{"paper_id":"4e428b83-742a-4fd6-a74f-5bba434bbe0c","body_text":"Infertility affects approximately 15% of couples worldwide, with male factors\ncontributing to nearly half of these cases. While conventional semen analysis\nremains a cornerstone in evaluating male fertility, it often falls short in\npredicting successful fertilization and pregnancy outcomes. Notably, sperm DNA\nfragmentation (SDF) has emerged as a critical parameter, with elevated SDF levels\nassociated with impaired embryo development, reduced blastocyst quality, and\nincreased miscarriage rates ( Conti  et\nal. , 2024 ;  Agarwal  et\nal. , 2020 ).\nTraditional sperm preparation techniques, such as density gradient centrifugation\n(DGC), are widely employed to isolate motile and morphologically normal spermatozoa.\nHowever, these methods involve multiple centrifugation steps and exposure to\ncolloidal silica media, which can generate reactive oxygen species (ROS),\npotentially compromising sperm membrane integrity and inducing DNA fragmentation\n( Muratori  et al. , 2019 ;\n Agarwal  et al. , 2020 ).\nConsequently, there is a growing interest in alternative, less invasive sperm\nselection methods that can minimize DNA damage and enhance assisted reproductive\ntechnology (ART) outcomes.\nMicrofluidic sperm sorting (MC) has emerged as a promising technique that mimics the\nnatural selection processes of the female reproductive tract. Utilizing laminar flow\nwithin microchannels, microfluidic devices facilitate the selection of highly motile\nsperm with intact DNA by leveraging mechanisms such as rheotaxis and chemotaxis.\nThese devices offer several advantages over conventional methods, including reduced\nprocessing time, minimal handling, and decreased operator dependency.\nThe concept of microfluidics was first introduced in the early 1990s in the context\nof analytical chemistry and biomedical diagnostics. Its application to reproductive\nmedicine began to gain traction in the early 2000s when researchers recognized that\nthe precise fluid dynamics of microchannels could be harnessed to mimic the\nphysiological environment of the female reproductive tract. By the 2010s,\ncommercially available sperm-sorting chips such as ZyMōt™, FERTILE™,\nand FERTILE PLUS™ began to appear, offering ART centers a more controlled,\nphysiologically relevant method for sperm preparation. These platforms aimed to\nreduce oxidative stress exposure and preserve DNA integrity in sperm selected for\nICSI ( Quinn  et al. , 2018 ;\n Anbari  et al. , 2021 ;\n Yildiz & Yuksel, 2019 ).\nSeveral studies have investigated the efficacy of MC in improving ART outcomes. For\ninstance, a retrospective cohort study by Pujol et al. demonstrated that the use of\na microfluidic sperm sorting device significantly reduced double-stranded DNA\nfragmentation by 46% compared to the swim-up method ( Pujol  et al ., 2022 ). Similarly, Vahidi et al. reported\nimprovements in sperm morphology, motility, and DNA integrity when using\nmicrofluidic sorting techniques ( Vahidi  et\nal. , 2025 ).\nClinical outcomes have also been evaluated in studies comparing MC to traditional\nmethods. A study by Banti et al. found that the use of the FERTILE PLUS™\nmicrofluidic sperm sorting chip resulted in higher blastocyst formation rates (76%\n vs . 56%) and euploidy rates (40%  vs . 20%)\ncompared to DGC ( Banti  et al .,\n2024 ). Although the increase in fertilization rates was not statistically\nsignificant, the findings suggest potential benefits of microfluidic sorting in\nenhancing embryo quality.\nDespite these promising results, some studies have reported marginal improvements\nwithout statistical significance. A meta-analysis concluded that while MC\ndemonstrates slight positive outcomes compared to standard techniques, the\ndifferences were not statistically significant across analyzed parameters ( Ferreira Aderaldo  et al. ,\n2023 ). The authors emphasized the need for larger, multicenter studies with\nstandardized protocols to validate the clinical benefits of MC.\nAdditional investigations have also echoed these findings. Ozcan et al., in a\ncomparative study involving 181 infertile males, reported a higher clinical\npregnancy rate in the MC group (49.5%) compared to the DGC group (40%), but the\ndifference was not statistically significant ( p =0.2) ( Ozcan  et al ., 2021 ).