Evaluation of DNA Double-Strand Breaks in Human Sperm Following Selection by Density Gradient Centrifugation, ZyMōt, and Felix Techniques

Evaluation of DNA Double-Strand Breaks in Human Sperm Following Selection by Density Gradient Centrifugation, ZyMōt, and Felix Techniques | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Evaluation of DNA Double-Strand Breaks in Human Sperm Following Selection by Density Gradient Centrifugation, ZyMōt, and Felix Techniques Mitsuru Nago, Akari Saito, Yuria Takahashi, Eri Kamioka, Ami Fujisawa, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8291848/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose: Density gradient centrifugation (DGC) is widely used for sperm preparation, but centrifugation-induced oxidative stress may cause DNA damage. This study compared sperm DNA double-strand breaks (DSBs) among DGC, ZyMōt, and Felix, and evaluated sperm recovery, motility, and processing time. Methods: Fifteen fresh semen samples collected from January to June 2025 were processed in parallel using DGC, ZyMōt, and Felix. Following sperm preparation, γH2AX immunostaining was performed, and at least 200 spermatozoa per sample were analyzed to determine the DSB-positive rate. Sperm recovery, motility, and processing time were also recorded. Statistical analyses were conducted using the Friedman test followed by Wilcoxon signed-rank tests with Bonferroni correction, and data were expressed as medians with interquartile ranges. Results: The DSB-positive rate was significantly lower in the ZyMōt (11.8%) and Felix (10.0%) groups compared with the DGC group (16.0%; p < 0.01). DGC yielded the highest sperm recovery, ZyMōt achieved the highest motility, and Felix required the shortest processing time, indicating that the three methods exhibit distinct performance characteristics. Conclusions: The non-centrifugal systems ZyMōt and Felix significantly reduced DSB-positive sperm compared with DGC while maintaining comparable overall performance, highlighting their potential usefulness as optimized sperm preparation approaches in assisted reproductive technology. DNA double-strand breaks Sperm DNA fragmentation Microfluidics Electrophoresis Male infertility Figures Figure 1 Figure 2 Figure 3 Capsule Summary Non-centrifugal sperm selection systems, ZyMōt and Felix, significantly reduced double-strand DNA breaks compared with conventional density gradient centrifugation. Each method also offered practical advantages, with enhanced motility observed in ZyMōt and shorter processing times in Felix, highlighting their potential value in assisted reproduction. Introduction Infertility affects approximately 8–15% of couples worldwide, and male factors are involved in nearly half of these cases [1,2]. The diagnosis of male infertility is primarily based on semen parameters such as sperm concentration, motility, and morphology, according to the World Health Organization (WHO) criteria [3]. However, even in assisted reproductive technology (ART) cycles using intracytoplasmic sperm injection (ICSI), where embryologists subjectively select spermatozoa for injection, some men may still experience poor embryo development or implantation failure [4,5]. This clinical discrepancy may occur because conventional semen analysis is primarily limited in its ability to evaluate the integrity of the paternal genome. Consequently, there is an increasing need for functional sperm quality biomarkers that more accurately reflect the fertilizing potential of sperm. One such indicator is sperm DNA fragmentation (SDF), which has been associated with increased miscarriage rates and adverse pregnancy outcomes [6,7]. SDF results from multiple causes, including abnormal chromatin condensation, incomplete apoptosis, and oxidative stress [8], and can be categorized into single-strand breaks (SSBs) and double-strand breaks (DSBs) [9]. Conventional SDF assays, such as the TUNEL assay, sperm chromatin structure assay, and sperm chromatin dispersion test, detect DNA strand breaks but do not distinguish between SSBs and DSBs [10]. This is a significant clinical distinction, as SSBs are often repairable by the robust DNA repair machinery of the oocyte. In contrast, DSBs are considered one of the more severe forms of DNA damage and may compromise genomic stability [11]. DSBs may arise from defective chromatin remodeling during spermatogenesis or from oxidative stress, and post-fertilization repair depends on error-prone non-homologous end joining. Consequently, DSBs can adversely affect embryo development and pregnancy outcomes and have been proposed as clinically meaningful biomarkers of sperm quality [10] . In addition, paternal age has been implicated in the accumulation of sperm DNA damage and adverse reproductive outcomes, including reduced success in ART and an increase in miscarriage risk, suggesting that it may influence the clinical relevance of DSBs in ART[12,13] . Density gradient centrifugation (DGC) is a widely used sperm preparation method that allows for the selection of morphologically normal and motile spermatozoa. However, centrifugation can induce reactive oxygen species (ROS), potentially increasing the risk of DNA damage [14]. In contrast, non-centrifugal approaches, including microfluidic and electrophoretic sperm selection, have been developed to minimize oxidative stress [15]. ZyMōt (DxNow Inc., USA) is a microfluidic device that employs a microchannel design, allowing motile sperm to traverse a microporous membrane while immotile sperm and cellular debris remain below. This approach enables sperm selection without centrifugation and is considered less mechanically manipulative than centrifugation-based methods. Felix (Memphasys Ltd., Australia) is an electrophoretic system that separates spermatozoa according to differences in surface charge. Mature, negatively charged sperm migrate toward the anode and pass through a microporous membrane, facilitating sperm recovery without centrifugation, which may reduce mechanical handling. Previous studies have compared DGC with either ZyMōt [16] or Felix [17,18], but such pairwise comparisons provide limited insight into relative performance under identical conditions. To our knowledge, no study has directly compared all three methods under identical conditions, particularly with respect to DSBs, which have greater clinical relevance than total DNA fragmentation. The lack of such a gold-standard comparative study assessing these three widely adopted preparation techniques based on this critical biomarker (DSB) hinders evidence-based decision-making in the laboratory. Furthermore, beyond DNA integrity, the practical considerations including sperm recovery yield, final sperm motility, and total processing time are vital for clinical application. Therefore, the present study quantitatively compared the proportion of DSB-positive spermatozoa obtained using DGC, ZyMōt, and Felix by immunostaining for phosphorylation of histone H2AX on serine 139 (γH2AX), a sensitive marker of DSBs [19]. This study provides preliminary data that may help inform future assessments of sperm preparation protocols in ART laboratories. Materials and methods Samples A total of 15 fresh semen samples collected at our clinic between January and June 2025 were included in this study. Only surplus specimens remaining after routine semen analysis and scheduled for disposal were used. Semen samples were obtained by masturbation following an abstinence period of 2–7 days. The exclusion criteria were as follows: (1) semen volume < 2.0 mL; (2) sperm concentration < 2 × 10⁶/mL; and (3) sperm motility < 10%. After liquefaction, each sample was thoroughly mixed and divided into three aliquots for parallel processing using DGC, the ZyMōt microfluidic system, and the Felix electrophoretic system. DGC ISolate Stock Solution (FUJIFILM Irvine Scientific, Santa Ana, CA, USA) was used as the density gradient medium. Liquefied semen was carefully layered onto the gradient and centrifuged at 400 × g for 15 min. The resulting pellet was resuspended in 0.5 mL of Gx-IVF Medium (Vitrolife, Gothenburg, Sweden), washed by centrifugation at 200 × g for 5 min, and finally adjusted to a volume of 0.5 mL. This centrifugation speed and duration have been selected as the standard protocol in our laboratory, chosen to optimize sperm separation while minimizing oxidative stress. ZyMōt microfluidic system Sperm separation was performed using the ZyMōt Multi 850 µL sperm separation device (DxNow Inc., USA) according to the manufacturer’s protocol. A total of 850 µL of liquefied semen was loaded into the inlet port, and 750 µL of Gx-IVF Medium was layered above the membrane. The outlet port was primed with 50 µL of the same medium. The device was incubated at 37°C with 6% CO₂ for 30 min, after which 0.5 mL of sperm suspension was carefully collected from the outlet port. Note that the final collection volume (0.5 mL) was matched across all three methods to ensure a fair comparison of sperm recovery and concentration. Felix electrophoretic system Sperm separation was performed using the Felix system (Memphasys Ltd., Sydney, Australia) according to the manufacturer’s protocol. A total of 4 mL of G-RINSE Medium (Vitrolife, Gothenburg, Sweden) was added to both the sample and harvest chambers of the separation cartridge, and an additional 1 mL of Gx-IVF Medium was added to the harvest chamber. Liquefied semen was mixed with Gx-IVF Medium, adjusted to a total volume of 1 mL, and loaded into the sample chamber. Electrophoresis was performed for 6 min, after which 0.5 mL of sperm suspension was carefully collected from the harvest chamber using a sterile glass pipette. As with the other methods, the final suspension was standardized to 0.5 mL for subsequent analysis. DSB assessment Following sperm preparation, spermatozoa were smeared onto poly-L-lysine–coated slides and fixed with 2% paraformaldehyde for 15 min. After washing with phosphate-buffered saline (PBS), the samples were permeabilized with 0.1% Triton X-100 for 15 min and blocked with 1% bovine serum albumin in PBS for 1 h. The samples were then incubated overnight at 4°C with a rabbit monoclonal anti–γH2AX (phospho S139) antibody [EP854(2)Y] (ab81299, Abcam, Cambridge, UK) diluted 1:250. After washing with PBS, a CoraLite 594-conjugated goat anti-rabbit IgG (H+L) secondary antibody (SA00013-4, Proteintech, Rosemont, IL, USA) diluted 1:250 was applied for 1 h at 24°C. Nuclei were counterstained with DAPI (4′,6-diamidino-2-phenylindole). Phosphorylation of histone H2AX at serine 139 (γH2AX) served as an established and highly sensitive indicator of DNA double-strand break repair foci formation, directly marking sites of DSBs in the nucleus. As a positive control, spermatozoa treated with 20 µM doxorubicin for 1 h exhibited distinct γH2AX signals, whereas omission of the primary antibody served as a negative control (Fig. 1a) . Fluorescence imaging was performed using an all-in-one fluorescence microscope (BZ-9000, Keyence, Osaka, Japan). At least 200 spermatozoa per sample were examined, and those showing a distinct γH2AX signal within the nucleus were classified as DSB-positive (Fig. 1a) . A spermatozoon was defined as DSB-positive if it exhibited one or more distinct γH2AX foci (visible fluorescent spots) within the DAPI-stained nuclear area, excluding non-specific background staining. All assessments were performed by a single blinded evaluator. Additional evaluations In addition to the DSB-positive rate, basic sperm characteristics and processing efficiency were evaluated for each preparation method. The parameters