Sequencing Matters: Comparative Effectiveness of Density-Gradient Centrifugation and MACS for Enhancing Sperm Quality and DNA Integrity

Methods and Literature Research

Statistical Analysis:

A. Density Gradient Centrifugation (DGC) Protocol and Efficacy

The usual DGC method includes layering semen on top of a discontinuous density gradient (often 40% and 80%), then spinning it at 300–500g for 15–20 minutes, which separates motile, normally shaped sperm from debris and dead cells. [1] Although DGC consistently reduces sperm DNA fragmentation relative to unprocessed semen, some samples paradoxically exhibit increased SDF after processing, particularly in men with high baseline SDF or poor semen quality. Thus, DGC reliably improves motility and morphology; however, its ability to reduce SDF is lower than that of the swim-up method in samples with <30% SDF. [1]When combined with MACS, the reduction in DNA fragmentation becomes significantly greater than when the technique is used alone. [2,3,4,5]

Figure 1. Sperm separation technique by DCG after centrifugation at 1600 rpm (400 g) [picture refined by AI-generated app using Google AI Studio Gemini 3, Nano Banana Pro: 28.12.2025].

Figure 1. Sperm separation technique by DCG after centrifugation at 1600 rpm (400 g) [picture refined by AI-generated app using Google AI Studio Gemini 3, Nano Banana Pro: 28.12.2025].

B. Swim-Up Technique Protocol and Efficacy

The swim-up procedure is performed by overlaying liquefied semen with culture medium and then incubating it for about 30–60 minutes at 37 °C. Sperm motility allows spermatozoa to swim into the medium, which is then collected. [1,6,7]The swim-up method has been reported to yield a higher proportion of motile sperm than DGC, with healthier and less fragmented DNA when applied to samples with a DFI below 30%. [1,8] However, the percentage that survives after 24 hours is lower in comparison with DGC. [1]Optimized protocols, such as the one-step swim-up/ICSI approach, which reduces, and in some cases even completely eliminates the need for centrifugation, can select a sperm population with near-zero SDF for ICSI. Therefore, such procedure can outperform conventional swim-up technique and in some instances even achieve better results than MACS. [7]

Figure 2. Demonstration for the 45° slope Swim-up technique for semen clearance. [picture refined by AI-generated app using Google AI Studio Gemini 3, Nano Banana Pro: 28.12.2025].

Figure 2. Demonstration for the 45° slope Swim-up technique for semen clearance. [picture refined by AI-generated app using Google AI Studio Gemini 3, Nano Banana Pro: 28.12.2025].

C. Magnetic-Activated Cell Sorting (MACS) Protocol and Efficacy

MACS uses annexin V-conjugated magnetic beads for the depletion of apoptotic sperm by binding phosphatidylserine-externalizing cells, thereby enriching the final preparation with non-apoptotic, DNA-intact sperm. [2,9]MACS is most effective in patients with high SDF, and when combined with DGC or swim-up it can further reduce DNA fragmentation beyond the levels achieved by conventional methods. [2,9]According to some studies MACS is considered most effective in patients with high SDF, and when combined with DGC or swim-up it can further reduce DNA fragmentation beyond the levels achieved by conventional methods. [2,9]They found that the protocol with greatest reduction in DNA fragmentation is the MACS-DGC-SU sequence, in which MACS is applied to raw semen, followed by DGC and then swim-up. [4]Although this approach provides the most substantial decrease in SDF, it may compromise sperm vitality and motility in samples with poor baseline parameters. [4]Nevertheless, the optimized protocol remains under experts review.

Figure 3. Principle for Magnetic-Activated Cell Sorting (MACS) technique.

Figure 3. Principle for Magnetic-Activated Cell Sorting (MACS) technique.

Results

Results

Factors Associated with Treatment Response

Several studies have identified factors predicting response to DGC-MACS treatment, as summarized in Table 5. However, there is conflicting data on the most effective framework and technical settings for achieving the highest fertilization and pregnancy rates, and these findings only partially translate into improved fertilization and ICSI outcomes. Bucar et al. (2015) compared five sperm-processing sequences and found that all protocols significantly reduced TUNEL-measured sperm DNA fragmentation. [4]The greatest mean reduction occurred with MACS–DGC–SU (83.3 ± 15.4%), whereas DGC–SU–MACS was least effective (53.8 ± 24.1%). In the MACS–DGC–SU group, the magnitude of reduction was inversely associated with vitality (r = −0.842), membrane integrity/ HOST (r = −0.799), and progressive motility (r = −0.528), suggesting this sequence may be particularly advantageous in poorer-quality samples. [4]Notably, in the same study, teratozoospermic patients tended to have lower SDF reduction rates than asthenozoospermic and asthenoteratozoospermic patients. [4]Berteli T.S. advocated starting with MACS to minimize handling of damaged sperm and reduce the risk of iatrogenic increases in SDF during later centrifugation steps, which can elevate ROS and promote DNA fragmentation during DGC. [23]Taken together, this sequence therefore combines complementary mechanisms—early removal of apoptotic (annexin V+) sperm with compromised membranes and higher ROS burden, followed by density- and morphology-based enrichment—thereby maximizing the recovery of motile sperm with lower SDF. [23]Said et al. demonstrated that the oocyte penetration rate was negatively correlated with apoptotic marker expression, while sperm chromatin decondensation following ICSI was associated only with apoptosis in sperm with damaged membranes. [24]

