Efficacy and Safety Evaluation of HyperSperm Treatment in Human Semen Samples

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Martinez-Vallejo, Gabriela Carrasquel-Martinez, Raúl Noblom Artigues, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7809501/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 Jan, 2026 Read the published version in Journal of Translational Medicine → Version 1 posted 4 You are reading this latest preprint version Abstract Background: Assisted reproductive technologies (ART) rely on the functional integrity of spermatozoa, which can be affected by in vitro handling and preparation procedures. HyperSperm is a novel sperm treatment medium developed to enhance sperm function and improve clinical outcomes. This study aimed to evaluate the efficacy and safety of HyperSperm in human semen samples from patients undergoing fertility treatment. Methods: A paired analysis was performed on 135 clinical semen samples, each divided into two equal fractions processed using either standard conditions or the HyperSperm protocol. Sperm motility and kinematic parameters were measured with computer-assisted analysis. Safety assessments included sperm viability at baseline and after 24 hours, DNA fragmentation using a fluorescence-based assay, and acrosomal integrity under spontaneous and progesterone-stimulated conditions. Comparisons between paired samples were analyzed using the Wilcoxon matched-pairs signed-rank test. Results: HyperSperm significantly enhanced sperm kinematic parameters, including curvilinear velocity and amplitude of lateral head displacement, resulting in higher levels of hyperactivated motility. Stratification by semen quality demonstrated that samples with reduced motility showed the greatest functional improvement. HyperSperm did not affect sperm viability or DNA integrity, even after 24 hours of incubation, and preserved acrosomal structure and responsiveness to progesterone. Conclusions: HyperSperm improves critical sperm functional parameters without compromising cellular viability, DNA stability, or acrosomal integrity. These findings support the safe and effective use of HyperSperm to optimize sperm performance and potentially improve outcomes in assisted reproduction. DNA fragmentation Assisted reproduction technologies Sperm infertility Figures Figure 1 Figure 2 Introduction Assisted reproductive technologies (ART) rely heavily on the functional integrity of spermatozoa, which are highly sensitive to environmental insults during handling and processing ( 1 ). The manipulation of sperm cells typically includes a series of steps such as centrifugation, incubation, density gradient, separation, and exposure to various culture conditions ( 2 ). While these procedures are essential for isolating, concentrating, and selecting motile and morphologically normal spermatozoa, they can inadvertently introduce a range of stressors that may impair sperm quality. Mechanical shear forces during centrifugation, osmotic shifts, pH imbalances, temperature fluctuations, and prolonged in vitro incubation are all known to adversely affect sperm physiology ( 3 ). These stressors can compromise key parameters such as membrane integrity, progressive motility, acrosomal status, and DNA fragmentation, ultimately reducing fertilization efficiency and developmental competence. Therefore, it is essential to ensure that any medium or treatment applied during sperm preparation does not exacerbate these effects and maintains a robust safety profile compatible with clinical use. Recently, we reported the development of HyperSperm, a novel sperm treatment medium formulated to promote capacitation-associated signaling pathways and enhance sperm function in vitro, which results in higher blastocyst rates in IVF ( 4 ). In our previous study, we demonstrated that HyperSperm effectively stimulates key molecular events linked to capacitation ( 5 ), including those leading the generation of hyperactivated motility, without compromising basal sperm parameters in healthy donors. These results suggested that HyperSperm could be a promising tool to optimize sperm functional competence during ART procedures. However, before its broader application in clinical settings, it is critical to comprehensively assess its safety profile, particularly in sperm from patients presenting various andrological conditions. These samples often display reduced motility, altered morphology, or increased susceptibility to stress, and may respond differently to in vitro treatments compared to normozoospermic controls. To this end, we extended our analysis to semen samples obtained from patients with a range of andrological diagnoses, including oligozoospermia, asthenozoospermia and teratozoospermia. These conditions often reflect underlying testicular dysfunction or oxidative imbalance that can influence the sperm’s response to in vitro manipulation ( 6 ). We systematically evaluated the impact of HyperSperm treatment on multiple safety and functionality endpoints, including sperm DNA integrity, survival, motility, and acrosomal status. These parameters are well-established indicators of fertilizing potential and embryo development and are routinely considered in ART labs when selecting viable sperm for insemination or injection ( 7 – 9 ). Here, we present evidence that supports the safety of HyperSperm in a clinically relevant context, reinforcing its potential as a valuable tool for enhancing sperm performance during ART, while maintaining critical aspects of sperm quality. This work provides an essential step toward the clinical translation of HyperSperm, supporting its use in routine practice for a wide range of male infertility diagnoses. Methods Semen samples and processing Semen samples included in this study were obtained from surplus material collected during assisted reproduction treatments and routine semen analyses performed at fertility clinics in Barcelona. Eligible participants were males between 18 and 60 years of age, able to obtain a semen sample by masturbation. In addition, the sample sent to the laboratory was accepted if it had at least 1 mL in volume. Exclusion criteria included: a current diagnosis of a sexually transmitted infection (STI); a previous diagnosis of hepatitis A, B, C, or D, or HIV; or prior participation in an interventional clinical trial within the past 3 months; or prior participation in this study. The study protocol was approved by the Clinical Research Ethics Committee of Hospital del Mar (CEIm PSMAR, Study #2024/11686/I), and the clinical trial was registered at ClinicalTrials.gov (NCT06742437). All patients provided written informed consent. Once in the lab, an initial macro- and microscopic evaluation of the semen sample was performed, including measurements of volume, assessment of appearance, pH determination, and analysis of motility, vitality, and concentration using a Neubauer chamber. Each sample was split into two equal fractions in order to carry out a comparative analysis. One fraction was designated as the Control and processed using the standard culture medium and incubation conditions, while the other fraction was processed using the HyperSperm method and media, enabling a side-by-side evaluation of outcomes and differences between both methods. Sperm Processing by Density Gradient Centrifugation A total of 61 semen samples were processed using density gradient centrifugation to isolate motile and morphologically normal spermatozoa, to effectively separate functional sperm from debris, leukocytes, and immotile or abnormal cells. Prior to processing, semen samples were allowed to liquefy at room temperature for a minimum of 30 minutes to ensure uniform viscosity and facilitate accurate handling. Then, samples were layered onto a discontinuous two-step gradient with the 40% layer over the 80% one (PureSperm, Nidacon, Sweden). The volume of each phase was adjusted according to the previously assessed concentration of the sample to maximize recovery efficiency: 0.5 mL per phase was used for concentrations < 30 million/mL, while 1 mL was used for higher concentrations. Approximately 1 mL of liquefied semen was placed on top of the gradient and centrifuged at 300g for 20 minutes. After centrifugation, motile spermatozoa were recovered from the pellet at the bottom of the tube by aspiration and washed once with 3 mL of media. For the Control fraction, the spermatozoa were washed and maintained in modified Human Tubal Fluid (HTF; FUJIFILM Irvine Scientific, Santa Ana, CA, USA) supplemented with 5% Human Serum Albumin (HSA; FUJIFILM Irvine Scientific); whereas samples in the experimental group were processed with HyperSperm media. Sperm Processing by Swim-Up Technique An additional set of 74 semen samples was processed by swim-up, separating motile spermatozoa from immotile or less viable spermatozoa that are left on the sperm layer. Following liquefaction at room temperature, approximately 1 mL of fresh semen was placed on a conical 15 mL falcon tube under 1 mL of culture medium (HTF for Control group and HyperSperm medium for the experimental group) and incubated at 37°C for 1 hour. After incubation, the upper fraction, now enriched with motile spermatozoa, was carefully aspirated avoiding any minimal disturbance of the lower layer to avoid contamination. Then, the supernatant portion was transferred to a new tube. Spermatozoa were then washed and resuspended in HTF for the Control group, or in HyperSperm medium for the HyperSperm group. HyperSperm Treatment After sperm selection by either density gradient or swim-up separation, control sperm were incubated in HTF at 37˚C for up to 4 hours. Spermatozoa in the experimental group were processed using the HyperSperm protocol ( 4 ), a proprietary method designed to optimize sperm performance. This protocol differs from conventional procedures in several key aspects: it employs three specialized culture media, an additional centrifugation step and a prolonged incubation period. Motility analysis Sperm motility was assessed at the beginning of the protocol and at the end of the procedure, for both HyperSperm-treated and Control spermatozoa. Sperm motility assessments were performed using the SCA CASA System-Microptic (Hamilton Thorne, Barcelona, Spain) a Computer-Assisted Sperm Analysis (CASA) system that provides objective and quantitative measurements. Motility was classified according to WHO criteria ( 10 ), classifying as fast progressive, slow progressive, non-progressive and non-motile. For each analysis a 10 uL drop of sperm suspension was placed on a clean microscope slide and covered with an 18 x 18 mm coverslip, creating a