assessed included sperm recovery, motility, and total processing time. Sperm recovery was determined by measuring the sperm concentration in the 0.5 mL suspension obtained after processing and expressed as the total sperm count (×10⁶ per 0.5 mL). Motility was calculated as the percentage of motile spermatozoa among the total sperm population observed under a microscope using a Makler chamber. The total processing time was defined as the duration (min) from the start of sperm preparation to the completion of sperm collection, including both hands-on manipulation and waiting periods such as centrifugation, incubation, or electrophoresis. This comprehensive metric was included to assess the practical efficiency of each method for routine clinical use in the ART laboratory. Statistical analysis Comparisons among the three sperm preparation methods were performed using the Friedman test, a nonparametric test for related samples. When significant differences were found, pairwise comparisons were conducted using the Wilcoxon signed-rank test with Bonferroni correction for multiple testing. In an exploratory analysis, Spearman’s rank correlation coefficient (ρ) was used to assess the relationship between patient age and the DSB-positive rate for each sperm preparation method. Statistical analyses were performed using EZR (version 1.61; Saitama Medical Center, Jichi Medical University, Japan) [20],which is a graphical user interface for R, and two-tailed p -values < 0.05 were considered statistically significant. All data are presented as medians with an interquartile range (IQR), as required for nonparametric statistical reporting. Processing time was excluded from statistical testing because it remained nearly constant for each method by design. Results Patient characteristics A total of 15 semen samples were analyzed in this study. The median age of the patients was 33 years (IQR, 31.0–34.0 years). The median time interval from ejaculation to sperm preparation was 130 min (IQR, 30.5–180.0 min). The median semen volume, sperm concentration, and motility were 3.2 mL (IQR, 2.5–4.6), 68.0 × 10⁶/mL (IQR, 46.5–89.0), and 48.9% (IQR, 45.9–60.7), respectively (Table 1). Table 1. Baseline characteristics of semen samples Variable Median (IQR) Age (years) 33.0 (31.0–34.0) Semen collection to preparation time (min) 130.0 (30.5–180.0) Volume (mL) 3.2 (2.5–4.6) Concentration (×10⁶/mL) 68.0 (46.5–89.0) Motility (%) 48.9 (45.9–60.7) Semen characteristics of the study samples (n = 15). Values are presented as median. IQR, interquartile range. Sperm DSB-positive rate The proportion of DSB-positive spermatozoa, as assessed by γH2AX immunostaining, differed significantly among the three preparation methods (Friedman χ² = 19.61, df = 2, p < 0.001). The median (IQR) DSB-positive rate was 16.0% (14.0–22.7) for DGC, 11.8% (8.4–15.4) for ZyMōt, and 10.0% (7.0–11.0) for Felix. Post-hoc Wilcoxon signed-rank tests with Bonferroni correction revealed that both ZyMōt ( p = 0.004) and Felix ( p = 0.002) yielded significantly lower DSB-positive rates than DGC, whereas there was no significant difference between ZyMōt and Felix ( p = 0.55; Fig. 1b) . Collectively, these data indicate that both non-centrifugal approaches were able to substantially reduce the proportion of spermatozoa harboring DSBs compared with DGC, despite being applied to the same ejaculate. Correlation between patient age and DSB-positive rate Spearman’s rank correlation analysis was performed to examine the association between patient age and the DSB-positive rate for each sperm preparation method. Sperm prepared by DGC showed a moderate positive correlation between age and the proportion of DSB-positive spermatozoa (ρ = 0.516, p = 0.049; Fig. 2a) . In contrast, no significant correlations were observed for ZyMōt (ρ = 0.229, p = 0.411; Fig. 2b) or Felix (ρ = −0.103, p = 0.714; Fig. 2c) . Visual inspection of the scatter plots suggested that the DSB-positive rate after DGC tended to increase with age, whereas DSB levels remained relatively low and stable across the age range for ZyMōt and Felix. Sperm recovery A significant overall difference in sperm recovery was observed among the three methods (Friedman χ² = 17.39, df = 2, p < 0.001). The number of recovered spermatozoa (median [IQR], ×10⁶) was 4.5 (3.2–11.5) for DGC, 4.0 (1.9–5.8) for ZyMōt, and 2.25 (0.83–3.65) for Felix. Post-hoc tests showed that DGC yielded significantly higher sperm recovery than ZyMōt ( p = 0.020) or Felix ( p < 0.001), whereas there was no significant difference between ZyMōt and Felix ( p = 0.27; Fig. 3a) . Sperm motility Sperm motility also differed significantly among the three methods (Friedman χ² = 19.97, df = 2, p < 0.001). The median (IQR) motility was 76.3% (65.7–86.8) for DGC, 84.6% (70.5–87.9) for Felix, and 100% (98.5–100.0) for ZyMōt. Post-hoc tests indicated that ZyMōt achieved significantly higher motility than both DGC ( p < 0.001) and Felix ( p = 0.006), whereas no significant difference was observed between DGC and Felix ( p = 1.00; Fig. 3b ). Processing time The total processing time differed markedly among the three methods: 8 min for Felix, 27 min for DGC, and 32 min for ZyMōt. Accordingly, Felix required a substantially shorter preparation time than the other two methods (Fig. 3c) . Discussion In this study, we compared three sperm selection methods, DGC, microfluidic system (ZyMōt), and electrophoretic system (Felix), focusing on the DSB-positive rate, sperm recovery, motility, and processing time. Both non-centrifugal systems, ZyMōt and Felix, showed significantly lower DSB-positive rates than DGC (Fig. 1b) . In relative terms, ZyMōt reduced the proportion of DSB-positive sperm by approximately 26%, whereas Felix achieved nearly a 38% reduction, suggesting that non-centrifugal systems may be associated with lower levels of DNA damage in this cohort. Furthermore, ZyMōt achieved higher motility (Fig. 3b) , whereas Felix offered a shorter processing time (Fig. 3c) , highlighting distinct practical advantages of each system. To our knowledge, few studies have directly compared these three methods under identical conditions, particularly with respect to DSBs. Normal embryo development after fertilization requires the genomic integrity of both sperm and oocytes. Considering the highly condensed chromatin, mature spermatozoa have minimal capacity to repair DNA damage; post-fertilization repair therefore depends on maternal factors [21]. However, the DNA repair capacity of oocytes declines with advancing maternal age. Horta et al. reported that aged mouse oocytes exhibited reduced expression of DNA repair–related genes and diminished repair responses, resulting in a markedly lower blastocyst formation rate when fertilized with sperm populations exhibiting high levels of DNA damage compared with young oocytes [22]. Setti et al. demonstrated that advanced maternal age is associated with lower blastocyst formation, pregnancy, and live birth rates, as well as increased miscarriage rates [23], suggesting that sperm DNA damage may contribute to adverse clinical outcomes. Moreover, because the repair response is substantially reduced after embryonic genome activation, unrepaired damage can manifest as “late paternal effects,” leading to impaired embryo development and adverse clinical outcomes [5,24]. Among types of DNA damage, DSBs are generally considered more challenging to repair and have been associated in some studies with adverse ART outcomes [4,10,25]. Thus, reducing DSB burden is clinically important, particularly for couples experiencing recurrent implantation failure, recurrent pregnancy loss, or borderline embryo development despite normal semen parameters. Notably, our findings are consistent with previous evidence indicating that sperm DSBs represent a distinct and clinically relevant form of sperm DNA damage. Lara-Cerrillo et al. reported that, although SSBs were markedly reduced after Swim-up and ICSI-based sperm selection, double-strand break levels remained unchanged between ejaculated, Swim-up, and ICSI-selected spermatozoa [26]. Their multimodal assessment, including the neutral Comet assay, demonstrated that DSBs are not associated with sperm motility and therefore cannot be efficiently eliminated simply by selecting morphologically normal and progressively motile spermatozoa. Importantly, recurrent miscarriage cases in that study exhibited significantly higher levels of DSBs despite comparatively normal semen parameters, underscoring the clinical relevance of DSBs as a biomarker of underlying genomic instability rather than conventional semen quality. These observations are in line with reports that DSBs, unlike SSBs, arise predominantly from enzymatic events during meiosis and chromatin remodeling and rely on limited, error-prone repair pathways after fertilization [10]. Taken together with our γH2AX-based evaluation of DSB-positive spermatozoa, these data support the concept that DSBs may represent a particularly relevant subset of sperm DNA damage in the context of ART, and that sperm preparation methods capable of reducing the DSB-positive rate may have relevance for embryo development and miscarriage risk, although clinical confirmation is needed. Among the available methods for detecting DSBs, γH2AX immunostaining allows highly sensitive detection, even at the level of a single DSB [27], and its association with clinical outcomes has also been reported [28]. The use of γH2AX in the present study is therefore appropriate, as it may better reflect clinical relevance than conventional assays such as TUNEL or the Comet assay. Given that DSBs rely on limited maternal repair capability, reducing the proportion of DSB-positive spermatozoa may be associated with improvements in embryo cleavage and blastocyst development and reductions in miscarriage risk. Although clinical outcomes were not assessed, the mechanistic links between DSBs and embryo competence suggest that improvements at the gamete level may translate into developmental benefits. Integrating γH2AX with established assays such as TUNEL or SCSA may help further clarify its utility as a complementary or potentially superior biomarker. Our findings are consistent with previous reports, which showed a reduction in the DNA fragmentation index with ZyMōt [16], and decreased DNA damage following Felix treatment, as reported by Shapouri et al. and Villeneuve et al. [17,18], thereby supporting the utility of non-centrifugal sperm selection methods. In DGC, spermatozoa are subjected to centrifugal forces, which can disrupt the delicate structure of the sperm membrane and promote excessive production of ROS within the pellet and surrounding medium [29]. Increased ROS levels can rapidly induce DNA damage, including DSBs, even in spermatozoa that appear morphologically normal and otherwise of good quality. In contrast, microfluidic and electrophoretic approaches avoid centrifugation and therefore may require fewer handling steps and impose less mechanical stress. The ZyMōt system is a microfluidic device that uses parallel microchannels connecting the inlet and outlet chambers; sperm separation relies on the intrinsic motility of higher-quality spermatozoa that actively swim through these channels into the collection chamber, thereby depleting less motile or damaged sperm without exposure to centrifugation or marked shear forces [30]. The Felix system is an electrophoretic device that separates spermatozoa in a gentle electric field according to their negative surface charge (zeta potential), a hallmark of mature sperm. By minimizing physical stress and maintaining