Societies guidelines do not endorse any specific order or routine for DGC vs MACS use and there is no study investigating in detail the sequence DGC followed by MACS. Nevertheless, as indirect study results, Tavalaee et al. directly compared DGC-MACS and MACS-DGC and found that combining MACS with DGC improved DNA integrity (TUNEL) and chromatin maturity (CMA3) compared with either method alone; they ultimately favored MACS-DGC, primarily because it yielded a lower proportion of caspase-positive sperm. They further noted that PS externalization may occur during capacitation independently of apoptosis/caspase activation, raising the possibility that initiating selection with MACS before DGC could mitigate depletion of capacitated sperm that might otherwise be lost with a DGC-first approach; however, fertilization outcomes were not evaluated in this study. [5]However, in a retrospective cohort of ICSI cycles involving men with high SDF (>20% by TUNEL), Pacheco et al. reported that DGC followed by MACS (n = 366) was associated with higher pregnancy and live-birth rates and a lower miscarriage rate than DGC alone (n = 358), while fertilization rates were similar (75.1% vs 73.3%; p = 0.133). Specifically, the pregnancy rate was higher in the group with DGC followed by MACS (60.7% vs 51.5%; p = 0.014), the miscarriage rate was lower (14.7% vs 20.6%; p = 0.034), and the live-birth rate was higher (47.4% vs 31.2%; p = 0.001)[2]. Again, Pacheco et al. did not perform a head-to-head comparison of MACS followed by DGC versus DGC followed by MACS; they compared DGC followed by MACS with DGC alone. In the literature there is one other randomized, prospective study comparing MACS-DGC vs DGC and reporting fertilization, although in this study the fertilization was not significantly different. [25]These findings were similarly reported in a clinical study comparing MACS-DGC vs DGC alone, whereby again there was no difference in the fertilization rate.[39]

To date, no larger randomized head-to-head trial has directly compared DGC followed by MACS with MACS followed by DGC and demonstrated superiority of either sequence for fertilization outcomes.

Table 6. Factors influencing treatment outcomes.

Table 6. Factors influencing treatment outcomes.

Factor	Findings	Study
Initial sperm quality	Samples with low progressive motility, vitality, and membrane integrity showed best SDF reduction	Bucar et al., 2014; Ventura Bucar et al., 2014
Semen diagnosis	Teratozoospermic patients showed lower SDF reduction rates than asthenozoospermic or asthenoteratozoospermic patients	Bucar et al., 2014
Baseline DFI	Higher baseline DFI associated with greater absolute reduction	Toishibekov et al., 2021
PLCζ expression	Higher PLCz1 expression associated with better treatment outcomes	Salehi Novin et al., 2023
Apoptotic markers	High motility, low caspase-3 activation, MMP integrity predicted good response	Said et al., 2006
Morphology baseline	Patients with values above reference for rapid progressive motility showed higher SDF reduction	Ventura Bucar et al., 2014

Own Study Algorithm and Results

The research was conducted as a prospective, blind observational study. It included a cohort of 67 male patients undergoing evaluation for infertility, with a mean age of 43.16 ± 7.4 years (range: 30-66 years). The primary inclusion criterion for participation was the presence of a pathological level of sperm DNA fragmentation, defined arbitrarily as a baseline value of ≥30% A standardized, two-step sperm preparation technique was applied to all semen samples, as shown in the graph 1. Initially, all samples were processed using a standard DGC technique to separate motile sperm from seminal plasma, debris, and non-motile cells.

The samples were stratified on a discontinuous density gradient (80% and 40%), centrifuged at 400g for 20 min, the supernatant discarded and the sperm pellet washed by centrifugation for 5 min at 500g.

