Flow cytometry sperm apoptosis test can predict sperm DNA fragmentation and can be used as an effective and easy-to-perform screening method for males from infertile couples

Introduction

Conventional sperm analysis has become the gold standard for male fertility assessment. The microscopic examination of fresh human semen is performed either manually or by a computer [computer-assisted sperm analysis (CASA)] (1). However, since a limited spectrum of parameters is being evaluated, some sperm pathologies might be missed during this examination. In other words, normal conventional sperm analysis parameters do not guarantee male fertility (2)—approximately 15% of normozoospermic men were shown to have lower sperm quality resulting in poor embryo quality (3). Conversely, as emphasized by the World Health Organization (WHO) (1), abnormalities in basic semen parameters are not synonymous with infertility.

In 2021, the 6^th edition of the WHO laboratory manual for the examination and processing of human semen (1) was published. This latest edition, in comparison to the previous ones (4), introduced a range of extended and advanced methods for more accurate semen evaluation, including sperm DNA fragmentation, genetic and genomic tests, tests related to immunology and immunological methods, assessment of interleukins, assessment of immature germ cells in the ejaculate, testing for antibody coating of spermatozoa, biochemical assays for accessory sex gland function, assessment of sequence of ejaculation, seminal oxidative stress and reactive oxygen species (ROS) testing, assessment of the acrosome reaction, assessment of sperm chromatin, and finally transmembrane ion flux and transport in sperm (1). While most of the methods are currently intended for research purposes and their use in clinical practice has not been standardized, sperm DNA fragmentation is regarded as the most precise indicator of the sperm potential to fertilize an oocyte both naturally and by assisted reproductive technologies (ART). Moreover, sperm DNA damage results in impaired embryo development (5), miscarriage, recurrent pregnancy loss (6,7), and birth defects (5).

It should be noted, however, that due to the lack of high-quality data and the heterogeneity of assays and cut-off values (8), several controversies persist (9). These include the ambiguous impact of test results on the final outcome in terms of live-birth rate (10), the uncertain effectiveness of the proposed therapeutic interventions (11), the varying effects across different ART methods (12-14), and, finally, inconsistent recommendations among expert societies (15).

The European Association of Urology (EAU) guidelines recommend determination of sperm DNA fragmentation in couples with recurrent pregnancy loss from natural conception and failure of ART or in men with unexplained infertility (strength rating: strong) (16).

Since the 1980s, several methods to measure DNA fragmentation have been developed. Sperm DNA defects, both single- and double-strand breaks, might be measured indirectly by sperm chromatin structure assay (SCSA) and sperm chromatin dispersion test (SCD; also called “Halo Test”), or directly by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL), and the alkaline comet test (COMET). While most data come from SCSA, which is currently the most commonly used technique to assess sperm DNA fragmentation (17), TUNEL is regarded as the most precise method (18). Irrespective of which method is used, sperm DNA fragmentation testing requires special technical equipment and chemicals together with skilled laboratory staff, and is usually time-consuming and costly (19). The combination of all these factors may negatively affect the availability of the examination to both physicians and their patients.

In our IVF centre, we introduced a flow cytometry sperm apoptosis test (FACS-APO) as a simple and inexpensive screening method to select patients who may benefit from a subsequent sperm DNA fragmentation test, which is currently not covered by the Czech public health care insurance. Here we describe the methods and results of this technique.

The primary aim of this study was to assess whether the FACS-APO can predict sperm DNA fragmentation as measured by the TUNEL assay. The secondary aims were (i) to evaluate correlations between sperm DNA fragmentation and conventional sperm analysis variables, and (ii) to develop and validate a simple prediction model that could serve as a clinical screening tool to identify males at higher risk of elevated sperm DNA fragmentation. We present this article in accordance with the STROBE reporting checklist (available at https://tau.amegroups.com/article/view/10.21037/tau-2026-0639/rc).

Methods

Experimental group

This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The requirement for ethics committee approval was waived by the Ethics Committee of IVF Clinic GENNET, given the retrospective and anonymized nature of the data obtained during routine clinical care. All participants provided written informed consent for the use of their anonymized clinical data for statistical analysis and publication.

