Despite shifts in conception patterns and maternal age, global infertility rates have remained relatively stable at approximately 1 in 8 to 1 in 12 couples over the past five decades [1]-[3]. However, recent global surveys suggest that male factor infertility has increased over this same time interval [1]-[5]. Together, these observations imply a shifting landscape in assisted reproductive technologies (ARTs) procedures to treat human infertility, where up to 50% or more of infertility cases now include a malefactor component [6][7].

While significant progress has been made in ARTs over the last fifty years, relatively few advances have directly improved the treatment of male infertility since the introduction of ICSI in the early 1990s. For much of this period, the presence of motile sperm in an ejaculate was assumed to be the primary requirement for successful fertilization [8][9]. However, recent studies demonstrate that motility—or even motility combined with normal morphology—cannot reliably predict positive outcomes or ultimate takehome baby rates [10]. These findings suggest that greater attention should be directed towards underlying biochemical and cellular processes, including DNA fragmentation [11]. [12], mitochondrial function [13][14], and membrane integrity [15], as well as towards techniques that preserve or enhance these functions during natural or assisted fertilization [10][16]-[18].

For the last twenty years, this laboratory has focused on the specimen-collection environment and its potential impact on sperm function [19][20]. A number of studies on this subject have led to the development of a collection system referred to as the Device for Improved Semen Collection (DISC); marketed as (ProteX) [RSI, Frisco, TX]. Several clinical trials have suggested improvements in semen parameters and clinical outcomes associated with the use of DISC for specimen collection [21][22]. The device incorporates physical design features that limit environmental extremes [19]-[22] and chemical components intended to maintain cellular physiological and biochemical properties. These chemical components were recently modified in an effort to further improve the collection environment. A recent equine study, conducted under extreme environmental conditions, demonstrated that both the original DISC and the updated DISC+ better maintained cellular biochemical function compared to traditional specimen collection methods [23]. The objective of the present study was to extend this equine evaluation to human samples under more controlled, clinically comparable conditions.

A total of 45 donor samples were recruited; fifteen failed one or more inclusion criteria and were excluded, leaving the 30 samples required for the 6 × 5 design. Figure 1 shows the baseline averages and ranges for motility (total motility and rapid cells), morphology, and biochemical measures (acrosomeintact and DNAintact cells). Although the experimental design minimized major differences between treatment groups (P = 0.52), natural variation in semen quality resulted in uneven distribution of high and lowparameter samples across the six

Figure 1. Starting values of samples across the six treatment conditions: standard specimen cup (SSC), SSC with media (SSCm), device for improved semen collection (DISC), DISC with media (DISCm), reformulated DISC (DISC+), and reformulated DISC with media (DISC + m). Bars represent starting means; lines indicate the corresponding ranges.

treatments. Therefore, all subsequent analyses were performed on data normalized to each sample’s own zerohour value. This approach reduces individual-sample effects and allows treatmentrelated differences to be evaluated more accurately.

It is important to note that these samples were not processed after collection and were maintained at room temperature for the full 48 hours. Focusing first on motility, Figure 2 shows the normalized motility over time (P < 0.002). As described in earlier studies, the DISCm combination extended motility longer than all other treatments. While the DISC+ maintained motility well for the first 9 hours, it declined rapidly thereafter (Figure 2).

Figure 2. Motility of semen samples over time collected in one of six treatment conditions: standard specimen cup (SSC), SSC with media (SSCm), device for improved semen collection (DISC), DISC with media (DISCm), reformulated DISC (DISC+), and reformulated DISC with media (DISC + m) (P < 0.002). Bars represent mean ± SEM.

Similar patterns were observed for rapid cells (Figure 3; P < 0.006). However, cells stored in either the DISCm or DISC + m maintained higher rapid cell number than either SSC devices between 1 - 9 hrs but all groups except the SSCm were similar at all times points after 12 hrs.

Unlike a previous study in the equine [23], no differences or changes in morphology were observed in the present study. Using WHO4 criteria [25], all samples had morphologies within one standard deviation of the mean (32.8%), regardless of treatment (P = 0.112) or time (P = 0.439).

Acrosome integrity and DNA intactness were used as indicators of biochemical activity. Previous studies have suggested that the DISC environment maintains acrosomeintact cells for longer periods compared to the traditional SSC environment. Data from the present study support this: samples collected in either DISC or DISC+, particularly those with media, maintained intact acrosomes significantly longer than samples collected in the SSC (Figure 4; P < 0.001). Interestingly, SSCm also maintained acrosome integrity in almost 50% of cells until approximately 24 hours.

Figure 3. Normalized Rapid Cell movement seen in semen samples over time collected in one of six treatment conditions: standard specimen cup (SSC), SSC with media (SSCm), device for improved semen collection (DISC), DISC with media (DISCm), reformulated DISC (DISC+), and reformulated DISC with media (DISC + m; P < 0.006). Bars represent mean with SEM.