As this step was followed by a MACS preparation, the post-gradient sample was washed with 2 ml of specific binding buffer for MACS. The binding buffer used in the Annexin V MACS kit (Miltenyi Biotech™) is calcium-free because Annexin V requires calcium to bind to PS residues on the sperm membrane. Keeping the buffer devoid of calcium prevents premature or nonspecific binding of Annexin V to PS. Calcium is added only at the appropriate step, ensuring controlled activation of the Annexin V–PS interaction and allowing a more precise removal of PS-positive/apoptotic sperm. Then sperm pellet was resuspended in 100 µl of Annexin V conjugated microbead reagent -allowing a maximal concentration of 10 ⁷ spermatozoa—and 400 µl of buffer were added making a final volume of 500ul. Annexin V represents the limiting reagent, and the 100-µL working volume is suitable for staining up to 10⁷ spermatozoa. The suspension, incubated for 15 min at room temperature, was finally loaded on a separation column, previously rinsed with 1 ml of binding buffer. The calcium required for Annexin V–PS interaction is provided directly within the Annexin V–conjugated MicroBeads, ensuring that binding occurs only during the controlled incubation step. This design improves the specificity and efficiency of removing PS-positive/apoptotic sperm during magnetic separation. Immature germ cells can also expose PS and therefore bind Annexin V. When present in high numbers, these cells may compete with apoptotic sperm for Annexin V MicroBeads, reducing the availability of binding sites and potentially decreasing the efficiency of the MACS separation.

For this reason, we believe that the sequential use of DGC followed by MACS could be more effective. When placed in a magnetic field, apoptotic sperm with externalized PS bind to the beads are retained, allowing the non-apoptotic fraction to be collected. A calcium-free medium such as the buffer provided in the Miltenyi MACS kit can temporarily reduce the observed progressive motility of the eluted spermatozoa. Calcium is essential for optimal flagellar activation, so progressive motility may appear diminished while the cells remain in a calcium-free environment. This effect is reversible once the sperm are transferred back into a physiological medium containing calcium.

For this reason, the eluted fraction was washed with 2 ml of HTF medium, centrifuged at 500 g for 5 minutes, and the resulting pellet was resuspended in fresh medium prior to analysis. For each patient, sperm DNA fragmentation (SDF) was evaluated using the Halosperm assay (Halotech™) before and after MACS treatment to allow direct comparison. A repeated semen analysis was performed according to the WHO 6th edition at both time points—prior to and following the dual sequential treatment. All assessments were conducted blindly to prevent observational bias.

Figure 4. Study algorithm.

Figure 4. Study algorithm.

The primary outcome measure was the change in SDF following the combined DGC-MACS treatment demonstrating a significant reduction in sperm DNA damage following the combined DGC-MACS protocol (P<0.0001), as shown in Table 7

As detailed in Table 7 the mean SDF in the native semen samples was 41.61%. After treatment, the mean absolute reduction in damaged cells was 19.42 ± 9.4%. A statistical analysis using the Wilcoxon-paired rank test confirmed that this decrease was highly significant (P<0.0001), underscoring the potent effect of the DGC-MACS intervention.

While the overall effect was positive, the study revealed significant variability in individual patient outcomes. The cohort could be stratified into distinct response groups:

Adequate Responders: The majority of patients (85.07%; 57 out of 67) responded favorably to the treatment. In this group, the DGC-MACS procedure successfully reduced SDF levels to below the 30% cut-off as pathological threshold. The mean SDF difference for this subgroup of responder was substantial with -20.65%± 9.45 (median 20%; range 4-43).

Inadequate Responders: A smaller subgroup of 10 patients (14.92%) did not achieve the desired outcome. Although their absolute SDF levels decreased, the reduction was inadequate, and their post-treatment SDF remained still above the 30% threshold after the dual sequential treatment procedure. This group exhibited a lower mean SDF difference of -12.4%± 5.4 (median 13%; range 5-19).

Exceptional Responders: Interestingly, the study noted that a few cases (6 patients; 8,95%) experienced an almost total elimination of fragmented sperm cells, demonstrating the protocol’s potential for profound impact in certain individuals.

To determine if baseline semen quality predicted the degree of SDF improvement, a Linear Regression analysis was performed. The results showed no correlation between the reduction in SDF and the native total sperm count or total motile sperm count. (P>0.1; n.s.). This is an expected finding and consistent with established literature[40,41], where it has demonstrated that infertile men with normospermia can still exhibit pathologically high levels of SDF. This reinforces SDF as an independent and crucial measure of sperm quality.

In summary, this study reported a mean absolute reduction in SDF of -19.42%. This result aligns robustly with the existing literature. A systematic review of 25 studies found that the absolute reduction in SDF ranged from 2.82% to 21.9%. In relative terms, this represents a 39% to 83% reduction in the number of sperm with damaged DNA. Therefore, the efficacy in SDF reduction after dual procedures—beginning with DGC and followed by MACS—observed in the present study falls comfortably within this established range.

