploid cells and pachytene spermatocytes. We explored publicly available databases [Human Cell Atlas-type references, UniProt, and Gene Expression Omnibus (GEO) datasets]; however, these resources did not provide testis-specific protein localization data with the same level of histological resolution as the Human Protein Atlas (HPA). Because our study focuses on cell-type-specific protein expression within seminiferous tubules, HPA was selected as the most appropriate reference database. The expression of ALOX15 and GPX4 in male germ cells was also confirmed by several important studies in humans and mice (24-27,34,35). The flow diagram outlining the grouping criteria for the patients included in our study is presented in Figure 1.

Figure 1 Flow diagram illustrating the grouping criteria for the patients included in our study. HS, hypospermatogenesis; NOA, non-obstructive azoospermia; OA, obstructive-azoospermia; TESE, testicular sperm extraction.

Parameters such as age, body mass index (BMI), testicular volume, hormonal profile, genetic evaluation, smoking habits, comorbidities, and previous surgeries were collected from clinical reports. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Ethics Committee of University of Split School of Medicine on May 10, 2020 (No. 003-08/20-03 I 0005), and all patients provided written informed consent.

Surgical procedure and tissue processing

cTESE was performed on patients with OA. A small incision (or multiple incisions) was made in the tunica albuginea at a location of the surgeon’s choice. The testis was squeezed to extrude tubules, and the biopsy specimen(s) were obtained using a pair of surgical scissors (36). In NOA patients, the microTESE procedure for direct microscopic identification of functioning seminiferous tubules was performed as previously described (37). A small incision was made in the scrotal skin, and the testicles were delivered from the vaginal tunica. Avascular regions of the upper, middle, and lower anterior surfaces of the tunica albuginea were incised over 0.5–1 cm. The testicular parenchyma was examined at higher magnification (×20) to identify typically dilated and more opaque seminiferous tubules. Small amounts of testicular tissue, including these tubules, were extracted by traction and sharply excised. If no morphologically normal tubules were found, the incision was extended to expose multiple areas, and any tubules that differed in size or large tissue strips where all tubules appeared identical morphologically were removed. The same surgeon performed all procedures. After harvesting, testicular tissue samples were transferred to 3-(N-morpholino) propane sulfonic acid (MOPS) buffer and examined by a skilled embryologist. Sperm retrieval was considered successful if at least one viable spermatozoon was microscopically visualized at ×200 magnification.

Each testicular biopsy specimen (3×3×3 mm³ per patient) was dissected into two fragments using microsurgical scissors. One fragment was fixed in Bouin’s solution (Sigma-Aldrich, St. Louis, USA) and embedded in paraffin for subsequent histological analysis and immunofluorescence, and the other was cryopreserved for use in medically assisted fertilization procedures.

Histologic procedures

Testicular biopsy fragments were fixed in Bouin’s solution (24 h, 4 °C), paraffin-embedded, and sectioned. 6-µm-thick sections were then deparaffinized, rehydrated, stained with hematoxylin and eosin (H&E), dehydrated, and mounted. Microscopy images were captured using a Nikon DS-U3 imaging system. On average, three sections and 35 seminiferous tubules per section were analyzed for each sample. Classification of spermatogenesis in histological biopsy specimens was described according to McLachlan et al. (32). Samples were classified as OA patients (N=14), showing normal spermatogenesis, and as NOA patients (N=31), classified as HS, exhibiting various levels of spermatogenic impairment. The quantitative degree of HS was defined in the same manner as previously determined by Hessel et al. (33). The prevalence of mild, moderate, and severe HS is presented in Figure 1.

Immunohistochemistry

Immunohistochemistry for ALOX15 and GPX4 protein localization in human testicular tissue was performed by deparaffinizing and rehydrating 6-µm-thick sections of the same testicular tissue fragments used for histological evaluation. ALOX15 and GPX4 were evaluated in separate staining experiments, so direct co-localization could not be assessed. The sections were then immersed in 10 mM citrate buffer (pH 6.0), heated at 120 °C for 7 minutes, cooled, and washed in phosphate-buffered saline (PBS). After encircling the tissue with a PAP pen and exposing it to blocking serum (4% goat serum) at 37 °C for 30 minutes, the sections were incubated with rabbit anti-ALOX15 polyclonal IgG (1:100; Sigma-Aldrich, St. Louis, MO, USA) and rabbit anti-GPX4 monoclonal IgG (1:100; Abcam, Cambridge, MA, USA) antibodies at 4 °C for 24 hours. Coverslips were washed (2× 5 minutes) in PBS before applying goat polyclonal secondary anti-rabbit IgG H&L (Alexa Fluor 488) (1:100, Abcam, Cambridge, MA, USA). Details on primary and secondary antibodies used are presented in Table 1. After a final wash in PBS, the slides were mounted with Shandon Immuno-Mount (Thermo Fisher Scientific, Boston, MA, USA) and visualized under an Olympus BX51 fluorescence microscope using appropriate filters to selectively detect green fluorescence, with an Olympus DP71 digital camera. Negative controls were prepared using the same procedure as the positive control, except that the primary antibody was replaced with PBS solution.