Figure 4. Influence of the collection environment on maintaining intact acrosomes over time in semen samples collected in one of six treatment conditions: standard specimen cup (SSC), SSC with media (SSCm), device for improved semen collection (DISC), DISC with media (DISCm), reformulated DISC (DISC+), and reformulated DISC with media (DISC + m) (P < 0.001). Bars represent mean ± SEM.

Finally, the HALO technique was used to assess the degree of DNA fragmentation over time. As described in earlier studies in both humans and other species [19][23] (Figure 5), samples collected in DISC with media maintained a higher proportion of DNAintact cells after the 6hour time point compared with the SSC conditions (P < 0.001).

Figure 5. Influence of the collection environment on the maintenance of DNAintact cells over time in semen samples collected in one of six treatment conditions: standard specimen cup (SSC), SSC with media (SSCm), device for improved semen collection (DISC), DISC with media (DISCm), reformulated DISC (DISC+), and reformulated DISC with media (DISC + m) (P < 0.001). Bars represent mean ± SEM.

A second focus of these experiments was to determine whether any differences could be detected within the first hour that might be associated with later manifestations of cell damage. As expected, normalized motility (94% - 103%) and the proportion of rapid cells (95% - 101%) remained very stable during the first hour postcollection regardless of treatment (P = 0.532). Morphology parameters were similarly consistent across all groups at these time points (P = 0.668). Not surprisingly, acrosome reaction—a process that naturally occurs over 3-7 hours—was also similar (P = 0.819), with only 0% - 3% of new cells having undergone the reaction at the zerohour time point. Further, no meaningful DNA fragmentation was detected during the first hour, ranging from 0% - 5% across all treatments at the five time points (P = 0.744).

Humanassisted reproductive technologies are nearly 50 years old, yet treatment of male infertility has changed surprisingly little over the past three decades. Although male factors contribute to approximately half of all infertility, the introduction of ICSI in the early 1990s largely eliminated the incentive to develop interventions aimed at improving sperm quality itself. Instead, the field focused on overcoming sperm deficiencies rather than addressing their underlying causes. Recent advances in preimplantation genetic testing [26][27] and growing evidence that lifestyle [28]-[30], environmental [31]-[33], and exposurerelated risk factors [34]-[36] influence sperm quality and reproductive outcomes [37][38] suggest that this paradigm may be shifting. As paternal contributions to fertility and offspring health become more widely recognized, greater emphasis should be placed on the collection, preservation, and selection of male gametes used in ART.

The DISC device represents one of the first efforts to optimize the sperm collection environment based on the premise that, beyond established patientlevel factors, conditions encountered during collection and early storage may influence sperm quality before laboratory processing even begins. To address these factors, the DISC was designed to maintain a stable microenvironment by regulating specimen temperature, supplying nutrients and buffering agents, and incorporating structurebased ROS scavengers intended to preserve, rather than restore, specimen stability.

This study was undertaken to better define the potential benefits of optimizing the sperm collection environment and to evaluate whether those benefits extend beyond conventional semen parameters. A secondary aim was to compare two different combinations of ROS scavengers to determine whether their effects could be further optimized.

Although declines in sperm quality were observed across all study groups over time, the underlying biological processes responsible for these changes may have differed between collection methods. Samples collected using standard approaches demonstrated evidence of progressive cellular dysfunction, including changes consistent with membrane instability and compromised DNA integrity. In contrast, sperm collected with the DISC maintained more favorable measures of cellular function throughout the observation period, suggesting that optimizing the collection environment may help mitigate some of the biochemical stresses experienced immediately after ejaculation.

Because oxidative stress was not measured directly, the contribution of reactive oxygen species can only be inferred from the downstream cellular changes observed in this study. Nevertheless, the preservation of membrane integrity and DNA stability in DISCcollected samples is consistent with the device’s proposed mechanism of maintaining a more physiologically stable environment during collection and early storage. The secondgeneration scavenger package, developed in consultation with plastics experts, appeared superior to both the SSC and the firstgeneration formulation in protecting biochemical processes. However, the rapid decline in physiological function suggests that this formulation may have been too efficient at removing ROS from culture media, potentially reducing ROS levels below those required for normal cellular function. These findings parallel recent equine data demonstrating preservation of membranes, DNA, and mitochondria [23] and are supported by previously described clinical observations [21][22].

Together, these findings continue to suggest that the semen collection environment may have a direct influence on clinical outcomes. However, the inability to detect changes within the first hour of specimen collection does not support this hypothesis. Because early changes may be subtle and only manifested over time, future studies incorporating more sensitive assays will be required to confirm or refute the role of the collection environment. Such studies should include direct measurement of ROS within the media at all time points.

The authors wish to thank the assistance RSI in manufacture of the DISC+ with the modified scavenger system particle funding for this study. Finally, the authors acknowledge the support of the Texas Tech University Innovation HUB for supplying additional funding used in support of this project.

S.P. and L.P. participated in all aspects of this project from conception to final manuscript preparation. K.A. and B.S. provided help with manuscript 6preparation and independent review of data. All authors have read and agreed to the published version of the manuscript.