We retrospectively analysed data from 688 men (age 15–66 years, mean 30.1 years, standard deviation 10.8 years) who, in the period from January 2019 to December 2024, underwent 971 TUNEL sperm DNA fragmentation tests performed together with FACS-APO and conventional sperm analysis as part of standard diagnostic work-up at IVF Clinic GENNET, Prague, Czech Republic. All men who underwent concurrent TUNEL, FACS-APO, and conventional sperm analysis testing during the study period were included in the analysis; no tests were excluded because of technical failure or incomplete measurement.

Among 688 men, three groups can be distinguished:

• 515 males from infertile couples referred to an assisted reproduction centre for evaluation of infertility, defined as the inability to achieve pregnancy after 12 months of regular unprotected intercourse (or earlier in women over 35 years of age) (age 23–66 years, mean 30.2 years; 564 samples);
• 42 males diagnosed with testicular cancer, with samples taken before and after oncological treatment (age 19–46 years, mean 32.1 years; 68 samples); and
• 131 adolescents—87 males diagnosed with clinically significant varicocele (diagnosed by physical examination; EAU grade classification was not systematically recorded) and 44 healthy age-matched volunteers without known urogenital pathology, not actively attempting conception, and with no chronic or systemic disease (age 15–20 years, mean 17.3 years; 339 samples).

The latter two groups (42 testicular cancer patients and 131 adolescents) were not recruited for the present retrospective analysis. They were examined as part of two separate, prospectively conducted research projects—one on sperm bioenergetics in patients with testicular germ cell tumours, and another on sperm functional variables in adolescents with varicocele (20,21).

The examinations were performed at the Laboratory for Immunology and the Laboratory for Embryology, respectively, both belonging to IVF Clinic GENNET, Prague, Czech Republic.

Conventional sperm analysis

Conventional sperm analyses were performed in the Laboratory for Embryology, IVF Clinic GENNET, Prague, Czech Republic. Semen samples were collected by masturbation after 2–7 days of sexual abstinence. Initially, the semen samples were weighed to establish precise sample volume. Physical characteristics such as macroscopic appearance, liquefaction, viscosity, odour, and pH were noted if abnormal. A 10 μL aliquot of each semen sample was loaded onto a Makler sperm counting chamber for determining sperm concentration, motility, and morphology assessment. The samples were assessed according to the 5^th (until August 2021) and the 6^th (from September 2021) editions of the WHO laboratory manual for the examination and processing of human semen, respectively. The semen samples were then transported to the Laboratory for Immunology, IVF Clinic GENNET, Prague, Czech Republic, for FACS-APO and sperm DNA fragmentation testing. FACS-APO analysis was performed within 1 hour after ejaculation. Semen samples for the TUNEL test were diluted in a phosphate-buffered saline (PBS) solution and cryopreserved at −20 ℃ according to standard WHO procedures for a maximum of 1 week prior to analysis.

FACS-APO

ApoFlowEx Kit (Exbio, Prague, Czech Republic) was used to detect phosphatidylserine externalization. All reagents were prepared according to the manufacturer’s instructions. 25 µL of sperm suspension was washed twice in PBS for 5 min at 150 g and then re-suspended in 1 mL Annexin V Binding Buffer (supplemented with Ca²⁺). 100 µL of sperm suspension was stained by Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI). After 15 min of incubation in the dark, the cells were diluted with binding buffer and analysed by the flow cytometer.

Analysis of sperm DNA fragmentation

Sperm DNA fragmentation was evaluated using the TUNEL assay as described earlier (7,17). All reagents were used according to the manufacturer’s instructions. Thawed aliquot of semen containing 3–5 million spermatozoa was washed in PBS (Sigma-Aldrich, St. Louis, MO, USA) for 5 minutes at 150 g. The sample fixation was performed by re-suspension in 1 mL of Intracellular Staining Perm Wash Buffer (BioLegend®) containing 3.7% paraformaldehyde and incubated for 30 minutes at 4 ℃. Paraformaldehyde was removed by centrifugation for 5 minutes at 150 g. Afterwards, the sample was washed twice with PBS for 5 minutes at 150 g. Subsequently, the sample was diluted in ice-cold 70% ethanol and incubated for at least 30 minutes at 4 ℃. ApoDirectTM Plus kit (Phoenix Flow Systems) was used to detect sperm DNA fragmentation.