Combining DGC with subsequent MACS significantly improves sperm quality by creating a synergistic selection process that targets different markers of sperm health. While DGC selects for mature spermatozoa with high DNA compaction, MACS utilizes Annexin V-conjugated microbeads to remove apoptotic (dying) sperm cells that exhibit PS externalization. Overall, the mean percentage reduction is approximately 47%—a 19.42% decrease from the baseline value of 41.61%. This estimate aligns well with the broader literature, which reports reductions ranging from 39% to 83%, providing an additional layer of validation. Indeed, the literature also reinforces the value of the combined DGC-MACS approach. Chi et al. similar demonstrated the synergistic effect of this two-step protocol. [14]While DGC alone reduced SDF from a baseline of 11.5% to 8.1% and MACS alone reduced it to 7.4%, the combined DGC-MACS technique achieved a greater reduction, lowering SDF to just 4.1%. This shows that the combined approach is more effective than either method alone.

Nonetheless, the existing literature is not entirely consistent, with some studies reporting contradictory results. Studies comparing MACS-DGC versus DGC-MACS sequences revealed that MACS performed before DGC (MACS- DGC) may achieve superior outcomes. Tavalaee et al. proposed MACS-DGC for clinical implementation based on higher efficiency in separating active caspase-positive sperm. Bucar et al. demonstrated that MACS-DGC-SU achieved the highest SDF reduction rate (83.3%) compared to DGC-SU-MACS (53.8%). [5] Berteli et al. confirmed that MACS-DGC led to significantly higher percentages of spermatozoa with progressive motility and normal morphology than DGC-MACS.[23] Notably, in this Berteli et al. study, the “calcium-free Miltenyi MACS buffer reduces motility” explanation is unlikely, since the Annexin V binding requires Ca²⁺ in the kit system and thus can temporarily reduce the observed progressive motility of the eluted spermatozoa. Calcium is essential for optimal flagellar activation, so progressive motility may appear diminished while the cells remain in a calcium-free environment. This effect is reversible once the sperm are transferred back into a physiological medium containing calcium. However, the paper does not clearly describe a standardized post-MACS wash/resuspension prior to motility scoring, and the results strongly track with which step was performed last (DGC last = high motility; MACS last = low motility. Therefore, the reported motility advantage of MACS-DGC over DGC-MACS may partially depend on buffer/handling (including how completely binding buffer was removed and what medium sperm were in at the time of assessment), not only on the biological “DGC-induced PS exposure removes functional sperm” mechanism. The more assumable mechanistic explanation for this observation relates to the fact that DGC may trigger membrane changes linked to capacitation/acrosome-related remodeling, which can expose PS on otherwise viable sperm. Since MACS targets PS-positive (annexin V–binding) cells, performing MACS after DGC could inadvertently remove capacitated, functional sperm in addition to apoptotic sperm.

Although this study focused on the laboratory outcome of SDF reduction and did not report on clinical results, the systematic review provides strong evidence of the likely downstream benefits for patients undergoing ART. The literature consistently shows that while fertilization rates are often comparable between DGC-MACS or MACS-DGC treated and control groups, the primary impact of the technique is observed in post-fertilization development. Indeed, clinical improvements had been reported in DGC-MACS sequencing protocol across multiple studies, whereby a significant increase in top-quality day-3 embryos (e.g., 72.5% vs. 51.47%), higher rates of successful development to the blastocyst stage (e.g., 69.69% vs. 48%), higher pregnancy and live birth rates had been shown (e.g., 60.7% vs. 51.5%, p=0.014 and 47.4% vs. 31.2%, p=0.001, respectively). Overall, a significant reduction in the rate of pregnancy loss (e.g., 14.7% vs. 20.6%, p=0.034) was additionally reported. Regarding benefit for boosting sperm motility and DFI, Toishibekov et al. reported that unprocessed semen had motility of 32.7 ± 5.9% and DFI of 32.4 ± 5.9%; after MACS, the annexin-negative fraction showed higher motility (47.2 ± 6.3%) and lower DFI (10.5 ± 3.8%) than the annexin-positive fraction (motility 3.5 ± 2.3%; DFI 67.8 ± 5.9%; P < 0.003 for both comparisons). MACS also reduced DFI from 32.4% in the original sample to 10.5% in the annexin-negative fraction (P < 0.01), [12]This was considered beneficial for IVF and ICSI cycles when selecting the best spermatozoa.

Summary

Figure 5. Potential therapeutic options for improving sperm quality.

Figure 5. Potential therapeutic options for improving sperm quality.

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