a near-physiological environment, it is regarded as a low-stress, non-centrifugal alternative [31]. The comparable reductions in DSB levels achieved by these two distinct systems are consistent with the broader applicability of these findings, although confirmation in larger studies is required. In the present cohort, a moderate positive correlation between patient age and the DSB-positive rate was observed only in sperm prepared by DGC (Fig. 2a) , whereas no significant associations were detected for ZyMōt (Fig. 2b) or Felix (Fig. 2c) . While this analysis is exploratory and limited by the small sample size and relatively narrow age range, it raises the possibility that centrifugation-based preparation could be more sensitive to age-related variation in DNA integrity, although this inference remains speculative. Aging is accompanied by increased oxidative stress and a decline in the efficiency of DNA repair pathways in the male germ line and semen [32–35]. In vitro manipulations that involve centrifugation have been suggested to generate additional oxidative stress [15], which could further increase the burden of DSBs in older men. By contrast, non-centrifugal systems such as ZyMōt and Felix may help to minimize exposure to age-related variation in sperm DNA integrity by avoiding high g-forces and reducing the generation of ROS during processing. Larger studies explicitly designed to investigate paternal age across different preparation methods will be required to confirm these hypotheses. Regarding sperm recovery, the DGC group yielded the highest number of spermatozoa, whereas ZyMōt and Felix resulted in lower recovery (Fig. 3a) . This difference is likely attributable to the inherent principles of each method. DGC recovers a broad sperm fraction by centrifugation, achieving high yield efficiently [15]. In contrast, ZyMōt employs a microchannel structure that enriches progressively motile sperm capable of migrating through microchannels, and previous studies have also reported lower absolute sperm recovery compared with DGC [16]. Similarly, Felix separates spermatozoa based on electrophoretic mobility, enriching mature cells characterized by negative surface charge, intact membranes, and appropriate size [17,18]. Regarding sperm motility, the ZyMōt yielded the highest motility, whereas Felix showed motility comparable to DGC (Fig. 3b) . This observation is consistent with the underlying principles of each method. ZyMōt selectively isolates sperm capable of actively migrating through microchannels, thereby enriching progressively motile spermatozoa [16]. In contrast, Felix separates sperm based on surface charge and membrane integrity rather than motility itself. Consequently, some spermatozoa that retain a negative surface charge but have lost motility may also be recovered, resulting in motility rates similar to those observed with DGC [17,18]. Regarding processing time, the Felix group exhibited the shortest duration among the three methods (Fig. 3c) . Felix is based on an electrophoretic principle and employs a standardized protocol that enables completion of sperm separation within a few minutes, offering a practical advantage that may improve workflow efficiency, particularly in contexts with workforce shortages such as embryology laboratories in Japan [36]. Taken together, these method-specific characteristics suggest that the three systems may be regarded as complementary approaches, depending on clinical priorities and laboratory workflow. When the primary aim is to minimize DSB burden in couples with recurrent implantation failure, recurrent pregnancy loss, or advanced maternal age, non-centrifugal approaches such as ZyMōt or Felix may be prioritized, with the choice between them guided by the relative importance of motility enrichment versus rapid and standardized processing. By contrast, in situations where high sperm yield is essential or access to newer devices is limited, conventional DGC remains a pragmatic option, as supported by our findings on sperm recovery. Future studies that prospectively assign sperm preparation methods according to these clinical scenarios may help to refine such individualized algorithms and clarify their impact on ART outcomes. A key strength of the present study is the within-sample design, in which each ejaculate was processed in parallel by all three sperm preparation methods. This approach effectively controls for inter-individual variability in baseline semen characteristics and allows more robust paired comparisons of DSB-positive rates, sperm recovery, motility, and processing time. Another strength is the focus on DSBs as quantified by γH2AX, a marker that is mechanistically linked to DNA double-strand break repair foci rather than to generic DNA fragmentation. By integrating a DSB-specific endpoint with practical laboratory metrics, this study provides information that may support method selection in ART laboratories. Furthermore, the use of commercially available devices and widely adopted media platforms enhances the generalizability of our findings across ART laboratories with comparable infrastructure. This study has several limitations. First, the number of cases was small, and the study was conducted at a single center. Second, the evaluated parameters were limited to laboratory outcomes and did not include direct assessment of clinical outcomes. Third, sperm motility was assessed by visual observation rather than by computer-assisted analysis. Nevertheless, this study provides new insights into the evaluation of sperm selection methods by directly comparing three different techniques under identical conditions, using highly sensitive γH2AX-based quantification of DSBs. Future large-scale, multicenter studies are warranted to determine how reducing sperm DNA damage can translate into improved fertilization, embryo development, and pregnancy outcomes in ART. The present findings also highlight opportunities to refine sperm preparation protocols. Adjusting centrifugation parameters or incorporating antioxidant buffering during DGC may reduce ROS generation and limit iatrogenic DNA damage. Similarly, microfluidic and electrophoretic systems may be further optimized through channel geometry refinement, voltage modulation, or integration with real-time sperm quality sensors. These technological advancements could eventually lead to hybrid selection systems that combine motility-driven and charge-based separation to maximize the recovery of high-quality spermatozoa. Conclusion To our knowledge, this study is among the first to quantitatively compare sperm populations obtained using three sperm preparation methods under identical conditions by assessing DSBs through γH2AX immunostaining. Both non-centrifugal systems (ZyMōt and Felix) were associated with lower proportions of DSB-positive sperm than DGC and showed broadly comparable effects, while each system retained distinct practical advantages. Future large-scale, multicenter prospective studies should determine whether reducing sperm DNA damage translates into improved fertilization, embryo development, pregnancy, and live birth, enabling optimization of sperm preparation according to patient profiles and laboratory settings. In the long term, incorporating DSB-focused sperm assessment into routine andrology workflows, together with appropriate selection of non-centrifugal preparation methods, may contribute to more personalized ART strategies. As additional evidence accumulates, these findings may help inform future evaluations of sperm preparation protocols that consider sperm DSBs. Their relevance to ART practice will need to be verified in larger studies that evaluate clinical outcomes. Declarations Ethical Approval This study was approved by the ethics committee of our institution and was conducted in accordance with the Declaration of Helsinki and relevant national regulations. Consent to Participate Informed consent for participation in this study was obtained using an opt-out procedure implemented via an in-clinic notice and the clinic’s website. Consent to Publish Not applicable. Funding The authors received no support from any organization for the submitted work. Conflict of Interest The authors have no competing interests to declare that are relevant to the content of this article. References Agarwal A, Mulgund A, Hamada A, Chyatte MR. A unique view on male infertility around the globe. Reprod Biol Endocrinol. 2015;13:37; https://doi.org/10.1186/s12958-015-0032-1 Vander Borght M, Wyns C. Fertility and infertility: definition and epidemiology. Clin Biochem. 2018;62:2–10; https://doi.org/10.1016/j.clinbiochem.2018.03.012 WHO laboratory manual for the examination and processing of human semen. 6th ed. Geneva: World Health Organization; 2021. https://www.who.int/publications/i/item/9789240030787. Accessed 21 Nov 2025. Casanovas A, Ribas-Maynou J, Lara-Cerrillo S, Jimenez-Macedo AR, Hortal O, Benet J, et al. Double-stranded sperm DNA damage is a cause of delay in embryo development and can impair implantation rates. Fertil Steril. 2019;111:699–707.e1; https://doi.org/10.1016/j.fertnstert.2018.11.035 Tesarik J, Greco E, Mendoza C. Late, but not early, paternal effect on human embryo development is related to sperm DNA fragmentation. Hum Reprod. 2004;19:611–5; https://doi.org/10.1093/humrep/deh127 Zhao J, Zhang Q, Wang Y, Li Y. Whether sperm deoxyribonucleic acid fragmentation has an effect on pregnancy and miscarriage after in vitro fertilization/intracytoplasmic sperm injection: a systematic review and meta-analysis. Fertil Steril. 2014;102:998–1005.e8; https://doi.org/10.1016/j.fertnstert.2014.06.033 Simon L, Zini A, Dyachenko A, Ciampi A, Carrell DT. A systematic review and meta-analysis to determine the effect of sperm DNA damage on in vitro fertilization and intracytoplasmic sperm injection outcome. Asian J Androl. 2017;19:80–90; https://doi.org/10.4103/1008-682X.182822 Muratori M, Marchiani S, Tamburrino L, Baldi E. Sperm DNA fragmentation: mechanisms of origin. In: Baldi E, Muratori M, editors. Genetic Damage in Human Spermatozoa. Advances in Experimental Medicine and Biology. Cham: Springer; 2019. pp. 75–85. Agarwal A, Majzoub A, Baskaran S, Panner Selvam MK, Cho CL, Henkel R, et al. Sperm DNA fragmentation: a new guideline for clinicians. World J Mens Health. 2020;38:412–71; https://doi.org/10.5534/wjmh.200128 Agarwal A, Barbăroșie C, Ambar R, Finelli R. The impact of single- and double-strand DNA breaks in human spermatozoa on assisted reproduction. Int J Mol Sci. 2020;21:3882; https://doi.org/10.3390/ijms21113882 Li N, Wang H, Zou S, Yu X, Li J. Perspective in the mechanisms for repairing sperm DNA damage. Reprod Sci. 2025;32:41–51. https://doi.org/10.1007/s43032-024-01714-5 Kaltsas A, Zikopoulos A, Vrachnis D, Skentou C, Symeonidis EN, Dimitriadis F, et al. Advanced Paternal Age in Focus: Unraveling Its Influence on Assisted Reproductive Technology Outcomes. J Clin Med. 2024;13:2731; https://doi.org/10.3390/jcm13102731 Halvaei I, Litzky J, Esfandiari N. Advanced paternal age: effects on sperm parameters, assisted reproduction outcomes and offspring health. Reprod Biol Endocrinol. 2020;18:110; https://doi.org/10.1186/s12958-020-00668-y Agarwal A, Ikemoto I, Loughlin KR. Effect of sperm washing on levels of reactive oxygen species in semen. Arch Androl. 1994;33:157–62; https://doi.org/10.3109/01485019408987819 Fleming S, Morroll D, Nijs M. Sperm separation and selection techniques to mitigate sperm DNA damage. Life (Basel). 