The sample was centrifuged for 5 minutes at 300 g to remove ethanol. Then the sample was washed twice in Wash buffer for 5 minutes at 300 g. The sperm pellet was resuspended in 50 µL of freshly prepared staining solution and then incubated for 60 minutes at 37 ℃. The staining solution contained terminal deoxynucleotidyl transferase (TdT) enzyme, TdT reaction buffer, FITC-tagged deoxyuridine triphosphate nucleotides (FITC-dUTP), and distilled water. After incubation, the sample was washed in the Rinse buffer for 5 minutes at 300 g. Afterwards, the pellet was resuspended in 0.5 mL of the ready-to-use PI/RNase Staining Buffer supplied with the ApoDirectTM Plus kit and then analysed by flow cytometry after 30 minutes of incubation at room temperature (20–25 ℃) in the dark. The exact PI concentration in this buffer is proprietary and not disclosed by the manufacturer. Additionally, a semen sample without the TdT enzyme (negative control) was used as a control of system validity.

Flow cytometry analyses

The FACS-APO and TUNEL tests were performed using the Navios flow cytometer (Beckman Coulter, Inc., Fullerton, CA, USA). The analyses were performed using the Kaluza software. Approximately 10,000 events were examined for each assay at a flow rate of 200–250 events per second. The sperm population was gated based on the measurement of forward scatter (FS) and side scatter (SS) to exclude electronic noise and debris.

The gating strategy is illustrated in Figures 1,2. The gating was based on granularity and size of the particles, respectively (Figures 1A,2A). In the next step, based on the area and height, the so-called singlets (single, non-aggregated cells passing individually through the flow cytometer, producing one clean signal per cell) were selected to exclude sperm aggregates (Figure 2B).

Figure 1 The FACS-APO analysis of a semen sample. The gating strategy used for sperm selection is shown for three different samples. Gating was based on the granularity and size of the particles (A). Based on the area and height, the singlets were selected to exclude sperm aggregates (not shown). FITC-labelled Annexin V-positive spermatozoa were measured in the FL1 channel (x axis), and PI-labelled spermatozoa were measured in the FL2 channel (y axis) of the flow cytometer (B). Cells were divided into three sperm populations: viable sperm (negative for both dyes; black), early apoptotic sperm (positive for Annexin V only; blue), and late apoptotic or necrotic sperm (positive for PI; red). The first sample in Panel B shows a high proportion of viable sperm; the second sample shows a low proportion of viable sperm and a high proportion of early apoptotic sperm; and the third sample shows a low proportion of viable sperm and a high proportion of late apoptotic or necrotic sperm. FACS-APO, flow cytometry sperm apoptosis test; FITC, fluorescein isothiocyanate; FS, forward scatter; INT, integral; PI, propidium iodide; SS, side scatter.

Figure 2 Sperm DNA Fragmentation Analysis. The gating strategy used for sperm selection is shown for three samples. Gating was based on the granularity and size of the particles (A). FITC-labelled spermatozoa dUTP-positive in the TUNEL assay were measured in the FL1 channel (y-axis), and PI-labelled spermatozoa were measured in the FL4 channel (x-axis) of the flow cytometer. In the next step, based on area and granularity, singlets were selected to exclude sperm aggregates (B). Fluorescence data were acquired using fixed gate settings in the logarithmic mode. (C) Objects were selected according to the main peak of PI fluorescence. The threshold for negativity was set according to the dUTP fluorescence of the negative control. Three samples were analysed: the first was a negative control—sample without the TdT enzyme (C1); the second was the same sample treated with the TdT enzyme, showing a normal proportion of sperm with DNA fragmentation (C2); and the third sample exhibited a higher proportion of sperm with DNA fragmentation (C3). dUTP, deoxyuridine triphosphate nucleotide; FITC, fluorescein isothiocyanate; FS, forward scatter; INT, integral; PI, propidium iodide; SS, side scatter; TdT, terminal deoxynucleotidyl transferase; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labelling.