space with 30 µm of depth to allow spermatozoa movements. Video recordings were captured at 50 and 60 frames per second (fps), with at least six independent microscopic fields and a minimum of 200 spermatozoa per evaluation. The following kinematic parameters of the spermatozoa were measured: curvilinear velocity (VCL, µm/s), straight line velocity (VSL, µm/s), average path velocity (VAP, µm/s), linearity (LIN: VSL/VCL × 100, %), straightness (STR: VSL/VAP × 100, %), wobble (WOB: VAP/VCL × 100, a measure of sperm head side to side movement, %), amplitude of lateral head displacement (ALH, µm, which measures the magnitude of the lateral displacement of the sperm head about the average path) and beat cross frequency (BCF, Hz). The drift correction threshold was set at 20 µm/s, to ensure accurate exclusion of non-progressive movements from the analysis. Spermatozoa were considered hyperactivated when presenting VCL ≥ 150 µm/s, LIN < 50% and ALH ≥ 3.5 µm ( 9 , 10 ), reflecting a vigorous, asymmetric motility pattern. Viability and survival assessment Sperm viability was assessed using 0.5% eosin Y (Sigma-Aldrich, Darmstadt, Germany) prepared according to the instructions given in the WHO manual ( 10 ). This assay is based on the principle that only spermatozoa with a compromised plasma membrane allow the dye to enter the cell, staining the cytoplasm pink, while intact spermatozoa remain unstained. The viability of Control and HyperSperm-treated spermatozoa were evaluated once the HyperSperm protocol was completed. In addition, a survival analysis was done by repeating the viability assessment 24 hours later. Morphology evaluation Sperm morphology was evaluated using a commercially available Diff-Quik staining kit (Cellavision, Lund, Switzerland) according to the manufacturer’s protocol. A thin semen smear was prepared on a clean glass slide and allowed to air-dry completely. The slide was sequentially immersed in the fixative solution (Solution I) for 1 minute, followed by the eosinophilic staining solution (Solution II) for 45 seconds, and the basophilic staining solution (Solution III) for 1 minute. Slides were rinsed briefly in distilled water and air dried. The stained smears were examined using bright-field microscopy under oil immersion at 1000× magnification. For each sample, at least 200 spermatozoa were evaluated, and morphology was classified according to WHO criteria into normal and abnormal forms ( 10 ). DNA fragmentation assessment (TUNEL assay) The Fluorescein In Situ Cell Death Detection Kit (Roche, Basel, Switzerland) was used for the evaluation of the percentage of spermatozoa with a high level of DNA fragmentation. This method enables the detection of DNA strand breaks by the incorporation of fluorescein-labeled nucleotides at free 3’-OH ends of DNA. After completion of the HyperSperm protocol, aliquots of Control and HyperSperm-treated sperm were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde (Thermo Scientific Chemicals, Waltham, Massachusetts, USA) and placed on a previously prepared coverslip with circles drawn with a Hydrophobic Barrier Pap Pen (Invitrogen, Waltham, Massachusetts, USA). Two additional wells were prepared for the positive control and the negative control. Slides were allowed to evaporate and stored cold until the day of staining. For the staining procedure, cells were permeabilized with 0.1% Triton X-100 in sodium citrate (Sigma Aldrich). The positive control was treated with RNase-free DNase solution (Thermo Scientific), to promote DNA-fragmentation, while the remaining wells were left with PBS to prevent drying. All wells except the negative control were then treated with the labeling solution and enzyme provided by the TUNEL kit. Only the labeling solution was added to the negative control. After this, glycerol with DAPI 1 µg/mL (Sigma-Aldrich) was added and covered with coverslips, leaving the slides ready for viewing under the fluorescence microscope. At least 200 cells were analyzed. Acrosomal status evaluation Aliquots of Control and HyperSperm-treated sperm were taken and divided for spontaneous or progesterone-induced acrosomal reaction evaluation. For the spontaneous reaction, spermatozoa were incubated in the corresponding medium, while for the induced reaction, cells were incubated for 30 minutes with 10 µM progesterone prior to fixation. After incubation, samples were washed with PBS and then fixed with paraformaldehyde on a previously prepared coverslip with circles drawn with a Hydrophobic Barrier Pap Pen. They were allowed to evaporate and stored cold until the day of staining. For analysis, spermatozoa were permeabilized with methanol (Sigma-Aldrich) to facilitate lectin access to acrosomal glycoproteins. Next, 10 µL of Pisum sativum agglutinin conjugated to fluorescein isothiocyanate (FITC-PSA, Sigma-Aldrich) were added and incubated for 1 hour in the dark. PSA binds selectively to acrosomal glycoproteins, enabling differentiation between acrosome-intact and acrosome-reacted spermatozoa. After this, glycerol with DAPI was added to visualize sperm nuclei and covered with coverslips to leave the slides ready for viewing under the fluorescence microscope. At least 200 cells were analyzed. Statistical analysis. Statistical analyses were performed using the GraphPad Prism 6 software (La Jolla, CA, USA). Comparisons between paired samples from the Control and HyperSperm groups were analyzed using Wilcoxon matched pairs signed rank test, a non-parametric test appropriate for paired datasets without normal distribution. Statistical significance was defined as p < 0.05 and the data is displayed as mean ± standard deviation, either in graph form or in table form. Results HyperSperm increases sperm motility parameters resulting in a higher percentage of hyperactivated human sperm A total of 135 semen samples were analyzed using a paired design, in which each sample was divided and processed with either the standard method (Control) or HyperSperm. The age of the patients who participated in this study ranges from 18 to 58 and the summary of the semen parameters is shown in Table 1 . Table 1 Summary of semen parameters of samples and patient characteristics, n = 135. Patient & sample parameters Mean ± Standard Deviation Age (years) 37.7 ± 7.4 BMI 25.3 ± 3.6 Abstinence days 3.1 ± 2.6 Sample volume (mL) 1.3 ± 0.7 pH 7.7 ± 0.3 Sample concentration (M/mL) 70.6 ± 52.3 Morphology (% normal forms) 5.3 ± 3.3 Initial total motility (%) 61.7 ± 21.2 Initial progressive motility (%) 47.1 ± 20.2 Both total (Density gradient - Control: 65.51% ± 23.74 vs. HyperSperm: 68.34% ± 22.42; p = 0.1334; Swim-Up - Control: 75.39% ± 21.07 vs. HyperSperm: 75.52% ± 18.71; p = 0.731) and progressive motility (Density gradient - Control: 55.60% ± 24.48 vs. HyperSperm: 59.44% ± 23.18; p = 0.0528; Swim-up - Control: 66.35% ± 21.81 vs. HyperSperm: 66.29% ± 19.59; p > 0.999) did not significantly differ between Control and HyperSperm groups, whether samples were processed by density gradient or swim-up (Fig. 1 ). In contrast, sperm kinematic parameters revealed significant enhancements following HyperSperm protocol. VCL (Gradient - Control: 89.27 µm/s ± 21.63 vs. HyperSperm: 101.80 µm/s ± 25.83; p < 0.0001 and Swim-Up - Control: 87.24 µm/s ± 15.55 vs. HyperSperm: 97.16 µm/s ± 18.65; p = 0.004) and ALH (Gradient - Control: 1.89 µm ± 0.29 vs. HyperSperm: 2.19 µm ± 0.45; p < 0.0001 and Swim-Up - Control: 1.87 µm ± 0.25 vs. HyperSperm: 2.10 µm ± 0.40; p = 0.0008) were significantly increased, reflecting faster and more vigorous movement. Additional parameters, such WOB, LIN and VAP, also exhibited a significant difference compared to HyperSperm (Fig. 1 ). The increase in VCL and ALH together with the decrease in LIN results leads to an increase in the percentage of hyperactivated motility (Fig. 1 ) in cells treated with HyperSperm. This was observed regardless the use of swim up or density gradient centrifugation (Gradient: Control: 3.07% ± 3.41 vs. HyperSperm: 8.70% ± 9.80; p < 0.0001 and Swim-Up: Control: 2.30% ± 2.79 vs. HyperSperm: 5.36% ± 7.10; p = 0.0004). HyperSperm increases Sperm Motility in Asthenozoospermic Samples While enhancing Hyperactivation Across Most Sample Types The samples were further categorized based on their baseline motility, morphology and concentration, following the WHO criteria, in: normozoospermic samples, oligozoospermic samples (< 15 M/mL), asthenozoospermic samples (< 30% progressive motility or < 42% total motility) and teratozoospermic samples (< 4% normal forms) (Table 2 ). This stratification enabled a clinically relevant evaluation of HyperSperm effects across distinct pathological conditions rather than relying solely on overall population averages. A total of 80 samples were analyzed and 37 of them were classified as normozoospermic, while the rest were categorized as oligozoospermic, asthenozoospermic, or teratozoospermic. Because abnormalities in semen quality often co-occur, the groups were not mutually exclusive. Within the oligozoospermic category (n = 7), 1 was oligozoospermic, 3 oligoasthenozoospermic, and 3 oligoasthenozoospermic. On the other hand, in the asthenozoospermic category (n = 22), 11 were asthenozoospermic, 5 asthenoteratozoospermic, 3 oligoasthenozoospermic, and 3 oligoasthenoteratozoospermic. Finally, of the teratozoospermic cases (n = 28), 20 were teratozoospermic, 5 asthenoteratozoospermic and 3 oligoasthenoteratozoospermic (Table 2 ). Table 2 Effect of HyperSperm treatment on motility parameters for samples with different diagnosis (Mean ± Standard Deviation): A. Normoozoospermic samples, n = 37; B. Oligozoospermic samples, n = 7; C. Asthenozoospermic samples, n = 22. and D. Teratozoospermic samples, n = 28 in total. Comparison between Control and HyperSperm with significance represented as: *, p < 0.05; **, p < 0.01; ***, p < 0.001. Normozoospermic Oligozoospermic Asthenozoospermic Teratozoospermic Parameters Control HyperSperm Control HyperSperm Control HyperSperm Control HyperSperm Motile cells (%) 77.7 ± 16.2 79.48 ± 14.3 35.3 ± 16.0 41.0 ± 17.6 51.9 ± 27.6 56.0 ± 25.0 * 63.3 ± 25.0 64.9 ± 22.6 Progressive motile cells (%) 68.5 ± 18.4 70.2 ± 16.9 25.3 ± 15.1 30.76 ± 18.8 41.7 ± 26.9 48.1 ± 25.2 ** 53.4 ± 24.8 56.0 ± 22.0 VCL (µm/s) 92.7 ± 16.2 107.1 ± 23.2 *** 63.8 ± 13.7 72.3 ± 18.6 * 76.5 ± 20.0 87.0 ± 22.0 ** 90.2 ± 22.9 98.6 ± 22.6 ** VAP (µm/s) 58.3 ± 13.8 61.8 ± 14.7 ** 35.7 ± 12.0 37.9 ± 15.3 46.9 ± 17.4 51.2 ± 17.1 * 55.9 ± 18.5 55.7 ± 16.0 VSL (µm/s) 43.6 ± 16.1 43.9 ± 16.7 27.0 ± 14.3 28.2 ± 18.7 35.4 ± 17.6 38.6 ± 16.4 43.4 ± 20.4 41.4 ± 19.5 STR (%) 60.9 ± 13.0 57.5 ± 11.4 50.0 ± 11.4 46.5 ± 12.5 58.0 ± 14.9 57.6 ± 13.6 