2025;15:302; https://doi.org/10.3390/life15020302 Hsu CT, Lee CI, Lin FS, Wang FZ, Chang HC, Wang TE, et al. Live motile sperm sorting device for enhanced sperm-fertilization competency: comparative analysis with density-gradient centrifugation and microfluidic sperm sorting. J Assist Reprod Genet. 2023;40:1855–64; https://doi.org/10.1007/s10815-023-02838-4 Shapouri F, Mahendran T, Govindarajan M, Xie P, Kocur O, Palermo GD, et al. A comparison between the Felix™ electrophoretic system of sperm isolation and conventional density gradient centrifugation: a multicentre analysis. J Assist Reprod Genet. 2023;40:83–95; https://doi.org/10.1007/s10815-022-02680-0 Villeneuve P, Saez F, Hug E, Chorfa A, Guiton R, Schubert B, et al. Spermatozoa isolation with Felix™ outperforms conventional density gradient centrifugation preparation in selecting cells with low DNA damage. Andrology. 2023;11:1593–604; https://doi.org/10.1111/andr.13384 Coban O, Serdarogullari M, Yarkiner Z, Serakinci N. Investigating the level of DNA double-strand break in human spermatozoa and its relation to semen characteristics and IVF outcome using phospho-histone H2AX antibody as a biomarker. Andrology. 2020;8:421–6; https://doi.org/10.1111/andr.12689 Kanda Y. Investigation of the freely available easy-to-use software “EZR” for medical statistics. Bone Marrow Transplant. 2013;48:452–8; https://doi.org/10.1038/bmt.2012.244 Newman H, Catt S, Vining B, Vollenhoven B, Horta F. DNA repair and response to sperm DNA damage in oocytes and embryos, and the potential consequences in ART: a systematic review. Mol Hum Reprod. 2022;28:gaab071; https://doi.org/10.1093/molehr/gaab071 Horta F, Catt S, Ramachandran P, Vollenhoven B, Temple-Smith P. Female ageing affects the DNA repair capacity of oocytes in IVF using a controlled model of sperm DNA damage in mice. Hum Reprod. 2020;35:529–44; https://doi.org/10.1093/humrep/dez308 Setti AS, Braga DP de AF, Provenza RR, Iaconelli A, Borges E. Oocyte ability to repair sperm DNA fragmentation: the impact of maternal age on intracytoplasmic sperm injection outcomes. Fertil Steril. 2021;116:123–9; https://doi.org/10.1016/j.fertnstert.2020.10.045 Pardiñas ML, de Celis C, Gil J, Ortega-Jaen D, Martin A, Mercader A, et al. Oocyte-mediated repair of sperm DNA fragmentation: a critical determinant of embryo viability. Reprod Biomed Online. 2025:105165; https://doi.org/10.1016/j.rbmo.2025.105165 García-Rodríguez A, Gosálvez J, Agarwal A, Roy R, Johnston S. DNA damage and repair in human reproductive cells. Int J Mol Sci. 2018;20:31; https://doi.org/10.3390/ijms20010031 Lara-Cerrillo S, Ribas-Maynou J, Rosado-Iglesias C, Lacruz-Ruiz T, Benet J, García-Peiró A. Sperm selection during ICSI treatments reduces single- but not double-strand DNA break values compared to the semen sample. J Assist Reprod Genet. 2021;38:1187–96; https://doi.org/10.1007/s10815-021-02129-w Heylmann D, Kaina B. The γH2AX DNA damage assay from a drop of blood. Sci Rep. 2016;6:22682; https://doi.org/10.1038/srep22682 Garolla A, Cosci I, Bertoldo A, Sartini B, Boudjema E, Foresta C. DNA double-strand breaks in human spermatozoa can be predictive for assisted reproductive outcome. Reprod Biomed Online. 2015;31:100–7; https://doi.org/10.1016/j.rbmo.2015.03.009 Aitken RJ, Clarkson JS. Significance of reactive oxygen species and antioxidants in defining the efficacy of sperm preparation techniques. J Androl. 1988;9:367–376. https://doi:10.1002/j.1939-4640.1988.tb01067.x. Gisbert Iranzo A, Cano-Extremera M, Hervás I, Falquet Guillem M, Gil Juliá M, Navarro-Gomezlechon A, et al. Sperm selection using microfluidic techniques significantly decreases sperm DNA fragmentation (SDF), enhancing reproductive outcomes: a systematic review and meta-analysis. Biology (Basel). 2025;14:792. https://doi.org/10.3390/biology14070792 Cariati F, Orsi MG, Bagnulo F, Del Mondo D, Vigilante L, De Rosa M, et al. Advanced sperm selection techniques for assisted reproduction. J Pers Med. 2024;14:726. https://doi.org/10.3390/jpm14070726 Nguyen-Powanda P, Robaire B. Oxidative stress and reproductive function in the aging male. Biology (Basel). 2020;9:1–15; https://doi.org/10.3390/biology9090282 Herati AS, Zhelyazkova BH, Butler PR, Lamb DJ. Age-related alterations in the genetics and genomics of the male germ line. Fertil Steril. 2017;107:319–23; https://doi.org/10.1016/j.fertnstert.2016.12.021 Elías-Llumbet A, Lira S, Manterola M. Male aging in germ cells: What are we inheriting? Genet Mol Biol. 2025;47:e20240052; https://doi.org/10.1590/1678-4685-gmb-2024-0052 Nago M, Arichi A, Omura N, Iwashita Y, Kawamura T, Yumura Y. Aging increases oxidative stress in semen. Investig Clin Urol. 2021;62:233–8; https://doi:10.4111/icu.20200066 Shirasawa H, Yamada M, Jwa SC, Kuroda K, Harada M, Osuga Y. Assessment of embryologist sufficiency and associated regional disparities in Japanese assisted reproduction facilities using nationwide survey data (IZANAMI project). J Obstet Gynaecol Res. 2024;50:1459–69; https://doi.org/10.1111/jog.16022 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8291848","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":558348545,"identity":"2e9d88ff-90bc-4087-b437-986d7c451fda","order_by":0,"name":"Mitsuru Nago","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEklEQVRIiWNgGAWjYPACGxDBeICBTQJIMzccYDAAchvwakkDk1AtjERpOQzTArYNv1rdBt6DH79UnM/n5z984MCPMgs5+faDjYcLCuwYmGdj12p2gC9ZWubMbcuZM9ISDvackzA2OJPYcHiGQTID45wDOLTwGEhLtt02MLjBY3CAt00icYMEY8NhIJuBcUYCLi3GvyXbzhkYnD//4eDfNon6+TMIazGT/Nh2wMDgQA7DYaAtCQw3CGk5zGNmzXAm2UByRprBYZlzEoYbQH7hMUjmwemX4z3GN39U2BkAQ+zhwzdldfLy7YcPf+b5YydniCPEGJiBiAebBI/hDOw6QIDxB1ZheQncWkbBKBgFo2BEAQCk+mJ7rVBR6AAAAABJRU5ErkJggg==","orcid":"","institution":"Rose Ladies Clinic","correspondingAuthor":true,"prefix":"","firstName":"Mitsuru","middleName":"","lastName":"Nago","suffix":""},{"id":558348546,"identity":"aa09dc76-d36b-479a-b74d-fea700e29815","order_by":1,"name":"Akari Saito","email":"","orcid":"","institution":"Rose Ladies Clinic","correspondingAuthor":false,"prefix":"","firstName":"Akari","middleName":"","lastName":"Saito","suffix":""},{"id":558348547,"identity":"733f4e82-cbfa-4145-97fc-b627f59089b1","order_by":2,"name":"Yuria Takahashi","email":"","orcid":"","institution":"Rose Ladies Clinic","correspondingAuthor":false,"prefix":"","firstName":"Yuria","middleName":"","lastName":"Takahashi","suffix":""},{"id":558348548,"identity":"3315616f-3456-431a-9c4b-ae1e8454c6cb","order_by":3,"name":"Eri Kamioka","email":"","orcid":"","institution":"Rose Ladies Clinic","correspondingAuthor":false,"prefix":"","firstName":"Eri","middleName":"","lastName":"Kamioka","suffix":""},{"id":558348549,"identity":"508107af-0841-4df4-84cc-118c577d77ba","order_by":4,"name":"Ami Fujisawa","email":"","orcid":"","institution":"Rose Ladies Clinic","correspondingAuthor":false,"prefix":"","firstName":"Ami","middleName":"","lastName":"Fujisawa","suffix":""},{"id":558348550,"identity":"d247b661-5757-4e19-a7bd-21986e7421c7","order_by":5,"name":"Mayuri Chino","email":"","orcid":"","institution":"Rose Ladies Clinic","correspondingAuthor":false,"prefix":"","firstName":"Mayuri","middleName":"","lastName":"Chino","suffix":""},{"id":558348551,"identity":"e883ee09-80cb-4c6e-b201-5b8c348a6d94","order_by":6,"name":"Manon Koyata","email":"","orcid":"","institution":"Rose Ladies Clinic","correspondingAuthor":false,"prefix":"","firstName":"Manon","middleName":"","lastName":"Koyata","suffix":""},{"id":558348552,"identity":"cc846125-726c-40bd-a41d-826b297d4cd8","order_by":7,"name":"Yuta Kawagoe","email":"","orcid":"","institution":"Rose Ladies Clinic","correspondingAuthor":false,"prefix":"","firstName":"Yuta","middleName":"","lastName":"Kawagoe","suffix":""},{"id":558348553,"identity":"b846c2c3-a50d-4087-b243-7c11bd1f4b2c","order_by":8,"name":"Bunpei Ishizuka","email":"","orcid":"","institution":"Rose Ladies Clinic","correspondingAuthor":false,"prefix":"","firstName":"Bunpei","middleName":"","lastName":"Ishizuka","suffix":""}],"badges":[],"createdAt":"2025-12-06 04:23:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8291848/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8291848/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":98206305,"identity":"0ff2cd8e-c960-4f74-a257-f55435611429","added_by":"auto","created_at":"2025-12-15 08:48:33","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":80073,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eγH2AX immunofluorescence staining and comparison of sperm DSB-positive rates among different selection methods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Immunofluorescence staining of human sperm nuclei with DAPI and γH2AX. DNA was counterstained with DAPI (blue), and DSBs were visualized by γH2AX immunofluorescence (red). The positive control (sperm treated with 20 µM doxorubicin for 1 h) exhibited clear γH2AX signals, whereas the negative control (untreated sperm) showed no detectable staining. Arrows indicate γH2AX-positive nuclei observed in the sample. Panels from left to right: DAPI, γH2AX, and merged images. Scale bar = 5 µm.\u003cbr\u003e\n(b) Comparison of sperm DSB-positive rates among different sperm selection methods. Box plots indicate the median, interquartile range (IQR), and individual data points. Asterisks indicate statistically significant differences (**\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01). DAPI, 4′,6-diamidino-2-phenylindole; DSB, double-strand breaks.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8291848/v1/aa564c390c25cb7175b149df.jpg"},{"id":98206307,"identity":"7041cb9a-a349-4665-bb1a-d279a1c26f01","added_by":"auto","created_at":"2025-12-15 08:48:33","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":80191,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCorrelation between patient age and sperm DSB-positive rate after different preparation methods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eScatter plots show the relationship between patient age and the DSB-positive rate (%) in sperm prepared by (a) DGC, (b) ZyMōt, and (c) Felix. Solid lines indicate the linear regression trend lines. Spearman’s rank correlation coefficient (ρ) and the corresponding \u003cem\u003ep\u003c/em\u003e-value are shown within each panel.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8291848/v1/6f24c150a1c0b23d17c5d1dd.jpg"},{"id":98431446,"identity":"5a4771e4-658e-4ce0-9910-5cdd300328d6","added_by":"auto","created_at":"2025-12-17 16:47:41","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":54858,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of outcomes among different sperm selection methods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Number of recovered sperm (×10⁶). (b) Sperm motility (%). (c) Preparation time (min). Box plots indicate the median, interquartile range (IQR), and individual data points for (a) and (b). Asterisks indicate significant differences after pairwise comparisons with Bonferroni correction (**\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). Preparation time (c) represents fixed values determined by device specifications, shown as bar graphs without statistical testing.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8291848/v1/2613fb92efbc01969fc34c0f.jpg"},{"id":98444872,"identity":"12b8f519-a596-4c71-a0c0-5610f724d232","added_by":"auto","created_at":"2025-12-17 17:17:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1040253,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8291848/v1/c861d2c8-af46-41ca-8476-3fb3c229ab28.