The FITC-labelled Annexin V-positive spermatozoa in FACS-APO test were measured in the FL1 channel, and the PI-labelled spermatozoa in the FL2 channel of the flow cytometer, respectively. Based on green and red fluorescence, cells were divided into three sperm populations: viable sperm (negative for both dyes), early apoptotic sperm (positive for Annexin V only), and late apoptotic or necrotic sperm (positive for PI), as seen in Figure 1.

The FITC-labelled dUTP-positive spermatozoa in the TUNEL test were measured in the FL1 channel, and the PI-labelled spermatozoa were measured in the FL4 channel of the flow cytometer, respectively. Fluorescent data were obtained at a fixed gain setting in logarithmic mode. The objects were selected according to the main peak of PI fluorescence as seen in Figure 2. The cut-off for negativity was established based on the dUTP fluorescence of the negative control.

No specific sperm markers were used to distinguish spermatozoa from other cell types (e.g., leukocytes or, in general, “round cells”). In samples with normal sperm concentrations (>16 million cells/mL), other cell types represented only an insignificant minority, and the use of such markers was, therefore, unnecessary.

In semen samples with elevated leukocyte concentrations, leukocytes were visualized in the FACS-APO analysis as a distinct cell population and excluded during data processing (Figure 3). When such a population was detected, the sample quality was verified by optical microscopy and, if required, the leukocyte concentration in the semen sample was determined using an anti-CD45 antibody. Leukocytes could thus be easily excluded from the analysis, as shown in Figure 3. Based on our laboratory experience, this correction was necessary in only a minority of samples, affecting approximately 1% of all analysed specimens.

Figure 3 In samples with elevated leukocyte concentrations (left; in this sample sperm concentration was 107 mil/mL, and leukocyte concentration was 5.3 mil/mL), these cells were visualized as a typical distinct cell population (middle; green). The cytometric finding of a significant amount of non-sperm cells was subsequently verified by optical microscopy. The exclusion of leukocytes did not markedly alter the final values (right). FITC, fluorescein isothiocyanate; FS, forward scatter; INT, integral; PI, propidium iodide; SS, side scatter.

Statistical analysis

Data distribution was assessed using the Shapiro-Wilk test and visual inspection of Q-Q plots. As none of the continuous variables followed a normal distribution, non-parametric tests were applied throughout. Correlation between continuous variables was computed using the non-parametric Spearman correlation coefficient. Differences in continuous variables among groups were assessed by the non-parametric Kruskal-Wallis test. The Kruskal-Wallis test compared the medians of the samples in the groups and returned a P value for the null hypothesis that all samples are drawn from populations with the same distribution. The Kruskal-Wallis test is a non-parametric version of the classical one-way ANOVA, and an extension of the Wilcoxon rank sum test for more than two groups (22). For prediction of sperm DNA fragmentation, a standard linear regression model was used. All tests were performed at the 0.05 level of significance. The statistical analysis and data visualisation were performed using MATLAB and Statistics Toolbox Release 7.5.0.342 (R2007b), The MathWorks, Inc., Natick, Massachusetts, United States.

Given the exploratory nature of the correlation analyses and the fact that several tested associations represent overlapping aspects of the same underlying relationship (sperm DNA fragmentation vs. sperm viability/apoptosis), no correction for multiple comparisons was applied. All reported associations remained significant after a post-hoc Bonferroni correction (α=0.05/19≈0.0026), with the exception of the weak correlation between sperm DNA fragmentation and early apoptotic sperm (ρ=0.10, P=0.002), which remained significant only narrowly.

When visualizing the data, many data points would lie on a line segment because the age of men was rounded to whole years. To avoid this, a small random perturbation (mean =0, standard deviation =0.2 years) was added to the age of the men. This augmentation was used only in visualization, not in the analyses. All 971 samples had complete data for the variables included in this analysis; no imputation was required. No additional sensitivity analyses were performed.