58.9 ± 15.8 53.3 ± 13.4 LIN (%) 46.4 ± 17.3 41.33 ± 17.8 * 41.4 ± 20.1 37.3 ± 23.3 43.9 ± 14.9 42.6 ± 14.6 46.4 ± 18.1 41.7 ± 18.9 WOB (%) 63.6 ± 8 58.3 ± 7.6 *** 60.7 ± 10.3 55.5 ± 10.9 * 62.8 ± 7.2 60.0 ± 5.9 * 63.4 ± 7.0 58.2 ± 7.5 ** ALH (µm) 1.9 ± 0.3 2.3 ± 0.5 *** 1.6 ± 0.2 1.7 ± 0.3 * 1.7 ± 0.2 1.9 ± 0.3 *** 1.9 ± 0.3 2.1 ± 0.4 *** BCF (Hz) 15.2 ± 3.1 15.3 ± 2.9 10.0 ± 2.6 10.4 ± 3.7 13.0 ± 3.9 14 ± 3.8 ** 14.7 ± 4.0 14.3 ± 3.3 Hyperactivation per motile cells (%) 3.3 ± 3.1 10.0 ± 10.6 *** 0.83 ± 1.3 2.5 ± 4.5 1.5 ± 2.5 3.5 ± 4.7 * 3.2 ± 3.8 7.1 ± 7.9 *** Motility assessment revealed that HyperSperm treatment did not significantly affect total or progressive motility compared to Control in the case of normozoospermic, oligozoospermic or teratozoospermic samples. In contrast, asthenozoospermic samples showed a significant improvement in motility following HyperSperm treatment compared to the Control, particularly in progressive motility, where the increase was more pronounced. HyperSperm produced a significant increase in several kinematic parameters across different semen categories, in particular in VCL, WOB and ALH (Table 2 ). These changes lead to a significantly increase in the proportion of hyperactivated sperm, a key functional parameter associated with capacitation and fertilizing potential, in all sample types except oligozoospermic samples, although a clear tendency in this latter can be observed (Table 2 ). These findings highlight the potential of HyperSperm as a clinically valuable tool for optimizing sperm function, particularly in patients with impaired motility. HyperSperm does not alter sperm viability and DNA integrity. Sperm viability was assessed using eosin staining after processing with either Control or HyperSperm. Immediately after treatment, no significant differences were observed between the two groups (Control: 81.92% ± 11.98 vs. HyperSperm: 82.92% ± 12.37; p = 0.216) (Fig. 2 A). To evaluate potential long-term effects, viability was reassessed after 24 hours of incubation at room temperature (Fig. 2 B), with no significant differences observed between the groups (Control: 75.84% ± 15.94 vs. HyperSperm: 76.37% ± 16.29; p = 0.756). These results indicate that HyperSperm does not have a detrimental effect on sperm viability when compared to the Control. The safety of HyperSperm treatment was further evaluated by measuring DNA fragmentation using the TUNEL assay. No significant differences in the percentage of DNA-fragmented spermatozoa were observed between the two groups (Control: 5.78% ± 8.04 vs. HyperSperm: 4.39% ± 5.57; p = 0.044) (Fig. 2 C). These findings demonstrate that HyperSperm does not induce DNA damage during sperm preparation. HyperSperm preserves acrosomal integrity. Acrosomal status was evaluated using FITC-PSA staining to assess potential effects on the ability of sperm to undergo the acrosome reaction. At the end of the protocol, Control and HyperSperm samples exhibited a similar proportion of acrosome-reacted spermatozoa (Control: 14.06% ± 6.12 vs. HyperSperm: 14.9% ± 7.51; p = 0.226) indicating that the treatment did not produce an abnormal loss or degeneration of the acrosome in the treated sperm (Fig. 2 D). In addition, after acrosome reaction induction with progesterone no significant differences were observed (Control: 24.42% ± 16.69 vs. HyperSperm: 25.02% ± 15.12; p = 0.754) (Fig. 2 E). These data support that HyperSperm does not affect acrosomal integrity and maintains sperm functionality for fertilization. Discussion The present study expands upon our previous findings on HyperSperm, a novel sperm treatment medium designed to enhance reproductive outcomes after ART. By evaluating its safety and efficacy in a diverse cohort of clinical semen samples, we observed consistent and statistically significant improvements in motility kinematic parameters such as VCL and ALH. Importantly, HyperSperm nearly doubled the percentage of hyperactivated spermatozoa compared to the standard method, a change consistent with the acquisition of capacitation-related motility ( 11 ). These results confirm that HyperSperm not only improves basic motility parameters but also promotes key functional hallmarks required for fertilization. Hyperactivation is critical for fertility because it enables sperm to generate the high-amplitude, asymmetric flagellar movements needed to detach from the oviductal epithelium, penetrate the viscoelastic cumulus matrix, and ultimately drill through the zona pellucida. At a clinical level, sperm that fail to undergo hyperactivation are unable to reach or fertilize the oocyte, and deficiencies in this motility pattern have been strongly associated with reduced success rates in both natural conception and assisted reproduction ( 12 , 13 ). A major strength of this study is the stratification of patient samples by underlying andrological condition, allowing for a nuanced understanding of HyperSperm’s effects across clinically relevant subpopulations. While normozoospermic and teratozoospermic samples showed no significant motility gains beyond those achieved with standard processing, HyperSperm produced marked improvements in semen samples with pathological conditions that often present challenges in ART. Additionally, hyperactivation levels were increased in most subgroups, suggesting that HyperSperm is broadly effective in enhancing functional competence regardless of initial sperm quality. These results highlight its potential clinical value, particularly in patients with suboptimal semen parameters. Beyond functional improvements, HyperSperm did not compromise cell viability or DNA integrity. These results suggest that reactive oxygen species (ROS) production is not exacerbated in comparison with the standard methods such as swim up or discontinuous gradient centrifugation. Viability is particularly sensitive to the aggression of ROS and that has an immediate impact on sperm motility ( 14 ), which is also unaltered in our conditions. This demonstration is a key safety requirement for any ART-related application. Excessive ROS generation is a major cause of male infertility ( 6 ), as it leads to oxidative damage of sperm membranes, impaired motility, and DNA fragmentation, all of which reduce fertilization potential. Therefore, maintaining ROS at physiological levels is essential to preserve sperm functionality and ensure optimal outcomes in ART. It is worth noting that some sperm selection methods have been reported to reduce DNA damage, adding potential value in cases with elevated sperm DNA fragmentation ( 15 ). In our study, this aspect could not be addressed because all samples analyzed presented low baseline levels of DNA damage; however, testing HyperSperm in samples with higher DNA fragmentation will be an important next step. The preservation of acrosomal integrity further reinforces the safety profile of HyperSperm. Neither spontaneous nor progesterone-induced acrosome reaction rates were altered by the treatment, indicating that the medium does not prematurely trigger or interfere with this critical step in fertilization. As discussed earlier, HyperSperm significantly enhanced hyperactivation, a hallmark of capacitation, without inducing acrosome exocytosis. This suggests that HyperSperm effectively stimulates upstream signaling pathways associated with capacitation, while maintaining control over downstream events such as the acrosome reaction. This balance is particularly important, given that while acrosome-reacted sperm are known to be capable of penetrating and fusing with the egg ( 16 , 17 ), premature acrosome reaction prior to encountering the oocyte can lead to a loss of fertilizing capacity. Thus, our findings support the idea that HyperSperm promotes capacitation in a physiologically regulated manner, enhancing functional competence without compromising fertilization potential. Conclusions This study provides strong evidence that HyperSperm enhances sperm motility and hyperactivation without compromising viability, DNA integrity, or acrosomal status. While further studies assessing fertilization and pregnancy outcomes are warranted, our findings support the clinical adoption of HyperSperm as a safe and effective tool for optimizing sperm function in ART settings. Declarations Acknowledgments: We would like to thank Nuria Correa Mañas and Júlia García Mulet for their technical assistance. Funding: This work has been carried out with the financial support of the Industrial Doctorates program, funded through a grant from the Agency for Management of University and Research Grants (AGAUR), Government of Catalonia. Competing interest: MGB and MDGE are shareholders of Fecundis. The rest of the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Authors' contributions: MJMV and GCM performed the experiments and analyzed data. RNA, KL, RLV, MRMFC, SRG discussed results and contributed with samples and resources. EI, MDGE, MJMV and MGB analyzed and interpreted the patient data. MGB and MJMV wrote the paper with contributions of all coauthors. MGB designed the study Ethics approval and consent to participate: The study protocol was approved by the Clinical Research Ethics Committee of Hospital del Mar (CEIm PSMAR, Study #2024/11686/I), and the clinical trial was registered at ClinicalTrials.gov (NCT06742437). Consent for publication : Not applicable Availability of data and materials: The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. References Said TM, Land JA. Effects of advanced selection methods on sperm quality and ART outcome: a systematic review. Hum Reprod Update. 2011;17(6):719–33. Pinto S, Carrageta DF, Alves MG, Rocha A, Agarwal A, Barros A, et al. Sperm selection strategies and their impact on assisted reproductive technology outcomes. Andrologia. 2021;53(2):e13725. Leahy T, Gadella BM. 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Am J Hum Genet. 2009;84(4):505–10. Drevet JR, Aitken RJ. Oxidative Damage to Sperm DNA: Attack and Defense. Adv Exp Med Biol. 2019;1166:107–17. Marinaro J, Schlegel PN. Sperm DNA Fragmentation and Fertility. Adv Exp Med Biol. 2025;1469:305–32. La Spina FA, Puga Molina LC, Romarowski A, Vitale AM, Falzone TL, Krapf D, et al. Mouse sperm begin to undergo acrosomal exocytosis in the upper isthmus of the oviduct. Dev Biol. 2016;411(2):172–82. Sosnik J, Miranda PV, Spiridonov NA, Yoon SY, Fissore RA, Johnson GR, et al. Tssk6 is required for Izumo relocalization and gamete fusion in the mouse. J Cell Sci. 2009;122(Pt 15):2741–9. Cite Share Download PDF Status: Published Journal Publication published 22 Jan, 2026 Read the published version in Journal of Translational Medicine → Version 1 posted Reviewers agreed at journal 15 Oct, 2025 Reviewers invited by journal 12 Oct, 2025 Editor assigned by journal 09 Oct, 2025 First submitted to journal 08 Oct, 2025 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-7809501","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":528233562,"identity":"d79a47ef-7d6c-4e05-b546-229602781e11","order_by":0,"name":"Maria J. 