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Evaluation of DNA Double-Strand Breaks in Human Sperm Following Selection by Density Gradient Centrifugation, ZyMōt, and Felix Techniques","fulltext":[{"header":"Capsule Summary","content":"\u003cp\u003eNon-centrifugal sperm selection systems, ZyMōt and Felix, significantly reduced double-strand DNA breaks compared with conventional density gradient centrifugation. Each method also offered practical advantages, with enhanced motility observed in ZyMōt and shorter processing times in Felix, highlighting their potential value in assisted reproduction.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eInfertility affects approximately 8–15% of couples worldwide, and male factors are involved in nearly half of these cases [1,2]. The diagnosis of male infertility is primarily based on semen parameters such as sperm concentration, motility, and morphology, according to the World Health Organization (WHO) criteria [3]. However, even in assisted reproductive technology (ART) cycles using intracytoplasmic sperm injection (ICSI), where embryologists subjectively select spermatozoa for injection, some men may still experience poor embryo development or implantation failure [4,5].\u0026nbsp;This clinical discrepancy may occur because conventional semen analysis is primarily limited in its ability to evaluate the integrity of the paternal genome. Consequently, there is an increasing need for functional sperm quality biomarkers that more accurately reflect the fertilizing potential of sperm.\u003c/p\u003e\n\u003cp\u003eOne such indicator is sperm DNA fragmentation (SDF), which has been associated with increased miscarriage rates and adverse pregnancy outcomes [6,7]. SDF results from multiple causes, including abnormal chromatin condensation, incomplete apoptosis, and oxidative stress [8], and can be categorized into single-strand breaks (SSBs) and double-strand breaks (DSBs) [9]. Conventional SDF assays, such as the TUNEL assay, sperm chromatin structure assay, and sperm chromatin dispersion test, detect DNA strand breaks but do not distinguish between SSBs and DSBs [10]. This is a significant clinical distinction, as SSBs are often repairable by the robust DNA repair machinery of the oocyte. In contrast, DSBs are considered one of the more severe forms of DNA damage and may compromise genomic stability [11]. DSBs may arise from defective chromatin remodeling during spermatogenesis or from oxidative stress, and post-fertilization repair depends on error-prone non-homologous end joining. Consequently, DSBs can adversely affect embryo development and pregnancy outcomes and have been proposed as clinically meaningful biomarkers of sperm quality [10]\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003eIn addition, paternal age has been implicated in the accumulation of sperm DNA damage and adverse reproductive outcomes, including reduced success in ART and an increase in miscarriage risk, suggesting that it may influence the clinical relevance of DSBs in ART[12,13]\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDensity gradient centrifugation (DGC) is a widely used sperm preparation method that allows for the selection of morphologically normal and motile spermatozoa. However, centrifugation can induce reactive oxygen species (ROS), potentially increasing the risk of DNA damage [14]. In contrast, non-centrifugal approaches, including microfluidic and electrophoretic sperm selection, have been developed to minimize oxidative stress [15]. ZyMōt (DxNow Inc., USA) is a microfluidic device that employs a microchannel design, allowing motile sperm to traverse a microporous membrane while immotile sperm and cellular debris remain below. This approach enables sperm selection without centrifugation and is considered less mechanically manipulative than centrifugation-based methods. Felix (Memphasys Ltd., Australia) is an electrophoretic system that separates spermatozoa according to differences in surface charge. Mature, negatively charged sperm migrate toward the anode and pass through a microporous membrane, facilitating sperm recovery without centrifugation, which may reduce mechanical handling.\u003c/p\u003e\n\u003cp\u003ePrevious studies have compared DGC with either ZyMōt [16] or Felix [17,18], but such pairwise comparisons provide limited insight into relative performance under identical conditions. To our knowledge, no study has directly compared all three methods under identical conditions, particularly with respect to DSBs, which have greater clinical relevance than total DNA fragmentation. The lack of such a gold-standard comparative study assessing these three widely adopted preparation techniques based on this critical biomarker (DSB) hinders evidence-based decision-making in the laboratory. Furthermore, beyond DNA integrity, the practical considerations including sperm recovery yield, final sperm motility, and total processing time are vital for clinical application. Therefore, the present study quantitatively compared the proportion of DSB-positive spermatozoa obtained using DGC, ZyMōt, and Felix by immunostaining for phosphorylation of histone H2AX on serine 139 (γH2AX), a sensitive marker of DSBs [19]. This study provides preliminary data that may help inform future assessments of sperm preparation protocols in ART laboratories.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eSamples\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 15 fresh semen samples collected at our clinic between January and June 2025 were included in this study. Only surplus specimens remaining after routine semen analysis and scheduled for disposal were used. Semen samples were obtained by masturbation following an abstinence period of 2–7 days. The exclusion criteria were as follows: (1) semen volume \u0026lt; 2.0 mL; (2) sperm concentration \u0026lt; 2 × 10⁶/mL; and (3) sperm motility \u0026lt; 10%. After liquefaction, each sample was thoroughly mixed and divided into three aliquots for parallel processing using DGC, the ZyMōt microfluidic system, and the Felix electrophoretic system.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDGC\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eISolate Stock Solution (FUJIFILM Irvine Scientific, Santa Ana, CA, USA) was used as the density gradient medium. Liquefied semen was carefully layered onto the gradient and centrifuged at 400 × g for 15 min. The resulting pellet was resuspended in 0.5 mL of Gx-IVF Medium (Vitrolife, Gothenburg, Sweden), washed by centrifugation at 200 × g for 5 min, and finally adjusted to a volume of 0.5 mL. This centrifugation speed and duration have been selected as the standard protocol in our laboratory, chosen to optimize sperm separation while minimizing oxidative stress.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eZyMōt microfluidic system\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSperm separation was performed using the ZyMōt Multi 850 µL sperm separation device (DxNow Inc., USA) according to the manufacturer’s protocol. A total of 850 µL of liquefied semen was loaded into the inlet port, and 750 µL of Gx-IVF Medium was layered above the membrane. The outlet port was primed with 50 µL of the same medium. The device was incubated at 37°C with 6% CO₂ for 30 min, after which 0.5 mL of sperm suspension was carefully collected from the outlet port. Note that the final collection volume (0.5 mL) was matched across all three methods to ensure a fair comparison of sperm recovery and concentration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFelix electrophoretic system\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSperm separation was performed using the Felix system (Memphasys Ltd., Sydney, Australia) according to the manufacturer’s protocol. A total of 4 mL of G-RINSE Medium (Vitrolife, Gothenburg, Sweden) was added to both the sample and harvest chambers of the separation cartridge, and an additional 1 mL of Gx-IVF Medium was added to the harvest chamber. Liquefied semen was mixed with Gx-IVF Medium, adjusted to a total volume of 1 mL, and loaded into the sample chamber. Electrophoresis was performed for 6 min, after which 0.5 mL of sperm suspension was carefully collected from the harvest chamber using a sterile glass pipette. As with the other methods, the final suspension was standardized to 0.5 mL for subsequent analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDSB assessment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollowing sperm preparation, spermatozoa were smeared onto poly-L-lysine–coated slides and fixed with 2% paraformaldehyde for 15 min. After washing with phosphate-buffered saline (PBS), the samples were permeabilized with 0.1% Triton X-100 for 15 min and blocked with 1% bovine serum albumin in PBS for 1 h. The samples were then incubated overnight at 4°C with a rabbit monoclonal anti–γH2AX (phospho S139) antibody [EP854(2)Y] (ab81299, Abcam, Cambridge, UK) diluted 1:250. After washing with PBS, a CoraLite 594-conjugated goat anti-rabbit IgG (H+L) secondary antibody (SA00013-4, Proteintech, Rosemont, IL, USA) diluted 1:250 was applied for 1 h at 24°C. Nuclei were counterstained with DAPI (4′,6-diamidino-2-phenylindole). Phosphorylation of histone H2AX at serine 139 (γH2AX) served as an established and highly sensitive indicator of DNA double-strand break repair foci formation, directly marking sites of DSBs in the nucleus.\u003cbr\u003eAs a positive control, spermatozoa treated with 20 µM doxorubicin for 1 h exhibited distinct γH2AX signals, whereas omission of the primary antibody served as a negative control \u003cstrong\u003e(Fig. 1a)\u003c/strong\u003e. Fluorescence imaging was performed using an all-in-one fluorescence microscope (BZ-9000, Keyence, Osaka, Japan). At least 200 spermatozoa per sample were examined, and those showing a distinct γH2AX signal within the nucleus were classified as DSB-positive\u003cstrong\u003e\u0026nbsp;(Fig. 1a)\u003c/strong\u003e. A spermatozoon was defined as DSB-positive if it exhibited one or more distinct γH2AX foci (visible fluorescent spots) within the DAPI-stained nuclear area, excluding non-specific background staining. All assessments were performed by a single blinded evaluator.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional evaluations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn addition to the DSB-positive rate, basic sperm characteristics and processing efficiency were evaluated for each preparation method. The parameters assessed included sperm recovery, motility, and total processing time.\u003cbr\u003e\u0026nbsp;Sperm recovery was determined by measuring the sperm concentration in the 0.5 mL suspension obtained after processing and expressed as the total sperm count (×10⁶ per 0.5 mL). Motility was calculated as the percentage of motile spermatozoa among the total sperm population observed under a microscope using a Makler chamber. The total processing time was defined as the duration (min) from the start of sperm preparation to the completion of sperm collection, including both hands-on manipulation and waiting periods such as centrifugation, incubation, or electrophoresis. This comprehensive metric was included to assess the practical efficiency of each method for routine clinical use in the ART laboratory.