Results

The results of the sperm apoptosis test correlate with the sperm DNA fragmentation assay

In total, 971 samples from 688 males were examined concurrently for TUNEL sperm DNA fragmentation, FACS-APO, and conventional sperm parameters. A significant but weak correlation was observed between sperm DNA fragmentation and age (Figure 4), which is in line with previous studies (23).

Figure 4 Visualisation of the 971 sperm DNA fragmentation results from 688 males. Each dot represents a unique measurement: 564 samples from 515 infertile males—blue; 68 samples from 42 males diagnosed with testicular cancer—red; 339 samples from 87 adolescents diagnosed with clinically significant varicocele and 44 healthy volunteers—green. A small perturbation was added to age to prevent the dots from overlapping. The correlation between age and sperm DNA fragmentation was weak (ρ=0.35) but significant (P<0.001).

A significant correlation was found between the percentage of sperm DNA fragmentation and the percentage of late apoptotic/necrotic sperm (ρ=0.38, P<0.001, Figure 5A), and a significant negative correlation was found between the percentage of sperm DNA fragmentation and the percentage of viable sperm (ρ=−0.39, P<0.001, Figure 5B). The correlation between the percentage of sperm DNA fragmentation and the percentage of early apoptotic sperm was weak, albeit significant (ρ=0.10, P=0.002, Figure 5C).

Figure 5 Correlation between sperm DNA fragmentation and sperm apoptosis parameters. There is a significant correlation (ρ=0.38, P<0.001) between the percentage of sperm DNA fragmentation and the percentage of late apoptotic/necrotic sperm (mean 41.9%; A). There is a significant negative correlation (ρ=−0.39, P<0.001) between the percentage of sperm DNA fragmentation and the percentage of viable sperm (mean 44.3%; B). There is a weak, albeit significant correlation (ρ=0.10, P=0.002) between the percentage of sperm DNA fragmentation and the percentage of early apoptotic sperm (mean 13.9%; C).

Figure 5A,5B reveal an important relation between sperm DNA fragmentation and the sperm apoptosis parameters. Both panels show a triangular pattern. That means that for a low fraction of viable sperm and for a high fraction of late apoptotic/necrotic sperm, respectively, any percentage of sperm DNA fragmentation is possible, however, for a high fraction of viable sperm and for a low fraction of late apoptotic/necrotic sperm, respectively, sperm DNA fragmentation is necessarily low. This observation will be important in the section “Sperm DNA fragmentation can be predicted from FACS-APO and age”.

Conventional sperm analysis parameters correlated with sperm DNA fragmentation

Conventional sperm analysis parameters including sperm concentration, total sperm count, and progressive motility were tested for correlation with sperm DNA fragmentation. A significant correlation was found in all the mentioned parameters (P<0.001; Figure 6). The limits were set (in accordance with the 6^th edition of WHO laboratory manual for the examination and processing of human semen as the 5^th and 50^th percentiles, respectively.

Figure 6 Sperm DNA fragmentation differed significantly among the predefined groups based on sperm concentration (A), total sperm count (B), and progressive motility (C). Pairwise comparisons revealed that each group differed significantly from the other two (P<0.001).

Sperm DNA fragmentation can be predicted from FACS-APO and age

Several regression models were tested to predict the percentage of sperm DNA fragmentation based on conventional sperm analysis parameters and the results of FACS-APO. Ultimately, the best predictor was a simple linear model that predicted the percentage of sperm DNA fragmentation from age and the percentage of viable sperm as follows:

DNAfrag=23+0.32×age−0.35×viablesperm[1]

where “DNA frag” stands for the percentage of sperm DNA fragmentation, “age” stands for the man’s age, and “viable sperm” stands for the percentage of viable sperm. Thus, sperm DNA fragmentation increases with age (10 years of age add 3 percentage points to sperm DNA fragmentation) and decreases with the percentage of viable sperm (a 10 percentage-points increase in viable sperm means approximately a 3.5 percentage-points decrease in sperm DNA fragmentation). The model can be illustrated with a simple nomogram (Figure 7).