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13:57:47","extension":"html","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":89088,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7809501/v1/06669d95191324aa7bb4b622.html"},{"id":94397933,"identity":"15967473-c7e1-4a4a-a290-95f064d36db4","added_by":"auto","created_at":"2025-10-27 13:56:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":112501,"visible":true,"origin":"","legend":"\u003cp\u003eTotal motility (progressive and non-progressive) and motility kinematic parameters measured by SCA CASA System by Hamilton Thorne, n=80. *, p\u0026lt;0.05; **, p\u0026lt;0.01; ***, p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7809501/v1/7fa05b1ebaabd245e8bc7c41.png"},{"id":94399098,"identity":"b70c83ec-ef2e-4926-8875-99aff904b136","added_by":"auto","created_at":"2025-10-27 13:57:21","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":74324,"visible":true,"origin":"","legend":"\u003cp\u003eComparative analysis of sperm viability, DNA fragmentation, and acrosome reaction in Control vs. HyperSperm treatment. A. Vitality immediately after treatment (n=122). B. Vitality after 24 hours (n=122). C. DNA fragmentation levels based on the percentage of TUNEL-positive cells (n=41). D. Spontaneous acrosome reaction (n =48). E. Induced acrosome reaction with progesterone (n = 17).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7809501/v1/877f13b8b3f7fc27f0f5a67b.png"},{"id":101153281,"identity":"2f86cde4-ca7e-4a52-9518-4d94c1c8ab86","added_by":"auto","created_at":"2026-01-26 16:14:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1032389,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7809501/v1/76343875-16ca-4209-a674-c3fc9be1c36e.pdf"}],"financialInterests":"","formattedTitle":"Efficacy and Safety Evaluation of HyperSperm Treatment in Human Semen Samples","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAssisted reproductive technologies (ART) rely heavily on the functional integrity of spermatozoa, which are highly sensitive to environmental insults during handling and processing (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). The manipulation of sperm cells typically includes a series of steps such as centrifugation, incubation, density gradient, separation, and exposure to various culture conditions (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). While these procedures are essential for isolating, concentrating, and selecting motile and morphologically normal spermatozoa, they can inadvertently introduce a range of stressors that may impair sperm quality. Mechanical shear forces during centrifugation, osmotic shifts, pH imbalances, temperature fluctuations, and prolonged in vitro incubation are all known to adversely affect sperm physiology (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). These stressors can compromise key parameters such as membrane integrity, progressive motility, acrosomal status, and DNA fragmentation, ultimately reducing fertilization efficiency and developmental competence. Therefore, it is essential to ensure that any medium or treatment applied during sperm preparation does not exacerbate these effects and maintains a robust safety profile compatible with clinical use.\u003c/p\u003e\u003cp\u003eRecently, we reported the development of HyperSperm, a novel sperm treatment medium formulated to promote capacitation-associated signaling pathways and enhance sperm function in vitro, which results in higher blastocyst rates in IVF (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). In our previous study, we demonstrated that HyperSperm effectively stimulates key molecular events linked to capacitation (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e), including those leading the generation of hyperactivated motility, without compromising basal sperm parameters in healthy donors. These results suggested that HyperSperm could be a promising tool to optimize sperm functional competence during ART procedures. However, before its broader application in clinical settings, it is critical to comprehensively assess its safety profile, particularly in sperm from patients presenting various andrological conditions. These samples often display reduced motility, altered morphology, or increased susceptibility to stress, and may respond differently to in vitro treatments compared to normozoospermic controls.\u003c/p\u003e\u003cp\u003e To this end, we extended our analysis to semen samples obtained from patients with a range of andrological diagnoses, including oligozoospermia, asthenozoospermia and teratozoospermia. These conditions often reflect underlying testicular dysfunction or oxidative imbalance that can influence the sperm\u0026rsquo;s response to in vitro manipulation (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). We systematically evaluated the impact of HyperSperm treatment on multiple safety and functionality endpoints, including sperm DNA integrity, survival, motility, and acrosomal status. These parameters are well-established indicators of fertilizing potential and embryo development and are routinely considered in ART labs when selecting viable sperm for insemination or injection (\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eHere, we present evidence that supports the safety of HyperSperm in a clinically relevant context, reinforcing its potential as a valuable tool for enhancing sperm performance during ART, while maintaining critical aspects of sperm quality. This work provides an essential step toward the clinical translation of HyperSperm, supporting its use in routine practice for a wide range of male infertility diagnoses.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eSemen samples and processing\u003c/h2\u003e\u003cp\u003eSemen samples included in this study were obtained from surplus material collected during assisted reproduction treatments and routine semen analyses performed at fertility clinics in Barcelona. Eligible participants were males between 18 and 60 years of age, able to obtain a semen sample by masturbation. In addition, the sample sent to the laboratory was accepted if it had at least 1 mL in volume. Exclusion criteria included: a current diagnosis of a sexually transmitted infection (STI); a previous diagnosis of hepatitis A, B, C, or D, or HIV; or prior participation in an interventional clinical trial within the past 3 months; or prior participation in this study. The study protocol was approved by the Clinical Research Ethics Committee of Hospital del Mar (CEIm PSMAR, Study #2024/11686/I), and the clinical trial was registered at ClinicalTrials.gov (NCT06742437).\u003c/p\u003e\u003cp\u003e All patients provided written informed consent. Once in the lab, an initial macro- and microscopic evaluation of the semen sample was performed, including measurements of volume, assessment of appearance, pH determination, and analysis of motility, vitality, and concentration using a Neubauer chamber. Each sample was split into two equal fractions in order to carry out a comparative analysis. One fraction was designated as the Control and processed using the standard culture medium and incubation conditions, while the other fraction was processed using the HyperSperm method and media, enabling a side-by-side evaluation of outcomes and differences between both methods.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eSperm Processing by Density Gradient Centrifugation\u003c/h3\u003e\n\u003cp\u003eA total of 61 semen samples were processed using density gradient centrifugation to isolate motile and morphologically normal spermatozoa, to effectively separate functional sperm from debris, leukocytes, and immotile or abnormal cells. Prior to processing, semen samples were allowed to liquefy at room temperature for a minimum of 30 minutes to ensure uniform viscosity and facilitate accurate handling. Then, samples were layered onto a discontinuous two-step gradient with the 40% layer over the 80% one (PureSperm, Nidacon, Sweden). The volume of each phase was adjusted according to the previously assessed concentration of the sample to maximize recovery efficiency: 0.5 mL per phase was used for concentrations\u0026thinsp;\u0026lt;\u0026thinsp;30\u0026nbsp;million/mL, while 1 mL was used for higher concentrations. Approximately 1 mL of liquefied semen was placed on top of the gradient and centrifuged at 300g for 20 minutes. After centrifugation, motile spermatozoa were recovered from the pellet at the bottom of the tube by aspiration and washed once with 3 mL of media. For the Control fraction, the spermatozoa were washed and maintained in modified Human Tubal Fluid (HTF; FUJIFILM Irvine Scientific, Santa Ana, CA, USA) supplemented with 5% Human Serum Albumin (HSA; FUJIFILM Irvine Scientific); whereas samples in the experimental group were processed with HyperSperm media.\u003c/p\u003e\n\u003ch3\u003eSperm Processing by Swim-Up Technique\u003c/h3\u003e\n\u003cp\u003eAn additional set of 74 semen samples was processed by swim-up, separating motile spermatozoa from immotile or less viable spermatozoa that are left on the sperm layer. Following liquefaction at room temperature, approximately 1 mL of fresh semen was placed on a conical 15 mL falcon tube under 1 mL of culture medium (HTF for Control group and HyperSperm medium for the experimental group) and incubated at 37\u0026deg;C for 1 hour. After incubation, the upper fraction, now enriched with motile spermatozoa, was carefully aspirated avoiding any minimal disturbance of the lower layer to avoid contamination. Then, the supernatant portion was transferred to a new tube. Spermatozoa were then washed and resuspended in HTF for the Control group, or in HyperSperm medium for the HyperSperm group.\u003c/p\u003e\n\u003ch3\u003eHyperSperm Treatment\u003c/h3\u003e\n\u003cp\u003eAfter sperm selection by either density gradient or swim-up separation, control sperm were incubated in HTF at 37˚C for up to 4 hours. Spermatozoa in the experimental group were processed using the HyperSperm protocol (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e), a proprietary method designed to optimize sperm performance. This protocol differs from conventional procedures in several key aspects: it employs three specialized culture media, an additional centrifugation step and a prolonged incubation period.