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003cbr\u003eComparisons among the three sperm preparation methods were performed using the Friedman test, a nonparametric test for related samples. When significant differences were found, pairwise comparisons were conducted using the Wilcoxon signed-rank test with Bonferroni correction for multiple testing. In an exploratory analysis, Spearman’s rank correlation coefficient (ρ) was used to assess the relationship between patient age and the DSB-positive rate for each sperm preparation method. Statistical analyses were performed using EZR (version 1.61; Saitama Medical Center, Jichi Medical University, Japan) [20],which is a graphical user interface for R, and two-tailed \u003cem\u003ep\u003c/em\u003e-values \u0026lt; 0.05 were considered statistically significant. All data are presented as medians with an interquartile range (IQR), as required for nonparametric statistical reporting. Processing time was excluded from statistical testing because it remained nearly constant for each method by design.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003ePatient characteristics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 15 semen samples were analyzed in this study. The median age of the patients was 33 years (IQR, 31.0\u0026ndash;34.0 years). The median time interval from ejaculation to sperm preparation was 130 min (IQR, 30.5\u0026ndash;180.0 min). The median semen volume, sperm concentration, and motility were 3.2 mL (IQR, 2.5\u0026ndash;4.6), 68.0 \u0026times; 10⁶/mL (IQR, 46.5\u0026ndash;89.0), and 48.9% (IQR, 45.9\u0026ndash;60.7), respectively (Table 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1. Baseline characteristics of semen samples\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"558\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 69.5341%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eVariable\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 30.4659%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMedian (IQR)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 69.5341%;\"\u003e\n \u003cp\u003eAge (years)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 30.4659%;\"\u003e\n \u003cp\u003e33.0 (31.0\u0026ndash;34.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 69.5341%;\"\u003e\n \u003cp\u003eSemen collection to preparation time (min)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 30.4659%;\"\u003e\n \u003cp\u003e130.0 (30.5\u0026ndash;180.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 69.5341%;\"\u003e\n \u003cp\u003eVolume (mL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 30.4659%;\"\u003e\n \u003cp\u003e3.2 (2.5\u0026ndash;4.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 69.5341%;\"\u003e\n \u003cp\u003eConcentration (\u0026times;10⁶/mL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 30.4659%;\"\u003e\n \u003cp\u003e68.0 (46.5\u0026ndash;89.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 69.5341%;\"\u003e\n \u003cp\u003eMotility (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 30.4659%;\"\u003e\n \u003cp\u003e48.9 (45.9\u0026ndash;60.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eSemen characteristics of the study samples (n = 15). Values are presented as median. IQR, interquartile range.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSperm DSB-positive rate\u003cbr\u003e\u003c/strong\u003eThe proportion of DSB-positive spermatozoa, as assessed by \u0026gamma;H2AX immunostaining, differed significantly among the three preparation methods (Friedman \u0026chi;\u0026sup2; = 19.61, df = 2, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001). The median (IQR) DSB-positive rate was 16.0% (14.0\u0026ndash;22.7) for DGC, 11.8% (8.4\u0026ndash;15.4) for ZyMōt, and 10.0% (7.0\u0026ndash;11.0) for Felix. Post-hoc Wilcoxon signed-rank tests with Bonferroni correction revealed that both ZyMōt (\u003cem\u003ep\u003c/em\u003e = 0.004) and Felix (\u003cem\u003ep\u003c/em\u003e = 0.002) yielded significantly lower DSB-positive rates than DGC, whereas there was no significant difference between ZyMōt and Felix (\u003cem\u003ep\u003c/em\u003e = 0.55;\u003cstrong\u003e\u0026nbsp;Fig. 1b)\u003c/strong\u003e. Collectively, these data indicate that both non-centrifugal approaches were able to substantially reduce the proportion of spermatozoa harboring DSBs compared with DGC, despite being applied to the same ejaculate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorrelation between patient age and DSB-positive rate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSpearman\u0026rsquo;s rank correlation analysis was performed to examine the association between patient age and the DSB-positive rate for each sperm preparation method. Sperm prepared by DGC showed a moderate positive correlation between age and the proportion of DSB-positive spermatozoa (\u0026rho; = 0.516, \u003cem\u003ep\u003c/em\u003e = 0.049; \u003cstrong\u003eFig. 2a)\u003c/strong\u003e. In contrast, no significant correlations were observed for ZyMōt (\u0026rho; = 0.229, \u003cem\u003ep\u003c/em\u003e = 0.411; \u003cstrong\u003eFig. 2b)\u0026nbsp;\u003c/strong\u003eor Felix (\u0026rho; = \u0026minus;0.103, \u003cem\u003ep\u003c/em\u003e = 0.714; \u003cstrong\u003eFig. 2c)\u003c/strong\u003e. Visual inspection of the scatter plots suggested that the DSB-positive rate after DGC tended to increase with age, whereas DSB levels remained relatively low and stable across the age range for ZyMōt and Felix.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSperm recovery\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA significant overall difference in sperm recovery was observed among the three methods (Friedman \u0026chi;\u0026sup2; = 17.39, df = 2, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001). The number of recovered spermatozoa (median [IQR], \u0026times;10⁶) was 4.5 (3.2\u0026ndash;11.5) for DGC, 4.0 (1.9\u0026ndash;5.8) for ZyMōt, and 2.25 (0.83\u0026ndash;3.65) for Felix. Post-hoc tests showed that DGC yielded significantly higher sperm recovery than ZyMōt (\u003cem\u003ep\u003c/em\u003e = 0.020) or Felix (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001), whereas there was no significant difference between ZyMōt and Felix (\u003cem\u003ep\u003c/em\u003e = 0.27; \u003cstrong\u003eFig. 3a)\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSperm motility\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSperm motility also differed significantly among the three methods (Friedman \u0026chi;\u0026sup2; = 19.97, df = 2, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001). The median (IQR) motility was 76.3% (65.7\u0026ndash;86.8) for DGC, 84.6% (70.5\u0026ndash;87.9) for Felix, and 100% (98.5\u0026ndash;100.0) for ZyMōt. Post-hoc tests indicated that ZyMōt achieved significantly higher motility than both DGC (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001) and Felix (\u003cem\u003ep\u003c/em\u003e = 0.006), whereas no significant difference was observed between DGC and Felix (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e= 1.00; \u003cstrong\u003eFig. 3b\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProcessing time\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe total processing time differed markedly among the three methods: 8 min for Felix, 27 min for DGC, and 32 min for ZyMōt. Accordingly, Felix required a substantially shorter preparation time than the other two methods\u003cstrong\u003e\u0026nbsp;(Fig. 3c)\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we compared three sperm selection methods, DGC, microfluidic system (ZyMōt), and electrophoretic system (Felix), focusing on the DSB-positive rate, sperm recovery, motility, and processing time. Both non-centrifugal systems, ZyMōt and Felix, showed significantly lower DSB-positive rates than DGC \u003cstrong\u003e(Fig. 1b)\u003c/strong\u003e. In relative terms, ZyMōt reduced the proportion of DSB-positive sperm by approximately 26%, whereas Felix achieved nearly a 38% reduction, suggesting that non-centrifugal systems may be associated with lower levels of DNA damage in this cohort. Furthermore, ZyMōt achieved higher motility\u003cstrong\u003e\u0026nbsp;(Fig. 3b)\u003c/strong\u003e, whereas Felix offered a shorter processing time\u003cstrong\u003e\u0026nbsp;(Fig. 3c)\u003c/strong\u003e, highlighting distinct practical advantages of each system. To our knowledge, few studies have directly compared these three methods under identical conditions, particularly with respect to DSBs.\u003c/p\u003e\n\u003cp\u003eNormal embryo development after fertilization requires the genomic integrity of both sperm and oocytes. Considering the highly condensed chromatin, mature spermatozoa have minimal capacity to repair DNA damage; post-fertilization repair therefore depends on maternal factors [21]. However, the DNA repair capacity of oocytes declines with advancing maternal age. Horta et al. reported that aged mouse oocytes exhibited reduced expression of DNA repair–related genes and diminished repair responses, resulting in a markedly lower blastocyst formation rate when fertilized with sperm populations exhibiting high levels of DNA damage compared with young oocytes [22]. Setti et al. demonstrated that advanced maternal age is associated with lower blastocyst formation, pregnancy, and live birth rates, as well as increased miscarriage rates [23], suggesting that sperm DNA damage may contribute to adverse clinical outcomes. Moreover, because the repair response is substantially reduced after embryonic genome activation, unrepaired damage can manifest as “late paternal effects,” leading to impaired embryo development and adverse clinical outcomes [5,24]. Among types of DNA damage, DSBs are generally considered more challenging to repair and have been associated in some studies with adverse ART outcomes [4,10,25]. Thus, reducing DSB burden is clinically important, particularly for couples experiencing recurrent implantation failure, recurrent pregnancy loss, or borderline embryo development despite normal semen parameters.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNotably, our findings are consistent with previous evidence indicating that sperm DSBs represent a distinct and clinically relevant form of sperm DNA damage. Lara-Cerrillo et al. reported that, although SSBs were markedly reduced after Swim-up and ICSI-based sperm selection, double-strand break levels remained unchanged between ejaculated, Swim-up, and ICSI-selected spermatozoa [26]. Their multimodal assessment, including the neutral Comet assay, demonstrated that DSBs are not associated with sperm motility and therefore cannot be efficiently eliminated simply by selecting morphologically normal and progressively motile spermatozoa. Importantly, recurrent miscarriage cases in that study exhibited significantly higher levels of DSBs despite comparatively normal semen parameters, underscoring the clinical relevance of DSBs as a biomarker of underlying genomic instability rather than conventional semen quality. These observations are in line with reports that DSBs, unlike SSBs, arise predominantly from enzymatic events during meiosis and chromatin remodeling and rely on limited, error-prone repair pathways after fertilization [10]. Taken together with our γH2AX-based evaluation of DSB-positive spermatozoa, these data support the concept that DSBs may represent a particularly relevant subset of sperm DNA damage in the context of ART, and that sperm preparation methods capable of reducing the DSB-positive rate may have relevance for embryo development and miscarriage risk, although clinical confirmation is needed.