Figure 7 A simple nomogram illustrating the linear model. The blue lines connect regions with the same predicted values of sperm DNA fragmentation. The blue numbers above the lines indicate the predicted percentage of sperm DNA fragmentation. The blue filled region in the top left corner of the diagram indicates the impossible region of DNA fragmentation less than 0%.

Table 1 presents the calculated percentages of viable sperm for different ages corresponding to 20% sperm DNA fragmentation, i.e., cut-off values for the abnormal sperm DNA fragmentation results. The presented prediction model is visualized in Figure 8.

Figure 8 Visualisation of the prediction model. The diagonal line indicates perfect prediction (y = x). Each dot represents one sample.

A total of 971 examinations were performed, and the prediction based on a DNA fragmentation cut-off value of 20% yielded the following results: 165 true positives, 158 false positives, 543 true negatives, and 105 false negatives. These values correspond to the following diagnostic accuracy measures: sensitivity 61.1%, specificity 77.5%, positive predictive value (PPV) 51.1%, and negative predictive value (NPV) 83.8%. The high NPV is consistent with the findings shown in Figure 5, indicating that this test represents a promising screening tool for identifying men at risk of high sperm DNA fragmentation.

Discussion

Apoptosis, or programmed cell death, is an irreversible and evolutionarily conserved process by which all cells in multicellular organisms physiologically terminate their life cycle (24). Dysregulation of apoptosis may lead to cell immortality and, eventually, to the development of cancer or autoimmune diseases (25). In the urogenital tract, apoptosis plays a pivotal role both in ontogenetic development to ensure the correct ratio between germinal and Sertoli cells (26), and in adulthood during spermatogenesis to eliminate defective cells—up to 75% of germ cells are eliminated to ensure proper quality control (27). However, if apoptosis is activated in the ejaculated sperm, it impairs the quality of the semen, and thus the ability to fertilize the oocyte (28,29).

Generally, apoptosis can be initiated by two different pathways—either by the mitochondrial (intrinsic) pathway triggered by nutrient deprivation, stress, or DNA damage, or by the death receptor (extrinsic) pathway activated by death receptors and death ligands. Both pathways lead ultimately to the activation of protease enzymes called caspases—in the extrinsic pathway directly, or in the intrinsic pathway through permeabilization of the outer mitochondrial membrane initiated by pro-apoptotic “BH-3 only” proteins belonging to the Bcl-2 protein family (30). This leads to the leak of cytochrome c and other substances from mitochondria to the cytoplasm, which in turn enables the formation of endonucleases that cleave DNA in a typical “laddered” manner (25). Thus, DNA fragmentation, or DNA damage in general, is regarded as both a cause and a consequence of apoptosis.

It should be noted, however, that some researchers have questioned the applicability of the classical mitochondrial apoptotic pathway to spermatozoa, arguing that in male germ cells the physical separation of the nucleus from the midpiece containing mitochondria, together with the highly condensed sperm chromatin, may prevent caspases and nucleases activated during apoptosis from triggering sperm nuclear DNA fragmentation. This issue remains debated and represents an important caveat when interpreting FACS-APO findings in the context of DNA damage (31).

An additional mechanism that has gained increasing attention is the concept of “abortive apoptosis”, whereby the apoptotic cascade is initiated but not completed in ejaculated sperm, resulting in cells that exhibit apoptotic markers (such as phosphatidylserine externalization) yet retain some degree of membrane integrity. This phenomenon is currently considered one of the accepted hypotheses explaining the origin of sperm DNA fragmentation, alongside oxidative stress and errors in chromatin remodeling during spermiogenesis (31).

Unlike necrosis, apoptosis is not accompanied by inflammation, and, thus, the manifestation of the early phases of apoptosis is usually very subtle and cannot be observed by native microscopy. One of the early signs of initiated apoptosis is the exposure of the phosphatidylserine molecule on the outer cell surface, which gives a unique possibility to use this feature as a reliable marker for early apoptotic cells. Late phases of apoptosis comprise cell shrinking, nuclear and DNA fragmentation, and production of apoptotic bodies (25). Moreover, professional and non-professional phagocytes remove apoptotic cells by efferocytosis, during which anti-inflammatory cytokines are produced and inflammatory reaction is actively inhibited (32).