The primary outcome measures were the number of ALOX15-positive germ cells per 1 mm² of seminiferous tubule surface (N/mm²) and the percentage (%) of GPX4-positive surface area relative to the tubule surface area, as measured using the ImageJ program. For each patient, one slide containing three testicular tissue sections for ALOX15 and one for GPX4 was selected for the immunodetection, and 12 seminiferous tubules were assessed. Tubules lacking germ cells were excluded from the analysis.

Statistical analysis

Statistical analysis was performed using GraphPad Prism 6 (GraphPad Software, San Diego, CA, USA). The t-test was used to compare the means of continuous numerical variables with a normal distribution. The Mann-Whitney non-parametric test was used to compare two groups, and the Kruskal-Wallis test was used to compare three or more independent groups. To test differences between any two groups, we used a Dunn’s multiple comparison post-hoc test. A simple chi-square test was used to assess the association between two categorical variables. In all cases, differences were considered significant at P<0.05. Data were expressed as mean ± standard deviation (SD).

Results

Clinical characteristics

The overall sperm retrieval rate following the TESE procedure was 62.5% (45/72). Among patients with SSR+, 14 belonged to the OA group and 31 to the NOA group (Figure 1). The histological features of the tubules in OA patients and in the different stages of the NOA group are shown in Figure 2. As expected, men with NOA had significantly higher levels of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) and considerably reduced testicular volume compared to those with OA (Table 2). Clinical parameters associated with SSR+ showed that a prior history of cryptozoospermia was more prevalent in the SSR+ group compared to the SSR− group (P≤0.001). Additionally, more patients in the SSR+ group had varicocele (P=0.002); meanwhile, significantly more patients from the SSR- group had allergies (P=0.02, Table S1).

Figure 2 Categories based on the severity of spermatogenic impairment. (A) Seminiferous tubules from patients with OA showing normal spermatogenesis; (B) tubules showing mild hypospermatogenesis; (C) tubules showing moderate hypospermatogenesis; (D) tubules showing severe hypospermatogenesis. Insets show higher-magnification views of the corresponding tubules. H&E staining; scale bar =20 µm. H&E, hematoxylin-eosin; OA, obstructive-azoospermia.

ALOX15 and GPX4 protein expression

Testicular tissue from 14 OA patients with normal spermatogenesis and 31 NOA patients with SSR+ classified as mild, moderate, and severe HS was used to analyze ALOX15 and GPX4 expression. 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. Dunn’s multiple comparison test revealed no significant difference (P<0.05) in ALOX15 expression levels between normal spermatogenesis and mild HS, as well as between moderate and severe HS. However, both normal spermatogenesis and mild HS showed significant differences when compared to moderate and severe HS (Figure 3A). Levels of expression of GPX4 did not differ between normal spermatogenesis and mild HS, nor between mild and moderate HS. However, in severe HS, GPX4 expression was significantly lower than in both normal spermatogenesis and mild HS (Figure 3B). Histological classification and immunofluorescence of ALOX15 and GPX4 in human testicular tissue of patients with OA and NOA are presented in Figure 4.

Figure 3 ALOX15 (A) and GPX4 (B) protein expression levels in testicular tissue samples of patients with OA exhibiting normal spermatogenesis and across NOA subgroups categorized as mild, moderate, and severe HS. The expression levels of ALOX15 and GPX4 protein localization in human testicular tissue were visualized under an Olympus BX51 fluorescence microscope and measured by the ImageJ program. The ALOX15 expression was significantly lower in moderate and severe HS, when compared to both normal spermatogenesis and mild HS. Significantly lower GPX4 expression was observed in patients with severe HS compared to both normal spermatogenesis and mild HS. The Kruskal-Wallis with a Dunn’s multiple comparison post-hoc test was performed to compare differences between any two groups. Each bar displays mean ± standard deviation. *, P<0.05. ALOX15, arachidonate 15-lipoxygenase; GPX4, glutathione peroxidase 4; HS, hypospermatogenesis; NOA, non-obstructive azoospermia; OA, obstructive azoospermia.