\u003c/p\u003e\n\u003ch3\u003eMotility analysis\u003c/h3\u003e\n\u003cp\u003eSperm motility was assessed at the beginning of the protocol and at the end of the procedure, for both HyperSperm-treated and Control spermatozoa. Sperm motility assessments were performed using the SCA CASA System-Microptic (Hamilton Thorne, Barcelona, Spain) a Computer-Assisted Sperm Analysis (CASA) system that provides objective and quantitative measurements. Motility was classified according to WHO criteria (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e), classifying as fast progressive, slow progressive, non-progressive and non-motile. For each analysis a 10 uL drop of sperm suspension was placed on a clean microscope slide and covered with an 18 x 18 mm coverslip, creating a space with 30 \u0026micro;m of depth to allow spermatozoa movements. Video recordings were captured at 50 and 60 frames per second (fps), with at least six independent microscopic fields and a minimum of 200 spermatozoa per evaluation. The following kinematic parameters of the spermatozoa were measured: curvilinear velocity (VCL, \u0026micro;m/s), straight line velocity (VSL, \u0026micro;m/s), average path velocity (VAP, \u0026micro;m/s), linearity (LIN: VSL/VCL \u0026times; 100, %), straightness (STR: VSL/VAP \u0026times; 100, %), wobble (WOB: VAP/VCL \u0026times; 100, a measure of sperm head side to side movement, %), amplitude of lateral head displacement (ALH, \u0026micro;m, which measures the magnitude of the lateral displacement of the sperm head about the average path) and beat cross frequency (BCF, Hz). The drift correction threshold was set at 20 \u0026micro;m/s, to ensure accurate exclusion of non-progressive movements from the analysis. Spermatozoa were considered hyperactivated when presenting VCL\u0026thinsp;\u0026ge;\u0026thinsp;150 \u0026micro;m/s, LIN\u0026thinsp;\u0026lt;\u0026thinsp;50% and ALH\u0026thinsp;\u0026ge;\u0026thinsp;3.5 \u0026micro;m (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e), reflecting a vigorous, asymmetric motility pattern.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eViability and survival assessment\u003c/h2\u003e\u003cp\u003eSperm viability was assessed using 0.5% eosin Y (Sigma-Aldrich, Darmstadt, Germany) prepared according to the instructions given in the WHO manual (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). This assay is based on the principle that only spermatozoa with a compromised plasma membrane allow the dye to enter the cell, staining the cytoplasm pink, while intact spermatozoa remain unstained. The viability of Control and HyperSperm-treated spermatozoa were evaluated once the HyperSperm protocol was completed. In addition, a survival analysis was done by repeating the viability assessment 24 hours later.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eMorphology evaluation\u003c/h3\u003e\n\u003cp\u003eSperm morphology was evaluated using a commercially available Diff-Quik staining kit (Cellavision, Lund, Switzerland) according to the manufacturer\u0026rsquo;s protocol. A thin semen smear was prepared on a clean glass slide and allowed to air-dry completely. The slide was sequentially immersed in the fixative solution (Solution I) for 1 minute, followed by the eosinophilic staining solution (Solution II) for 45 seconds, and the basophilic staining solution (Solution III) for 1 minute. Slides were rinsed briefly in distilled water and air dried. The stained smears were examined using bright-field microscopy under oil immersion at 1000\u0026times; magnification. For each sample, at least 200 spermatozoa were evaluated, and morphology was classified according to WHO criteria into normal and abnormal forms (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eDNA fragmentation assessment (TUNEL assay)\u003c/h3\u003e\n\u003cp\u003eThe Fluorescein In Situ Cell Death Detection Kit (Roche, Basel, Switzerland) was used for the evaluation of the percentage of spermatozoa with a high level of DNA fragmentation. This method enables the detection of DNA strand breaks by the incorporation of fluorescein-labeled nucleotides at free 3\u0026rsquo;-OH ends of DNA. After completion of the HyperSperm protocol, aliquots of Control and HyperSperm-treated sperm were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde (Thermo Scientific Chemicals, Waltham, Massachusetts, USA) and placed on a previously prepared coverslip with circles drawn with a Hydrophobic Barrier Pap Pen (Invitrogen, Waltham, Massachusetts, USA). Two additional wells were prepared for the positive control and the negative control. Slides were allowed to evaporate and stored cold until the day of staining. For the staining procedure, cells were permeabilized with 0.1% Triton X-100 in sodium citrate (Sigma Aldrich). The positive control was treated with RNase-free DNase solution (Thermo Scientific), to promote DNA-fragmentation, while the remaining wells were left with PBS to prevent drying. All wells except the negative control were then treated with the labeling solution and enzyme provided by the TUNEL kit. Only the labeling solution was added to the negative control. After this, glycerol with DAPI 1 \u0026micro;g/mL (Sigma-Aldrich) was added and covered with coverslips, leaving the slides ready for viewing under the fluorescence microscope. At least 200 cells were analyzed.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eAcrosomal status evaluation\u003c/h2\u003e\u003cp\u003eAliquots of Control and HyperSperm-treated sperm were taken and divided for spontaneous or progesterone-induced acrosomal reaction evaluation. For the spontaneous reaction, spermatozoa were incubated in the corresponding medium, while for the induced reaction, cells were incubated for 30 minutes with 10 \u0026micro;M progesterone prior to fixation. After incubation, samples were washed with PBS and then fixed with paraformaldehyde on a previously prepared coverslip with circles drawn with a Hydrophobic Barrier Pap Pen. They were allowed to evaporate and stored cold until the day of staining. For analysis, spermatozoa were permeabilized with methanol (Sigma-Aldrich) to facilitate lectin access to acrosomal glycoproteins. Next, 10 \u0026micro;L of \u003cem\u003ePisum sativum agglutinin\u003c/em\u003e conjugated to fluorescein isothiocyanate (FITC-PSA, Sigma-Aldrich) were added and incubated for 1 hour in the dark. PSA binds selectively to acrosomal glycoproteins, enabling differentiation between acrosome-intact and acrosome-reacted spermatozoa. After this, glycerol with DAPI was added to visualize sperm nuclei and covered with coverslips to leave the slides ready for viewing under the fluorescence microscope. At least 200 cells were analyzed.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis.\u003c/h2\u003e\u003cp\u003eStatistical analyses were performed using the GraphPad Prism 6 software (La Jolla, CA, USA). Comparisons between paired samples from the Control and HyperSperm groups were analyzed using Wilcoxon matched pairs signed rank test, a non-parametric test appropriate for paired datasets without normal distribution. Statistical significance was defined as \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and the data is displayed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation, either in graph form or in table form.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eHyperSperm increases sperm motility parameters resulting in a higher percentage of hyperactivated human sperm\u003c/h2\u003e\u003cp\u003eA total of 135 semen samples were analyzed using a paired design, in which each sample was divided and processed with either the standard method (Control) or HyperSperm. The age of the patients who participated in this study ranges from 18 to 58 and the summary of the semen parameters is shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eSummary of semen parameters of samples and patient characteristics, n\u0026thinsp;=\u0026thinsp;135.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePatient \u0026amp; sample parameters\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;Standard Deviation\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAge (years)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e37.7\u0026thinsp;\u0026plusmn;\u0026thinsp;7.4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBMI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e25.3\u0026thinsp;\u0026plusmn;\u0026thinsp;3.6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAbstinence days\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e3.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSample volume (mL)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e1.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003epH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e7.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSample concentration (M/mL)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e70.6\u0026thinsp;\u0026plusmn;\u0026thinsp;52.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMorphology (% normal forms)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e5.3\u0026thinsp;\u0026plusmn;\u0026thinsp;3.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eInitial total motility (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e61.7\u0026thinsp;\u0026plusmn;\u0026thinsp;21.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eInitial progressive motility (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e47.1\u0026thinsp;\u0026plusmn;\u0026thinsp;20.