Several factors have been identified to cause sperm DNA damage. These include aging (33,34), hormonal dysbalances (35), urogenital infections (36), varicocele (37,38), impact of various gonadotoxic agents in a patient’s history, and poor lifestyle including smoking (39) and unhealthy diet (40). If no factor can be identified during examination, professional or environmental exposure to so far unknown endocrine disruptors is expected (41). It is also assumed that ROS are the common denominator for most of the above-mentioned causes (42).

In comparison to other cells, sperm are very sensitive to ROS (43). This vulnerability is given by several mechanisms accounting for (I) low capacity to repair DNA damage (44); (II) high proportion of polyunsaturated fatty acids in the membranes which makes them susceptible to lipid peroxidation and, consequently, leads to the loss of membrane integrity (45); and (III) limited availability of antioxidant systems such as superoxide dismutase and catalase (46). In addition, mitochondria are essential for energy production necessary for sperm motility, but they produce a high amount of ROS as by-products.

The integrity of sperm DNA is vital for physiological fertilisation, embryo development, and successful implantation (47). The chance of spontaneous conception declines as sperm DNA fragmentation exceeds 20% and values higher than 30–40% (measured by TUNEL) are associated with nearly zero chance of conception after ART (48).

Males with high levels of sperm DNA fragmentation should undergo a thorough diagnostic workup to identify a possible underlying cause. Reversible causes, as mentioned above, should be treated accordingly. If no clear cause can be identified and high levels of sperm DNA fragmentation persist despite empiric antioxidant treatment, various techniques enabling the selection or harvest of sperm with lower sperm DNA damage might be offered to the infertile couple. The precise sperm selection methods account for microfluidic sperm sorting (MFSS) and magnetic-activated sperm sorting (MACS), based on the ability of sperm to pass through a chip containing a complex artificial labyrinth mimicking the female genital tract (49-51), or on removing abnormal sperm after their labelling with magnetic particles (52). Since testicular sperm usually have lower sperm DNA damage than ejaculated sperm, testicular sperm extraction (TESE) followed by immediate intracytoplasmic sperm injection (ICSI) might also be an option. However, more randomized controlled trials are needed to support this approach (14).

Under ideal conditions, the sperm DNA fragmentation test would be performed as a standard examination together with conventional sperm analysis in all males to assess their fertile potential (19). However, it is neither technically nor economically feasible to perform sperm DNA fragmentation testing in all patients, since it requires special technical equipment and chemicals together with skilled laboratory staff, and is usually time-consuming and costly – particularly for flow-cytometry-based methods such as TUNEL, which is considered the most precise direct assay. Alternative methods such as the SCD test, recommended by WHO (1), require only a standard light microscope; however, their per-sample reagent cost is comparably high and their diagnostic accuracy may be lower than that of TUNEL. For those reasons, there remains the crucial question of how to distinguish males who might benefit from advanced sperm DNA testing from those in whom the benefit is unlikely. In our laboratory, we perform an inexpensive (in our setting, 25 € compared with 140 € for the TUNEL-based sperm DNA fragmentation test), accurate, and easy-to-perform flow-cytometry-based apoptosis test as a standard examination of males from infertile couples. FACS-APO can be performed in any laboratory already equipped with a flow cytometer, which is standard infrastructure in assisted reproduction centres. Based on the results, we select the subgroup of those at higher risk of elevated sperm DNA fragmentation. This information is extremely valuable in couples with unexplained infertility and/or in males with normal conventional sperm analysis values.

To our knowledge, this is the first comparison of sperm DNA fragmentation measured directly by TUNEL with sperm apoptosis and morphological parameters in such a large cohort of samples.

The present study found a significant correlation between the percentage of sperm DNA fragmentation and the percentage of late apoptotic/necrotic sperm (ρ=0.38, P<0.001) and a significant negative correlation between the percentage of sperm DNA fragmentation and the percentage of viable sperm (ρ=−0.39, P<0.001).