Figure 4 Histological classification and immunofluorescence of ALOX15 and GPX4 in human testicular tissue of patients with OA and NOA. Classification of spermatogenesis in histological biopsy was described according to McLachlan et al. (32). The quantitative degree of HS was defined in the same manner as previously determined by Hessel et al. (33). (A) Normal spermatogenesis in a patient with OA; (B) ALOX15 expression in a patient with normal spermatogenesis; (C) GPX4 expression in a patient with normal spermatogenesis; (D) mild HS in a patient with NOA; (E) ALOX15 expression in a patient mild HS; (F) GPX4 expression in a patient with mild HS; (G) moderate HS in patient with NOA; (H) ALOX15 in a patient with moderate HS; (I) GPX4 in a patient with moderate HS; (J) severe HS in a patient with NOA; (K) ALOX15 in a patient with severe HS; (L) GPX4 in a patient with severe HS. H&E black scale bar =20 µm; immunofluorescence white scale bar =50 µm. ALOX15, arachidonate 15-lipoxygenase; GPX4, glutathione peroxidase 4; H&E, hematoxylin-eosin; HS, hypospermatogenesis; NOA, non-obstructive azoospermia; OA, obstructive azoospermia.

Association of clinical parameters with expression levels of ALOX15 and GPX4

No significant differences were observed in the expression levels of ALOX15 and GPX4 when analyzed by age group (<35 vs. >35 years; ALOX15: P=0.77, GPX4: P=0.717). Similarly, expression levels remained comparable across BMI categories [healthy weight (18.5–24.9 kg/m²); overweight (25.0–29.9 kg/m²); and obese (30.0 or higher kg/m²)], and between smokers and non-smokers (ALOX15: P>0.99, GPX4: P=0.6). The association of clinical parameters with ALOX15 and GPX4 expression levels is presented in Table S2.

Expression levels of ALOX15 and GPX4 in NOA patients with testis-related pathologies and comorbidities unrelated to the reproductive system

We observed no significant differences in ALOX15 and GPX4 expression when comparing patients with common testis-related pathologies such as varicocele (ALOX15: P=0.98; GPX4: P=0.79) or cryptorchidism (ALOX15: P=0.56; GPX4: P=0.6) to those without these conditions (Table S2). In contrast, patients presenting with non-reproductive comorbidities (including cardiovascular, kidney/urinary tract, epilepsy, allergies, and gastrointestinal diseases) demonstrated significantly elevated GPX4 expression (P=0.04), whereas ALOX15 expression was not significantly altered (P=0.054, Table S2).

Discussion

Our study revealed distinct patterns in the expression levels of ALOX15 and GPX4 between OA and NOA, with both enzymes showing higher expression in the OA group than in the NOA group. The results showed that ALOX15 and GPX4 enzyme expression levels declined in a consistent, gradual manner with increasing severity of spermatogenic impairment.

The highest levels of ALOX15 expression were observed in patients with normal spermatogenesis and mild HS, and significantly lower in those with moderate and severe HS. Nonetheless, expression of ALOX15 remained present in all phenotypes, emphasizing its stable role in spermatogenic pathways regardless of impairment level (38-40).

Similar GPX4 expression levels were observed in OA and NOA patients with mild and moderate HS, suggesting that, despite a lower percentage of functional tubules exhibiting spermatogenesis, the antioxidant capacity of functional testicular tubules in these NOA cases may be equally preserved. To confirm such a finding, additional studies with a larger sample size are needed. In contrast, a significantly lower expression was observed in severe HS, suggesting a possible link between decreased GPX4 levels and the severity of spermatogenic impairment. However, reduced ALOX15 and GPX4 expression observed in severe HS may partly reflect a decreased number of germ cells expressing these proteins, rather than a direct demonstration of altered OS activity. This finding is consistent with a study reporting that GPX4 expression at both the RNA and protein levels decreased across various idiopathic NOA subtypes, along with a reduction in germ cell types (26). Aside from the reduced activity of GPX4, which may indicate testicular failure caused by ferroptosis triggered by the loss of GPX4’s phospholipid peroxidase activity (14), the authors also observed significantly higher levels of ferrous ions and malondialdehyde (MDA), a marker of lipid peroxidation, in idiopathic NOA samples compared to the control group (26). Another study also found an association between GPX4 and the pathological alterations in the azoospermic testis. Although the sample size was small, it was shown that in testes with sloughing and disorganization of germ cells, GPX4 expression had disappeared (25).

The present study was designed primarily as a mechanistic analysis of ferroptosis-related protein expression across varying degrees of HS in patients with SSR+, rather than as a predictive clinical biomarker study. Patients with maturation arrest were few in number and, therefore, not included in subgroup analyses due to insufficient statistical power. Sertoli cell-only syndrome was excluded because the absence of germ cells precludes biologically meaningful assessment of ALOX15 and GPX4 expression, which are predominantly germ cell-associated proteins. Larger studies, including all NOA subtypes, will be necessary to determine whether these markers have predictive value for clinical outcomes, such as sperm retrieval success.