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eBoth total (Density gradient - Control: 65.51% \u0026plusmn; 23.74 vs. HyperSperm: 68.34% \u0026plusmn; 22.42; p\u0026thinsp;=\u0026thinsp;0.1334; Swim-Up - Control: 75.39% \u0026plusmn; 21.07 vs. HyperSperm: 75.52% \u0026plusmn; 18.71; p\u0026thinsp;=\u0026thinsp;0.731) and progressive motility (Density gradient - Control: 55.60% \u0026plusmn; 24.48 vs. HyperSperm: 59.44% \u0026plusmn; 23.18; p\u0026thinsp;=\u0026thinsp;0.0528; Swim-up - Control: 66.35% \u0026plusmn; 21.81 vs. HyperSperm: 66.29% \u0026plusmn; 19.59; p\u0026thinsp;\u0026gt;\u0026thinsp;0.999) did not significantly differ between Control and HyperSperm groups, whether samples were processed by density gradient or swim-up (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn contrast, sperm kinematic parameters revealed significant enhancements following HyperSperm protocol. VCL (Gradient - Control: 89.27 \u0026micro;m/s\u0026thinsp;\u0026plusmn;\u0026thinsp;21.63 vs. HyperSperm: 101.80 \u0026micro;m/s\u0026thinsp;\u0026plusmn;\u0026thinsp;25.83; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 and Swim-Up - Control: 87.24 \u0026micro;m/s\u0026thinsp;\u0026plusmn;\u0026thinsp;15.55 vs. HyperSperm: 97.16 \u0026micro;m/s\u0026thinsp;\u0026plusmn;\u0026thinsp;18.65; p\u0026thinsp;=\u0026thinsp;0.004) and ALH (Gradient - Control: 1.89 \u0026micro;m\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29 vs. HyperSperm: 2.19 \u0026micro;m\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 and Swim-Up - Control: 1.87 \u0026micro;m\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25 vs. HyperSperm: 2.10 \u0026micro;m\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40; p\u0026thinsp;=\u0026thinsp;0.0008) were significantly increased, reflecting faster and more vigorous movement. Additional parameters, such WOB, LIN and VAP, also exhibited a significant difference compared to HyperSperm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe increase in VCL and ALH together with the decrease in LIN results leads to an increase in the percentage of hyperactivated motility (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) in cells treated with HyperSperm. This was observed regardless the use of swim up or density gradient centrifugation (Gradient: Control: 3.07% \u0026plusmn; 3.41 vs. HyperSperm: 8.70% \u0026plusmn; 9.80; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 and Swim-Up: Control: 2.30% \u0026plusmn; 2.79 vs. HyperSperm: 5.36% \u0026plusmn; 7.10; p\u0026thinsp;=\u0026thinsp;0.0004).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eHyperSperm increases Sperm Motility in Asthenozoospermic Samples While enhancing Hyperactivation Across Most Sample Types\u003c/h2\u003e\u003cp\u003eThe samples were further categorized based on their baseline motility, morphology and concentration, following the WHO criteria, in: normozoospermic samples, oligozoospermic samples (\u0026lt;\u0026thinsp;15 M/mL), asthenozoospermic samples (\u0026lt;\u0026thinsp;30% progressive motility or \u0026lt;\u0026thinsp;42% total motility) and teratozoospermic samples (\u0026lt;\u0026thinsp;4% normal forms) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This stratification enabled a clinically relevant evaluation of HyperSperm effects across distinct pathological conditions rather than relying solely on overall population averages. A total of 80 samples were analyzed and 37 of them were classified as normozoospermic, while the rest were categorized as oligozoospermic, asthenozoospermic, or teratozoospermic. Because abnormalities in semen quality often co-occur, the groups were not mutually exclusive. Within the oligozoospermic category (n\u0026thinsp;=\u0026thinsp;7), 1 was oligozoospermic, 3 oligoasthenozoospermic, and 3 oligoasthenozoospermic. On the other hand, in the asthenozoospermic category (n\u0026thinsp;=\u0026thinsp;22), 11 were asthenozoospermic, 5 asthenoteratozoospermic, 3 oligoasthenozoospermic, and 3 oligoasthenoteratozoospermic. Finally, of the teratozoospermic cases (n\u0026thinsp;=\u0026thinsp;28), 20 were teratozoospermic, 5 asthenoteratozoospermic and 3 oligoasthenoteratozoospermic (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eEffect of HyperSperm treatment on motility parameters for samples with different diagnosis (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;Standard Deviation): \u003cb\u003eA.\u003c/b\u003e Normoozoospermic samples, n\u0026thinsp;=\u0026thinsp;37; \u003cb\u003eB.\u003c/b\u003e Oligozoospermic samples, n\u0026thinsp;=\u0026thinsp;7; \u003cb\u003eC.\u003c/b\u003e Asthenozoospermic samples, n\u0026thinsp;=\u0026thinsp;22. and \u003cb\u003eD.\u003c/b\u003e Teratozoospermic samples, n\u0026thinsp;=\u0026thinsp;28 in total. Comparison between Control and HyperSperm with significance represented as: *, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; ***, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"9\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003e\u003cem\u003eNormozoospermic\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003e\u003cem\u003eOligozoospermic\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003e\u003cem\u003eAsthenozoospermic\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003e\u003cem\u003eTeratozoospermic\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eParameters\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eControl\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003eHyperSperm\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003eControl\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003eHyperSperm\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003eControl\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003eHyperSperm\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003eControl\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u003cb\u003eHyperSperm\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMotile cells (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e77.7\u0026thinsp;\u0026plusmn;\u0026thinsp;16.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e79.48\u0026thinsp;\u0026plusmn;\u0026thinsp;14.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e35.3\u0026thinsp;\u0026plusmn;\u0026thinsp;16.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e41.0\u0026thinsp;\u0026plusmn;\u0026thinsp;17.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e51.9\u0026thinsp;\u0026plusmn;\u0026thinsp;27.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e56.0\u0026thinsp;\u0026plusmn;\u0026thinsp;25.0\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e63.3\u0026thinsp;\u0026plusmn;\u0026thinsp;25.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e64.9\u0026thinsp;\u0026plusmn;\u0026thinsp;22.6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eProgressive motile cells (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e68.5\u0026thinsp;\u0026plusmn;\u0026thinsp;18.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e70.2\u0026thinsp;\u0026plusmn;\u0026thinsp;16.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e25.3\u0026thinsp;\u0026plusmn;\u0026thinsp;15.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e30.76\u0026thinsp;\u0026plusmn;\u0026thinsp;18.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e41.7\u0026thinsp;\u0026plusmn;\u0026thinsp;26.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e48.1\u0026thinsp;\u0026plusmn;\u0026thinsp;25.2\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e53.4\u0026thinsp;\u0026plusmn;\u0026thinsp;24.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e56.0\u0026thinsp;\u0026plusmn;\u0026thinsp;22.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVCL (\u0026micro;m/s)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e92.7\u0026thinsp;\u0026plusmn;\u0026thinsp;16.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e107.1\u0026thinsp;\u0026plusmn;\u0026thinsp;23.2\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e63.8\u0026thinsp;\u0026plusmn;\u0026thinsp;13.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e72.3\u0026thinsp;\u0026plusmn;\u0026thinsp;18.6\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e76.5\u0026thinsp;\u0026plusmn;\u0026thinsp;20.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e87.0\u0026thinsp;\u0026plusmn;\u0026thinsp;22.0\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e90.2\u0026thinsp;\u0026plusmn;\u0026thinsp;22.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e98.6\u0026thinsp;\u0026plusmn;\u0026thinsp;22.6\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVAP (\u0026micro;m/s)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e58.3\u0026thinsp;\u0026plusmn;\u0026thinsp;13.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e61.8\u0026thinsp;\u0026plusmn;\u0026thinsp;14.7\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e35.7\u0026thinsp;\u0026plusmn;\u0026thinsp;12.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e37.9\u0026thinsp;\u0026plusmn;\u0026thinsp;15.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e46.9\u0026thinsp;\u0026plusmn;\u0026thinsp;17.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e51.2\u0026thinsp;\u0026plusmn;\u0026thinsp;17.1\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e55.9\u0026thinsp;\u0026plusmn;\u0026thinsp;18.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e55.7\u0026thinsp;\u0026plusmn;\u0026thinsp;16.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVSL (\u0026micro;m/s)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e43.6\u0026thinsp;\u0026plusmn;\u0026thinsp;16.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e43.9\u0026thinsp;\u0026plusmn;\u0026thinsp;16.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e27.0\u0026thinsp;\u0026plusmn;\u0026thinsp;14.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e28.2\u0026thinsp;\u0026plusmn;\u0026thinsp;18.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e35.4\u0026thinsp;\u0026plusmn;\u0026thinsp;17.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e38.6\u0026thinsp;\u0026plusmn;\u0026thinsp;16.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e43.4\u0026thinsp;\u0026plusmn;\u0026thinsp;20.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e41.4\u0026thinsp;\u0026plusmn;\u0026thinsp;19.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSTR (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e60.9\u0026thinsp;\u0026plusmn;\u0026thinsp;13.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e57.5\u0026thinsp;\u0026plusmn;\u0026thinsp;11.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e50.0\u0026thinsp;\u0026plusmn;\u0026thinsp;11.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e46.5\u0026thinsp;\u0026plusmn;\u0026thinsp;12.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e58.0\u0026thinsp;\u0026plusmn;\u0026thinsp;14.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e57.6\u0026thinsp;\u0026plusmn;\u0026thinsp;13.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e58.9\u0026thinsp;\u0026plusmn;\u0026thinsp;15.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e53.3\u0026thinsp;\u0026plusmn;\u0026thinsp;13.4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLIN (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e46.4\u0026thinsp;\u0026plusmn;\u0026thinsp;17.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e41.33\u0026thinsp;\u0026plusmn;\u0026thinsp;17.8\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e41.4\u0026thinsp;\u0026plusmn;\u0026thinsp;20.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e37.3\u0026thinsp;\u0026plusmn;\u0026thinsp;23.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e43.9\u0026thinsp;\u0026plusmn;\u0026thinsp;14.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e42.6\u0026thinsp;\u0026plusmn;\u0026thinsp;14.