In addition, we found significantly higher sperm DNA fragmentation in patients with a low sperm concentration (<16 mil/mL), low total sperm count (<39 mil sperm), and progressive motility (<30%). This agrees with the results of previous studies, which found a strong and significant negative correlation between sperm DNA fragmentation on the one hand and total sperm count (53) and sperm concentration on the other (54).

By proper setting of cut-offs, data obtained from FACS-APO together with age may serve for accurate prediction of sperm DNA fragmentation value and could be, thus, used as a simple screening method in both infertile males and even apparently healthy young sperm donors to select the individuals who may benefit from further sperm DNA fragmentation test. Given its high NPV but modest PPV, FACS-APO is best used as a rule-out screening step—a normal (low-apoptosis) result reliably identifies men who can safely omit TUNEL testing—rather than as a stand-alone diagnostic predictor of high sperm DNA fragmentation.

Limitations

Several limitations of the present study should be acknowledged. First, the study cohort is heterogeneous, comprising three clinically distinct groups (males from infertile couples, males with testicular cancer, and adolescents with varicocele or healthy volunteers). While this heterogeneity enhances the generalizability of the FACS-APO method across different clinical settings, it limits the direct applicability of the prediction model to any single population. Future studies should validate the model separately in homogeneous clinical cohorts. Second, TUNEL testing was not performed routinely in all men evaluated for infertility. It was recommended by the treating physician in men considered at higher risk of elevated sperm DNA fragmentation, but since the test is not covered by public health insurance in the Czech Republic, only men willing to pay out-of-pocket ultimately underwent it. This may have introduced a selection bias toward men perceived as higher-risk and/or with greater financial means, limiting the representativeness of the clinical cohort. Third, varicocele grading was performed clinically but was not systematically recorded, which precludes a subgroup analysis by grade. Fourth, the prediction model was developed and validated in the same single-centre dataset; external validation in independent cohorts is required before clinical implementation. Fifth, the FACS-APO test requires access to a flow cytometer, which, while standard in assisted reproduction centres, may not be available in all andrology laboratories.

Conclusions

Based on our results and the results of previous studies, conventional sperm analysis is not sufficient to reliably differentiate between fertile and infertile males. Sperm DNA fragmentation has become an evidence-based tool to assess the real fertility potential of a male. However, direct assays of sperm DNA fragmentation—particularly the most precise flow-cytometry-based TUNEL method—are expensive, time-consuming, and require specialized equipment and trained staff, and therefore have not become a standard part of routine examination. We suggest that an inexpensive and easy-to-perform FACS-APO—performed on the same flow cytometer already used in assisted reproduction laboratories—can be used as a first-choice screening method to identify males at higher risk of elevated sperm DNA fragmentation. Those identified as high-risk may then be referred for confirmatory TUNEL testing, further andrological evaluation, and appropriate treatment. By enabling earlier detection of at-risk individuals, this approach may help prevent recurrent pregnancy losses after natural conception or repeated failures of assisted reproductive techniques—conditions that currently represent the main indications for sperm DNA fragmentation testing.

Acknowledgments

Preliminary results of this study were presented at the World Meeting on Sexual Medicine, Porto, Portugal, 27 February 2026 (E-poster Session – Male 2), and at the 41st Annual EAU Congress (Abstract Session 22, Reconstruction & Andrology), London, UK, 14 March 2026.

Footnote

Reporting Checklist: The authors have completed the STROBE reporting checklist. Available at https://tau.amegroups.com/article/view/10.21037/tau-2026-0639/rc

Data Sharing Statement: Available at https://tau.amegroups.com/article/view/10.21037/tau-2026-0639/dss

Peer Review File: Available at https://tau.amegroups.com/article/view/10.21037/tau-2026-0639/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-2026-0639/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 requirement for ethics committee approval was waived by the Ethics Committee of IVF Clinic GENNET, given the retrospective and anonymized nature of the data obtained during routine clinical care. All participants provided written informed consent for the use of their anonymized clinical data for statistical analysis and publication.

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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