Additionally, we observed that GPX4 levels were higher in patients with non-reproductive comorbidities. Poor general health status has been associated with dysfunction in the male reproductive system (41) and medical comorbidities like hypertension, diabetes, and kidney disease with increased OS that could potentially lead to additional damage to distant tissues (42,43). However, a large-scale study conducted in Japan revealed that treating medical comorbidities in infertile men led to a significant improvement in sperm parameters (44), which may help clarify the elevated GPX4 levels observed among patients with non-reproductive comorbidities in our study. We also observed the expression levels of ALOX15 and GPX4 in patients with varicocele and cryptorchidism, the most common testis-related pathologies included in this study. Despite the number of studies indicating that spermatogenic dysfunction, common to both varicocele (45-47) and cryptorchidism-associated infertility (48), has been linked to OS, we did not find any difference between these patients. Expression levels of ALOX15 and GPX4 also remained similar between smokers and non-smokers, despite the well-known harmful effect of tobacco smoking on male fertility (49,50). The lack of significant differences in smokers or patients with varicocele may, similarly to the reduced ALOX15 and GPX4 expression seen in severe HS, reflect the cellular composition of the analyzed tissue rather than the overall oxidative status, since our measurements were limited to germ cell-containing tubules. Although varicocele has been strongly associated with increased OS and lipid peroxidation, most varicocele cases in our cohort were mild or moderate, and the two severe cases had undergone varicocelectomy prior to tissue sampling. This could have reduced the oxidative burden at the time of sampling, as several studies have suggested (46,51-53). Furthermore, OS in varicocele is influenced by additional factors such as smoking and systemic comorbidities. Together with the small sample size and clinical heterogeneity in varicocele grade and prior treatment, these factors may explain the absence of detectable differences in ALOX15 and GPX4 expression in our cohort.

ALOX15 has been proposed as a potential biomarker of sperm pathophysiology and a promising target for preventing OS (27,51,54). Pharmacological inhibition of ALOX15 decreased 4HNE levels under OS in human spermatozoa, resulting in significantly improved sperm motility, sperm-egg recognition, and the ability to complete the acrosome reaction (27). However, extensive research is needed to understand the physiological role of lipoxygenase enzymes in both developing and mature germ cells, before considering ALOX15 inhibition as a strategy to prevent OS-related pathologies in the testis.

Several limitations of this study should be acknowledged. First, the sample size was relatively small, particularly within specific NOA subgroups, which may limit statistical power and generalizability. Second, only patients with SSR+ were included in the main immunohistochemical analysis, which may introduce selection bias and limit applicability to the broader NOA population. Third, due to the cross-sectional design, causal relationships between ALOX15/GPX4 expression and spermatogenic impairment cannot be established. Additionally, reduced protein expression observed by immunohistochemistry in severe HS may reflect a reduced number of germ cells expressing these markers rather than altered per-cell expression. Finally, we did not directly measure OS biomarkers in tissue or sperm; therefore, conclusions regarding OS involvement remain indirect. Future prospective studies should include parallel, contemporaneous functional OS assessment (in freshly processed tissue and/or sperm) together with protein expression analyses to better link molecular expression patterns to OS status.

Conclusions

In conclusion, this study provides evidence of ALOX15 and GPX4 expression in human testicular tissue from men with OA and NOA. Enzyme expression levels varied according to the severity of spermatogenic impairment, with the highest levels observed in normal and mildly impaired tissue, and the lowest in severely affected seminiferous epithelium. The consistent expression of GPX4 in patients with mild and moderate HS, compared to those with normal spermatogenesis, suggests that the antioxidant capacity in their seminiferous epithelium was preserved, highlighting the crucial role of GPX4 in maintaining cell membrane integrity and preventing cell death.

ALOX15 and GPX4 protein expression analyses provide insights into spermatogenic health and the effects of systemic conditions on testicular function.

Acknowledgments

We would like to thank all staff of the Department of Anatomy, Histology, and Embryology, as well as the Women’s Health Clinic, Polyclinic Cito, Split, Croatia, for providing access to the necessary infrastructure for this research project.

Footnote

Reporting Checklist: The authors have completed the STROBE reporting checklist. Available at https://tau.amegroups.com/article/view/10.21037/tau-2025-1-1005/rc

Data Sharing Statement: Available at https://tau.amegroups.com/article/view/10.21037/tau-2025-1-1005/dss

Peer Review File: Available at https://tau.amegroups.com/article/view/10.21037/tau-2025-1-1005/prf

Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tau.amegroups.com/article/view/10.21037/tau-2025-1-1005/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study received approval from the Ethics Committee of University of Split School of Medicine on May 10, 2020 (No. 003-08/20-03 I 0005). All patients provided written informed consent.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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