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e46.4\u0026thinsp;\u0026plusmn;\u0026thinsp;18.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e41.7\u0026thinsp;\u0026plusmn;\u0026thinsp;18.9\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWOB (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e63.6\u0026thinsp;\u0026plusmn;\u0026thinsp;8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e58.3\u0026thinsp;\u0026plusmn;\u0026thinsp;7.6\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e60.7\u0026thinsp;\u0026plusmn;\u0026thinsp;10.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e55.5\u0026thinsp;\u0026plusmn;\u0026thinsp;10.9\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e62.8\u0026thinsp;\u0026plusmn;\u0026thinsp;7.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e60.0\u0026thinsp;\u0026plusmn;\u0026thinsp;5.9\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e63.4\u0026thinsp;\u0026plusmn;\u0026thinsp;7.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e58.2\u0026thinsp;\u0026plusmn;\u0026thinsp;7.5\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eALH (\u0026micro;m)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e2.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBCF (Hz)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e15.2\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e15.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e10.4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e13.0\u0026thinsp;\u0026plusmn;\u0026thinsp;3.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e14\u0026thinsp;\u0026plusmn;\u0026thinsp;3.8\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e14.7\u0026thinsp;\u0026plusmn;\u0026thinsp;4.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e14.3\u0026thinsp;\u0026plusmn;\u0026thinsp;3.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHyperactivation per motile cells (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.3\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10.0\u0026thinsp;\u0026plusmn;\u0026thinsp;10.6\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.83\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.5\u0026thinsp;\u0026plusmn;\u0026thinsp;4.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e3.5\u0026thinsp;\u0026plusmn;\u0026thinsp;4.7\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e3.2\u0026thinsp;\u0026plusmn;\u0026thinsp;3.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e7.1\u0026thinsp;\u0026plusmn;\u0026thinsp;7.9\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eMotility assessment revealed that HyperSperm treatment did not significantly affect total or progressive motility compared to Control in the case of normozoospermic, oligozoospermic or teratozoospermic samples. In contrast, asthenozoospermic samples showed a significant improvement in motility following HyperSperm treatment compared to the Control, particularly in progressive motility, where the increase was more pronounced. HyperSperm produced a significant increase in several kinematic parameters across different semen categories, in particular in VCL, WOB and ALH (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These changes lead to a significantly increase in the proportion of hyperactivated sperm, a key functional parameter associated with capacitation and fertilizing potential, in all sample types except oligozoospermic samples, although a clear tendency in this latter can be observed (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These findings highlight the potential of HyperSperm as a clinically valuable tool for optimizing sperm function, particularly in patients with impaired motility.\u003c/p\u003e\u003cp\u003e\u003cb\u003eHyperSperm does not alter sperm viability and DNA integrity.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSperm viability was assessed using eosin staining after processing with either Control or HyperSperm. Immediately after treatment, no significant differences were observed between the two groups (Control: 81.92% \u0026plusmn; 11.98 vs. HyperSperm: 82.92% \u0026plusmn; 12.37; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.216) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo evaluate potential long-term effects, viability was reassessed after 24 hours of incubation at room temperature (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), with no significant differences observed between the groups (Control: 75.84% \u0026plusmn; 15.94 vs. HyperSperm: 76.37% \u0026plusmn; 16.29; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.756). These results indicate that HyperSperm does not have a detrimental effect on sperm viability when compared to the Control.\u003c/p\u003e\u003cp\u003eThe safety of HyperSperm treatment was further evaluated by measuring DNA fragmentation using the TUNEL assay. No significant differences in the percentage of DNA-fragmented spermatozoa were observed between the two groups (Control: 5.78% \u0026plusmn; 8.04 vs. HyperSperm: 4.39% \u0026plusmn; 5.57; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.044) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). These findings demonstrate that HyperSperm does not induce DNA damage during sperm preparation.\u003c/p\u003e\u003cp\u003e\u003cb\u003eHyperSperm preserves acrosomal integrity.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAcrosomal status was evaluated using FITC-PSA staining to assess potential effects on the ability of sperm to undergo the acrosome reaction. At the end of the protocol, Control and HyperSperm samples exhibited a similar proportion of acrosome-reacted spermatozoa (Control: 14.06% \u0026plusmn; 6.12 vs. HyperSperm: 14.9% \u0026plusmn; 7.51; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.226) indicating that the treatment did not produce an abnormal loss or degeneration of the acrosome in the treated sperm (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). In addition, after acrosome reaction induction with progesterone no significant differences were observed (Control: 24.42% \u0026plusmn; 16.69 vs. HyperSperm: 25.02% \u0026plusmn; 15.12; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.754) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). These data support that HyperSperm does not affect acrosomal integrity and maintains sperm functionality for fertilization.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present study expands upon our previous findings on HyperSperm, a novel sperm treatment medium designed to enhance reproductive outcomes after ART. By evaluating its safety and efficacy in a diverse cohort of clinical semen samples, we observed consistent and statistically significant improvements in motility kinematic parameters such as VCL and ALH. Importantly, HyperSperm nearly doubled the percentage of hyperactivated spermatozoa compared to the standard method, a change consistent with the acquisition of capacitation-related motility (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). These results confirm that HyperSperm not only improves basic motility parameters but also promotes key functional hallmarks required for fertilization. Hyperactivation is critical for fertility because it enables sperm to generate the high-amplitude, asymmetric flagellar movements needed to detach from the oviductal epithelium, penetrate the viscoelastic cumulus matrix, and ultimately drill through the zona pellucida. At a clinical level, sperm that fail to undergo hyperactivation are unable to reach or fertilize the oocyte, and deficiencies in this motility pattern have been strongly associated with reduced success rates in both natural conception and assisted reproduction (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eA major strength of this study is the stratification of patient samples by underlying andrological condition, allowing for a nuanced understanding of HyperSperm\u0026rsquo;s effects across clinically relevant subpopulations. While normozoospermic and teratozoospermic samples showed no significant motility gains beyond those achieved with standard processing, HyperSperm produced marked improvements in semen samples with pathological conditions that often present challenges in ART. Additionally, hyperactivation levels were increased in most subgroups, suggesting that HyperSperm is broadly effective in enhancing functional competence regardless of initial sperm quality. These results highlight its potential clinical value, particularly in patients with suboptimal semen parameters.\u003c/p\u003e\u003cp\u003eBeyond functional improvements, HyperSperm did not compromise cell viability or DNA integrity. These results suggest that reactive oxygen species (ROS) production is not exacerbated in comparison with the standard methods such as swim up or discontinuous gradient centrifugation. Viability is particularly sensitive to the aggression of ROS and that has an immediate impact on sperm motility (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e), which is also unaltered in our conditions. This demonstration is a key safety requirement for any ART-related application. Excessive ROS generation is a major cause of male infertility (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e), as it leads to oxidative damage of sperm membranes, impaired motility, and DNA fragmentation, all of which reduce fertilization potential. Therefore, maintaining ROS at physiological levels is essential to preserve sperm functionality and ensure optimal outcomes in ART. It is worth noting that some sperm selection methods have been reported to reduce DNA damage, adding potential value in cases with elevated sperm DNA fragmentation (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). In our study, this aspect could not be addressed because all samples analyzed presented low baseline levels of DNA damage; however, testing HyperSperm in samples with higher DNA fragmentation will be an important next step.\u003c/p\u003e\u003cp\u003eThe preservation of acrosomal integrity further reinforces the safety profile of HyperSperm. Neither spontaneous nor progesterone-induced acrosome reaction rates were altered by the treatment, indicating that the medium does not prematurely trigger or interfere with this critical step in fertilization. As discussed earlier, HyperSperm significantly enhanced hyperactivation, a hallmark of capacitation, without inducing acrosome exocytosis. This suggests that HyperSperm effectively stimulates upstream signaling pathways associated with capacitation, while maintaining control over downstream events such as the acrosome reaction. This balance is particularly important, given that while acrosome-reacted sperm are known to be capable of penetrating and fusing with the egg (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e), premature acrosome reaction prior to encountering the oocyte can lead to a loss of fertilizing capacity. Thus, our findings support the idea that HyperSperm promotes capacitation in a physiologically regulated manner, enhancing functional competence without compromising fertilization potential.