\u003c/p\u003e\n\u003cp\u003eAmong the available methods for detecting DSBs, γH2AX immunostaining allows highly sensitive detection, even at the level of a single DSB [27],\u0026nbsp;and its association with clinical outcomes has also been reported [28]. The use of γH2AX in the present study is therefore appropriate, as it may better reflect clinical relevance than conventional assays such as TUNEL or the Comet assay. Given that DSBs rely on limited maternal repair capability, reducing the proportion of DSB-positive spermatozoa may be associated with improvements in embryo cleavage and blastocyst development and reductions in miscarriage risk. Although clinical outcomes were not assessed, the mechanistic links between DSBs and embryo competence suggest that improvements at the gamete level may translate into developmental benefits. Integrating γH2AX with established assays such as TUNEL or SCSA may help further clarify its utility as a complementary or potentially superior biomarker.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOur findings are consistent with previous reports, which showed a reduction in the DNA fragmentation index with ZyMōt [16], and decreased DNA damage following Felix treatment, as reported by Shapouri et al. and Villeneuve et al. [17,18], thereby supporting the utility of non-centrifugal sperm selection methods. In DGC, spermatozoa are subjected to centrifugal forces, which can disrupt the delicate structure of the sperm membrane and promote excessive production of ROS within the pellet and surrounding medium [29]. Increased ROS levels can rapidly induce DNA damage, including DSBs, even in spermatozoa that appear morphologically normal and otherwise of good quality. In contrast, microfluidic and electrophoretic approaches avoid centrifugation and therefore may require fewer handling steps and impose less mechanical stress. The ZyMōt system is a microfluidic device that uses parallel microchannels connecting the inlet and outlet chambers; sperm separation relies on the intrinsic motility of higher-quality spermatozoa that actively swim through these channels into the collection chamber, thereby depleting less motile or damaged sperm without exposure to centrifugation or marked shear forces [30]. The Felix system is an electrophoretic device that separates spermatozoa in a gentle electric field according to their negative surface charge (zeta potential), a hallmark of mature sperm. By minimizing physical stress and maintaining a near-physiological environment, it is regarded as a low-stress, non-centrifugal alternative [31]. The comparable reductions in DSB levels achieved by these two distinct systems are consistent with the broader applicability of these findings, although confirmation in larger studies is required.\u003c/p\u003e\n\u003cp\u003eIn the present cohort, a moderate positive correlation between patient age and the DSB-positive rate was observed only in sperm prepared by DGC \u003cstrong\u003e(Fig. 2a)\u003c/strong\u003e, whereas no significant associations were detected for ZyMōt \u003cstrong\u003e(Fig. 2b)\u003c/strong\u003e or Felix \u003cstrong\u003e(Fig. 2c)\u003c/strong\u003e. While this analysis is exploratory and limited by the small sample size and relatively narrow age range, it raises the possibility that centrifugation-based preparation could be more sensitive to age-related variation in DNA integrity, although this inference remains speculative. Aging is accompanied by increased oxidative stress and a decline in the efficiency of DNA repair pathways in the male germ line and semen [32–35]. In vitro manipulations that involve centrifugation have been suggested to generate additional oxidative stress [15], which could further increase the burden of DSBs in older men. By contrast, non-centrifugal systems such as ZyMōt and Felix may help to minimize exposure to age-related variation in sperm DNA integrity by avoiding high g-forces and reducing the generation of ROS during processing. Larger studies explicitly designed to investigate paternal age across different preparation methods will be required to confirm these hypotheses.\u003c/p\u003e\n\u003cp\u003eRegarding sperm recovery, the DGC group yielded the highest number of spermatozoa, whereas ZyMōt and Felix resulted in lower recovery \u003cstrong\u003e(Fig. 3a)\u003c/strong\u003e. This difference is likely attributable to the inherent principles of each method. DGC recovers a broad sperm fraction by centrifugation, achieving high yield efficiently [15]. In contrast, ZyMōt employs a microchannel structure that enriches progressively motile sperm capable of migrating through microchannels, and previous studies have also reported lower absolute sperm recovery compared with DGC [16]. Similarly, Felix separates spermatozoa based on electrophoretic mobility, enriching mature cells characterized by negative surface charge, intact membranes, and appropriate size [17,18].\u0026nbsp;Regarding sperm motility, the ZyMōt yielded the highest motility, whereas Felix showed motility comparable to DGC \u003cstrong\u003e(Fig. 3b)\u003c/strong\u003e. This observation is consistent with the underlying principles of each method. ZyMōt selectively isolates sperm capable of actively migrating through microchannels, thereby enriching progressively motile spermatozoa [16]. In contrast, Felix separates sperm based on surface charge and membrane integrity rather than motility itself. Consequently, some spermatozoa that retain a negative surface charge but have lost motility may also be recovered, resulting in motility rates similar to those observed with DGC [17,18].\u0026nbsp;Regarding processing time, the Felix group exhibited the shortest duration among the three methods \u003cstrong\u003e(Fig. 3c)\u003c/strong\u003e. Felix is based on an electrophoretic principle and employs a standardized protocol that enables completion of sperm separation within a few minutes, offering a practical advantage that may improve workflow efficiency, particularly in contexts with workforce shortages such as embryology laboratories in Japan [36].\u003c/p\u003e\n\u003cp\u003eTaken together, these method-specific characteristics suggest that the three systems may be regarded as complementary approaches, depending on clinical priorities and laboratory workflow. When the primary aim is to minimize DSB burden in couples with recurrent implantation failure, recurrent pregnancy loss, or advanced maternal age, non-centrifugal approaches such as ZyMōt or Felix may be prioritized, with the choice between them guided by the relative importance of motility enrichment versus rapid and standardized processing. By contrast, in situations where high sperm yield is essential or access to newer devices is limited, conventional DGC remains a pragmatic option, as supported by our findings on sperm recovery. Future studies that prospectively assign sperm preparation methods according to these clinical scenarios may help to refine such individualized algorithms and clarify their impact on ART outcomes.\u003c/p\u003e\n\u003cp\u003eA key strength of the present study is the within-sample design, in which each ejaculate was processed in parallel by all three sperm preparation methods. This approach effectively controls for inter-individual variability in baseline semen characteristics and allows more robust paired comparisons of DSB-positive rates, sperm recovery, motility, and processing time. Another strength is the focus on DSBs as quantified by γH2AX, a marker that is mechanistically linked to DNA double-strand break repair foci rather than to generic DNA fragmentation. By integrating a DSB-specific endpoint with practical laboratory metrics, this study provides information that may support method selection in ART laboratories. Furthermore, the use of commercially available devices and widely adopted media platforms enhances the generalizability of our findings across ART laboratories with comparable infrastructure.\u003c/p\u003e\n\u003cp\u003eThis study has several limitations. First, the number of cases was small, and the study was conducted at a single center. Second, the evaluated parameters were limited to laboratory outcomes and did not include direct assessment of clinical outcomes. Third, sperm motility was assessed by visual observation rather than by computer-assisted analysis. Nevertheless, this study provides new insights into the evaluation of sperm selection methods by directly comparing three different techniques under identical conditions, using highly sensitive γH2AX-based quantification of DSBs. Future large-scale, multicenter studies are warranted to determine how reducing sperm DNA damage can translate into improved fertilization, embryo development, and pregnancy outcomes in ART. The present findings also highlight opportunities to refine sperm preparation protocols. Adjusting centrifugation parameters or incorporating antioxidant buffering during DGC may reduce ROS generation and limit iatrogenic DNA damage. Similarly, microfluidic and electrophoretic systems may be further optimized through channel geometry refinement, voltage modulation, or integration with real-time sperm quality sensors. These technological advancements could eventually lead to hybrid selection systems that combine motility-driven and charge-based separation to maximize the recovery of high-quality spermatozoa.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eTo our knowledge, this study is among the first to quantitatively compare sperm populations obtained using three sperm preparation methods under identical conditions by assessing DSBs through \u0026gamma;H2AX immunostaining. Both non-centrifugal systems (ZyMōt and Felix) were associated with lower proportions of DSB-positive sperm than DGC and showed broadly comparable effects, while each system retained distinct practical advantages. Future large-scale, multicenter prospective studies should determine whether reducing sperm DNA damage translates into improved fertilization, embryo development, pregnancy, and live birth, enabling optimization of sperm preparation according to patient profiles and laboratory settings. In the long term, incorporating DSB-focused sperm assessment into routine andrology workflows, together with appropriate selection of non-centrifugal preparation methods, may contribute to more personalized ART strategies. As additional evidence accumulates, these findings may help inform future evaluations of sperm preparation protocols that consider sperm DSBs. Their relevance to ART practice will need to be verified in larger studies that evaluate clinical outcomes.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the ethics committee of our institution and was conducted in accordance with the Declaration of Helsinki and relevant national regulations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInformed consent for participation in this study was obtained using an opt-out procedure implemented via an in-clinic notice and the clinic’s website.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors received no support from any organization for the submitted work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no competing interests to declare that are relevant to the content of this article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAgarwal A, Mulgund A, Hamada A, Chyatte MR. A unique view on male infertility around the globe. Reprod Biol Endocrinol. 2015;13:37; https://doi.org/10.1186/s12958-015-0032-1\u003c/li\u003e\n\u003cli\u003eVander Borght M, Wyns C. Fertility and infertility: definition and epidemiology. Clin Biochem. 2018;62:2\u0026ndash;10; https://doi.org/10.1016/j.clinbiochem.2018.03.012\u003c/li\u003e\n\u003cli\u003eWHO laboratory manual for the examination and processing of human semen. 6th ed. Geneva: World Health Organization; 2021. https://www.who.int/publications/i/item/9789240030787. Accessed 21 Nov 2025.\u003c/li\u003e\n\u003cli\u003eCasanovas A, Ribas-Maynou J, Lara-Cerrillo S, Jimenez-Macedo AR, Hortal O, Benet J, et al. Double-stranded sperm DNA damage is a cause of delay in embryo development and can impair implantation rates. Fertil Steril. 2019;111:699\u0026ndash;707.e1; https://doi.org/10.1016/j.fertnstert.2018.11.035\u003c/li\u003e\n\u003cli\u003eTesarik J, Greco E, Mendoza C. Late, but not early, paternal effect on human embryo development is related to sperm DNA fragmentation. Hum Reprod. 2004;19:611\u0026ndash;5; https://doi.org/10.1093/humrep/deh127\u003c/li\u003e\n\u003cli\u003eZhao J, Zhang Q, Wang Y, Li Y. Whether sperm deoxyribonucleic acid fragmentation has an effect on pregnancy and miscarriage after in vitro fertilization/intracytoplasmic sperm injection: a systematic review and meta-analysis. Fertil Steril. 2014;102:998\u0026ndash;1005.e8; https://doi.org/10.1016/j.fertnstert.2014.06.033\u003c/li\u003e\n\u003cli\u003eSimon L, Zini A, Dyachenko A, Ciampi A, Carrell DT. A systematic review and meta-analysis to determine the effect of sperm DNA damage on in vitro fertilization and intracytoplasmic sperm injection outcome. Asian J Androl. 2017;19:80\u0026ndash;90; https://doi.org/10.4103/1008-682X.182822\u003c/li\u003e\n\u003cli\u003eMuratori M, Marchiani S, Tamburrino L, Baldi E. Sperm DNA fragmentation: mechanisms of origin. In: Baldi E, Muratori M, editors. Genetic Damage in Human Spermatozoa. Advances in Experimental Medicine and Biology. Cham: Springer; 2019. pp. 75\u0026ndash;85.\u003c/li\u003e\n\u003cli\u003eAgarwal A, Majzoub A, Baskaran S, Panner Selvam MK, Cho CL, Henkel R, et al. Sperm DNA fragmentation: a new guideline for clinicians. World J Mens Health. 2020;38:412\u0026ndash;71; https://doi.org/10.5534/wjmh.200128\u003c/li\u003e\n\u003cli\u003eAgarwal A, Barbăroșie C, Ambar R, Finelli R. The impact of single- and double-strand DNA breaks in human spermatozoa on assisted reproduction. Int J Mol Sci. 2020;21:3882; https://doi.org/10.3390/ijms21113882\u003c/li\u003e\n\u003cli\u003eLi N, Wang H, Zou S, Yu X, Li J. Perspective in the mechanisms for repairing sperm DNA damage. Reprod Sci. 2025;32:41\u0026ndash;51. https://doi.org/10.1007/s43032-024-01714-5\u003c/li\u003e\n\u003cli\u003eKaltsas A, Zikopoulos A, Vrachnis D, Skentou C, Symeonidis EN, Dimitriadis F, et al. Advanced Paternal Age in Focus: Unraveling Its Influence on Assisted Reproductive Technology Outcomes. J Clin Med. 2024;13:2731; https://doi.org/10.3390/jcm13102731\u003c/li\u003e\n\u003cli\u003eHalvaei I, Litzky J, Esfandiari N. Advanced paternal age: effects on sperm parameters, assisted reproduction outcomes and offspring health. Reprod Biol Endocrinol. 2020;18:110; https://doi.org/10.1186/s12958-020-00668-y\u003c/li\u003e\n\u003cli\u003eAgarwal A, Ikemoto I, Loughlin KR. Effect of sperm washing on levels of reactive oxygen species in semen. Arch Androl. 1994;33:157\u0026ndash;62; https://doi.org/10.3109/01485019408987819\u003c/li\u003e\n\u003cli\u003eFleming S, Morroll D, Nijs M. Sperm separation and selection techniques to mitigate sperm DNA damage. Life (Basel). 2025;15:302; https://doi.org/10.3390/life15020302\u003c/li\u003e\n\u003cli\u003eHsu CT, Lee CI, Lin FS, Wang FZ, Chang HC, Wang TE, et al. Live motile sperm sorting device for enhanced sperm-fertilization competency: comparative analysis with density-gradient centrifugation and microfluidic sperm sorting. J Assist Reprod Genet. 2023;40:1855\u0026ndash;64; https://doi.org/10.1007/s10815-023-02838-4\u003c/li\u003e\n\u003cli\u003eShapouri F, Mahendran T, Govindarajan M, Xie P, Kocur O, Palermo GD, et al. A comparison between the Felix\u0026trade; electrophoretic system of sperm isolation and conventional density gradient centrifugation: a multicentre analysis. J Assist Reprod Genet. 2023;40:83\u0026ndash;95; https://doi.org/10.1007/s10815-022-02680-0\u003c/li\u003e\n\u003cli\u003eVilleneuve P, Saez F, Hug E, Chorfa A, Guiton R, Schubert B, et al. Spermatozoa isolation with Felix\u0026trade; outperforms conventional density gradient centrifugation preparation in selecting cells with low DNA damage. Andrology. 2023;11:1593\u0026ndash;604; https://doi.org/10.1111/andr.13384\u003c/li\u003e\n\u003cli\u003eCoban O, Serdarogullari M, Yarkiner Z, Serakinci N. Investigating the level of DNA double-strand break in human spermatozoa and its relation to semen characteristics and IVF outcome using phospho-histone H2AX antibody as a biomarker. Andrology. 2020;8:421\u0026ndash;6; https://doi.org/10.1111/andr.12689\u003c/li\u003e\n\u003cli\u003eKanda Y. Investigation of the freely available easy-to-use software \u0026ldquo;EZR\u0026rdquo; for medical statistics. Bone Marrow Transplant. 2013;48:452\u0026ndash;8; https://doi.org/10.1038/bmt.2012.244\u003c/li\u003e\n\u003cli\u003eNewman H, Catt S, Vining B, Vollenhoven B, Horta F. DNA repair and response to sperm DNA damage in oocytes and embryos, and the potential consequences in ART: a systematic review. Mol Hum Reprod. 2022;28:gaab071; https://doi.org/10.1093/molehr/gaab071\u003c/li\u003e\n\u003cli\u003eHorta F, Catt S, Ramachandran P, Vollenhoven B, Temple-Smith P. Female ageing affects the DNA repair capacity of oocytes in IVF using a controlled model of sperm DNA damage in mice. Hum Reprod. 2020;35:529\u0026ndash;44; https://doi.org/10.1093/humrep/dez308\u003c/li\u003e\n\u003cli\u003eSetti AS, Braga DP de AF, Provenza RR, Iaconelli A, Borges E. Oocyte ability to repair sperm DNA fragmentation: the impact of maternal age on intracytoplasmic sperm injection outcomes. Fertil Steril. 2021;116:123\u0026ndash;9; https://doi.org/10.1016/j.fertnstert.2020.10.045\u003c/li\u003e\n\u003cli\u003ePardi\u0026ntilde;as ML, de Celis C, Gil J, Ortega-Jaen D, Martin A, Mercader A, et al. Oocyte-mediated repair of sperm DNA fragmentation: a critical determinant of embryo viability. Reprod Biomed Online. 2025:105165; https://doi.org/10.1016/j.rbmo.2025.105165\u003c/li\u003e\n\u003cli\u003eGarc\u0026iacute;a-Rodr\u0026iacute;guez A, Gos\u0026aacute;lvez J, Agarwal A, Roy R, Johnston S. DNA damage and repair in human reproductive cells. Int J Mol Sci. 2018;20:31; https://doi.org/10.3390/ijms20010031\u003c/li\u003e\n\u003cli\u003eLara-Cerrillo S, Ribas-Maynou J, Rosado-Iglesias C, Lacruz-Ruiz T, Benet J, Garc\u0026iacute;a-Peir\u0026oacute; A. Sperm selection during ICSI treatments reduces single- but not double-strand DNA break values compared to the semen sample. J Assist Reprod Genet. 2021;38:1187\u0026ndash;96; https://doi.org/10.1007/s10815-021-02129-w\u003c/li\u003e\n\u003cli\u003eHeylmann D, Kaina B. The \u0026gamma;H2AX DNA damage assay from a drop of blood. Sci Rep. 2016;6:22682; https://doi.org/10.1038/srep22682\u003c/li\u003e\n\u003cli\u003eGarolla A, Cosci I, Bertoldo A, Sartini B, Boudjema E, Foresta C. DNA double-strand breaks in human spermatozoa can be predictive for assisted reproductive outcome. Reprod Biomed Online. 2015;31:100\u0026ndash;7; https://doi.org/10.1016/j.rbmo.2015.03.009\u003c/li\u003e\n\u003cli\u003eAitken RJ, Clarkson JS. Significance of reactive oxygen species and antioxidants in defining the efficacy of sperm preparation techniques. J Androl. 1988;9:367\u0026ndash;376. https://doi:10.1002/j.1939-4640.1988.tb01067.x. \u003c/li\u003e\n\u003cli\u003eGisbert Iranzo A, Cano-Extremera M, Herv\u0026aacute;s I, Falquet Guillem M, Gil Juli\u0026aacute; M, Navarro-Gomezlechon A, et al. Sperm selection using microfluidic techniques significantly decreases sperm DNA fragmentation (SDF), enhancing reproductive outcomes: a systematic review and meta-analysis. Biology (Basel). 2025;14:792. https://doi.org/10.3390/biology14070792\u003c/li\u003e\n\u003cli\u003eCariati F, Orsi MG, Bagnulo F, Del Mondo D, Vigilante L, De Rosa M, et al. Advanced sperm selection techniques for assisted reproduction. J Pers Med. 2024;14:726. https://doi.org/10.3390/jpm14070726\u003c/li\u003e\n\u003cli\u003eNguyen-Powanda P, Robaire B. Oxidative stress and reproductive function in the aging male. Biology (Basel). 2020;9:1\u0026ndash;15; https://doi.org/10.3390/biology9090282\u003c/li\u003e\n\u003cli\u003eHerati AS, Zhelyazkova BH, Butler PR, Lamb DJ. Age-related alterations in the genetics and genomics of the male germ line. Fertil Steril. 2017;107:319\u0026ndash;23; https://doi.org/10.1016/j.fertnstert.2016.12.021\u003c/li\u003e\n\u003cli\u003eEl\u0026iacute;as-Llumbet A, Lira S, Manterola M. Male aging in germ cells: What are we inheriting? Genet Mol Biol. 2025;47:e20240052; https://doi.org/10.1590/1678-4685-gmb-2024-0052\u003c/li\u003e\n\u003cli\u003eNago M, Arichi A, Omura N, Iwashita Y, Kawamura T, Yumura Y. Aging increases oxidative stress in semen. Investig Clin Urol. 2021;62:233\u0026ndash;8; https://doi:10.4111/icu.20200066\u003c/li\u003e\n\u003cli\u003eShirasawa H, Yamada M, Jwa SC, Kuroda K, Harada M, Osuga Y. Assessment of embryologist sufficiency and associated regional disparities in Japanese assisted reproduction facilities using nationwide survey data (IZANAMI project). J Obstet Gynaecol Res. 2024;50:1459\u0026ndash;69; https://doi.org/10.1111/jog.16022\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"DNA double-strand breaks, Sperm DNA fragmentation, Microfluidics, Electrophoresis, Male infertility","lastPublishedDoi":"10.21203/rs.3.rs-8291848/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8291848/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003ePurpose:\u003c/strong\u003eDensity gradient centrifugation (DGC) is widely used for sperm preparation, but centrifugation-induced oxidative stress may cause DNA damage. This study compared sperm DNA double-strand breaks (DSBs) among DGC, ZyMōt, and Felix, and evaluated sperm recovery, motility, and processing time.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003eFifteen fresh semen samples collected from January to June 2025 were processed in parallel using DGC, ZyMōt, and Felix. Following sperm preparation, γH2AX immunostaining was performed, and at least 200 spermatozoa per sample were analyzed to determine the DSB-positive rate. Sperm recovery, motility, and processing time were also recorded. Statistical analyses were conducted using the Friedman test followed by Wilcoxon signed-rank tests with Bonferroni correction, and data were expressed as medians with interquartile ranges.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003eThe DSB-positive rate was significantly lower in the ZyMōt (11.8%) and Felix (10.0%) groups compared with the DGC group (16.0%; \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01). DGC yielded the highest sperm recovery, ZyMōt achieved the highest motility, and Felix required the shortest processing time, indicating that the three methods exhibit distinct performance characteristics.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003eThe non-centrifugal systems ZyMōt and Felix significantly reduced DSB-positive sperm compared with DGC while maintaining comparable overall performance, highlighting their potential usefulness as optimized sperm preparation approaches in assisted reproductive technology.\u003c/p\u003e","manuscriptTitle":"Evaluation of DNA Double-Strand Breaks in Human Sperm Following Selection by Density Gradient Centrifugation, ZyMōt, and Felix Techniques","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-15 08:48:28","doi":"10.21203/rs.3.rs-8291848/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f614620f-8dd6-48f2-b65d-4a745534e48f","owner":[],"postedDate":"December 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-15T08:48:28+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-15 08:48:28","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8291848","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8291848","identity":"rs-8291848","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}