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study provides strong evidence that HyperSperm enhances sperm motility and hyperactivation without compromising viability, DNA integrity, or acrosomal status. While further studies assessing fertilization and pregnancy outcomes are warranted, our findings support the clinical adoption of HyperSperm as a safe and effective tool for optimizing sperm function in ART settings.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u0026nbsp;\u003c/strong\u003eWe would like to thank Nuria Correa Mañas and Júlia García Mulet for their technical assistance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis work has been carried out with the financial support of the Industrial Doctorates program, funded through a grant from the Agency for Management of University and Research Grants (AGAUR), Government of Catalonia.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest:\u0026nbsp;\u003c/strong\u003eMGB and MDGE are shareholders of Fecundis. The rest of the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions:\u003c/strong\u003e MJMV and GCM performed the experiments and analyzed data. RNA, KL, RLV, MRMFC, SRG discussed results and contributed with samples and resources.\u003c/p\u003e\n\u003cp\u003eEI, MDGE, MJMV and MGB analyzed and interpreted the patient data. MGB and MJMV wrote the paper with contributions of all coauthors. MGB designed the study\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate:\u0026nbsp;\u003c/strong\u003eThe study protocol was approved by the Clinical Research Ethics Committee of Hospital del Mar (CEIm PSMAR, Study #2024/11686/I), and the clinical trial was registered at ClinicalTrials.gov (NCT06742437).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e: Not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials:\u003c/strong\u003e The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSaid TM, Land JA. Effects of advanced selection methods on sperm quality and ART outcome: a systematic review. Hum Reprod Update. 2011;17(6):719\u0026ndash;33.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePinto S, Carrageta DF, Alves MG, Rocha A, Agarwal A, Barros A, et al. Sperm selection strategies and their impact on assisted reproductive technology outcomes. Andrologia. 2021;53(2):e13725.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLeahy T, Gadella BM. Sperm surface changes and physiological consequences induced by sperm handling and storage. Reprod Camb Engl. 2011;142(6):759\u0026ndash;78.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eG\u0026oacute;mez-El\u0026iacute;as MD, Luque GM, Oscoz-Susino N, Novero AG, Briski O, K\u0026aacute;sparas I et al. Increased reproductive outcomes after optimized sperm preparation. Front Cell Dev Biol [Internet]. 2025 May 13 [cited 2025 Oct 3];13. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.frontiersin.org/journals/cell-and-developmental-biology/articles/\u003c/span\u003e\u003cspan address=\"https://www.frontiersin.org/journals/cell-and-developmental-biology/articles/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fcell.2025.1596421/full\u003c/span\u003e\u003cspan address=\"10.3389/fcell.2025.1596421/full\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePuga Molina LC, Luque GM, Balestrini PA, Mar\u0026iacute;n-Briggiler CI, Romarowski A, Buffone MG. Molecular Basis of Human Sperm Capacitation. Front Cell Dev Biol. 2018;6:72.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAgarwal A, Rana M, Qiu E, AlBunni H, Bui AD, Henkel R. Role of oxidative stress, infection and inflammation in male infertility. Andrologia. 2018;50(11):e13126.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBungum M. Sperm DNA integrity assessment: a new tool in diagnosis and treatment of fertility. Obstet Gynecol Int. 2012;2012:531042.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTello-Mora P, Hern\u0026aacute;ndez-Cadena L, Pedraza J, L\u0026oacute;pez-Bayghen E, Quintanilla-Vega B. Acrosome reaction and chromatin integrity as additional parameters of semen analysis to predict fertilization and blastocyst rates. Reprod Biol Endocrinol RBE. 2018;16(1):102.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKızılay F, Altay B. Sperm function tests in clinical practice. Turk J Urol. 2017;43(4):393\u0026ndash;400.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWorld Health Organization. WHO laboratory manual for the examination of human sperm. 6th Edition. 2021.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHo K, Wolff CA, Suarez SS. CatSper-null mutant spermatozoa are unable to ascend beyond the oviductal reservoir. Reprod Fertil Dev. 2009;21(2):345\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWilliams HL, Mansell S, Alasmari W, Brown SG, Wilson SM, Sutton KA, et al. Specific loss of CatSper function is sufficient to compromise fertilizing capacity of human spermatozoa. Hum Reprod Oxf Engl. 2015;30(12):2737\u0026ndash;46.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAvenarius MR, Hildebrand MS, Zhang Y, Meyer NC, Smith LLH, Kahrizi K, et al. Human male infertility caused by mutations in the CATSPER1 channel protein. Am J Hum Genet. 2009;84(4):505\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDrevet JR, Aitken RJ. Oxidative Damage to Sperm DNA: Attack and Defense. Adv Exp Med Biol. 2019;1166:107\u0026ndash;17.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMarinaro J, Schlegel PN. Sperm DNA Fragmentation and Fertility. Adv Exp Med Biol. 2025;1469:305\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLa Spina FA, Puga Molina LC, Romarowski A, Vitale AM, Falzone TL, Krapf D, et al. Mouse sperm begin to undergo acrosomal exocytosis in the upper isthmus of the oviduct. Dev Biol. 2016;411(2):172\u0026ndash;82.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSosnik J, Miranda PV, Spiridonov NA, Yoon SY, Fissore RA, Johnson GR, et al. Tssk6 is required for Izumo relocalization and gamete fusion in the mouse. J Cell Sci. 2009;122(Pt 15):2741\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-translational-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jtrm","sideBox":"Learn more about [Journal of Translational Medicine](http://translational-medicine.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jtrm/default.aspx","title":"Journal of Translational Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"DNA fragmentation, Assisted reproduction technologies, Sperm, infertility","lastPublishedDoi":"10.21203/rs.3.rs-7809501/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7809501/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eAssisted reproductive technologies (ART) rely on the functional integrity of spermatozoa, which can be affected by in vitro handling and preparation procedures. HyperSperm is a novel sperm treatment medium developed to enhance sperm function and improve clinical outcomes. This study aimed to evaluate the efficacy and safety of HyperSperm in human semen samples from patients undergoing fertility treatment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eA paired analysis was performed on 135 clinical semen samples, each divided into two equal fractions processed using either standard conditions or the HyperSperm protocol. Sperm motility and kinematic parameters were measured with computer-assisted analysis. Safety assessments included sperm viability at baseline and after 24 hours, DNA fragmentation using a fluorescence-based assay, and acrosomal integrity under spontaneous and progesterone-stimulated conditions. Comparisons between paired samples were analyzed using the Wilcoxon matched-pairs signed-rank test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eHyperSperm significantly enhanced sperm kinematic parameters, including curvilinear velocity and amplitude of lateral head displacement, resulting in higher levels of hyperactivated motility. Stratification by semen quality demonstrated that samples with reduced motility showed the greatest functional improvement. HyperSperm did not affect sperm viability or DNA integrity, even after 24 hours of incubation, and preserved acrosomal structure and responsiveness to progesterone.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eHyperSperm improves critical sperm functional parameters without compromising cellular viability, DNA stability, or acrosomal integrity. These findings support the safe and effective use of HyperSperm to optimize sperm performance and potentially improve outcomes in assisted reproduction.\u003c/p\u003e","manuscriptTitle":"Efficacy and Safety Evaluation of HyperSperm Treatment in Human Semen Samples","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-26 01:02:03","doi":"10.21203/rs.3.rs-7809501/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-10-15T11:14:42+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-12T05:16:45+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-09T14:59:53+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Translational Medicine","date":"2025-10-08T11:36:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-translational-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jtrm","sideBox":"Learn more about [Journal of Translational Medicine](http://translational-medicine.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jtrm/default.aspx","title":"Journal of Translational Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"41af94cd-7e55-481e-af17-3e4c5b06b031","owner":[],"postedDate":"October 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-01-26T16:12:59+00:00","versionOfRecord":{"articleIdentity":"rs-7809501","link":"https://doi.org/10.1186/s12967-025-07656-z","journal":{"identity":"journal-of-translational-medicine","isVorOnly":false,"title":"Journal of Translational Medicine"},"publishedOn":"2026-01-22 15:58:42","publishedOnDateReadable":"January 22nd, 2026"},"versionCreatedAt":"2025-10-26 01:02:03","video":"","vorDoi":"10.1186/s12967-025-07656-z","vorDoiUrl":"https://doi.org/10.1186/s12967-025-07656-z","workflowStages":[]},"version":"v1","identity":"rs-7809501","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7809501","identity":"rs-7809501","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}