\nSimilarly, Quinn et al. found no significant improvement in embryo quality or\nclinical pregnancy rates between MC and DGC in ICSI cycles ( Quinn  et al ., 2022 ). These findings suggest\nthat while MC technologies hold promise in improving sperm DNA integrity, their\ntranslation into enhanced clinical outcomes remains inconsistent.\nIn light of current evidence, MC presents a compelling alternative to conventional\nsperm preparation methods, particularly in cases with high SDF levels. However,\nfurther research is necessary to establish its efficacy in improving clinical\noutcomes consistently. This study aims to compare sperm quality and ART outcomes\nbetween MC and DGC, providing insights into the practical applications of MC\ntechnology in routine IVF procedures. Moreover, unlike most previous studies, our\nresearch was designed to extend follow-up beyond fertilization and early embryo\ndevelopment, with particular emphasis on evaluating live birth outcomes as the\nultimate endpoint of assisted reproduction success.\n\nThis randomized controlled study was conducted on 119 couples who underwent IVF\ncycles at the Center of IVF and Tissue engineering - Hanoi Medical University\nHospital, from March 2023 to March 2024, following ethical approval by the Hanoi\nMedical University Institutional Ethical Review Board (HMU IRB). All\nparticipants used autologous oocytes and ejaculated sperm, with no use of donor\ngametes. On the male side, exclusion criteria included patients requiring\ntesticular sperm extraction or those diagnosed with oligoasthenozoospermia (OA)\n( WHO, 2021 ). On the female side,\ncouples were excluded if the female partner was classified as having a poor\novarian prognosis according to the POSEIDON 2016 criteria. This includes women\nwith reduced ovarian reserve, defined by anti-Müllerian hormone (AMH)\nlevels <1.2 ng/mL or antral follicle count (AFC) <5, or those with normal\nreserve but a history of poor ovarian response (≤9 oocytes retrieved) in\nprevious cycles. Preimplantation genetic testing (PGT) cycles were also excluded\nfrom the study.\nCouples who met inclusion criteria were randomized into two groups: the\nintervention group (MC group), in which semen was processed using a microfluidic\nsperm sorting device and the control group (DGC group), in which sperm was\nprepared using traditional density gradient centrifugation.\nBefore patient recruitment began, a list of 120 random numbers was generated\nusing the RAND() function in Microsoft Excel. The list was then sorted in\nascending order. The first 60 positions were allocated to the intervention group\n(MC), and the remaining 60 to the control group (DGC). During the study,\neligible patients were consecutively enrolled according to the actual order of\nrecruitment and assigned to the corresponding group based on the pre-determined\nrandomization list. However, one couple in the control group withdrew from\ntreatment before oocyte retrieval, resulting in a final sample size of 60\ncouples in the MC group and 59 in the DGC group.\nBoth groups followed the same controlled ovarian stimulation protocol and\nlaboratory workflow for IVF treatment. On the day of oocyte retrieval, semen\nsamples were processed according to the assigned technique and used for\nintracytoplasmic sperm injection (ICSI). Embryologists performing ICSI and\nembryo assessment were blinded to the group allocation to minimize bias.\nThe primary outcomes evaluated were the sperm DNA fragmentation index (DFI) and\nthe live birth rate. Secondary outcomes included sperm quality after preparation\n(motility and morphology), fertilization rate, number and quality of embryos on\nday 2, and pregnancy outcomes, including biochemical, clinical, and ongoing\npregnancies.\nSemen samples in the control group were processed using a discontinuous\ndensity gradient system. Briefly, 1 mL of 90% gradient medium\n(SpermGrad™, Vitrolife, Sweden) was layered beneath 1 mL of 45%\ngradient medium in a centrifuge tube. Then, 1 mL of liquefied semen was\ngently added on top. The sample was centrifuged at 345 × g for 8\nminutes. The resulting pellet was washed with 4 mL of sperm washing medium\n(SpermRinse™, Vitrolife, Sweden) and centrifuged again at the same\nspeed for 5 minutes. The final pellet was resuspended in 0.3 - 0.5 mL of\nwashing medium.\nFor the intervention group, semen was processed using the ZyMōt Multi Sperm\nSeparation Device (850 µL; DxNow Inc., USA). A total of 850 µL\nof liquefied semen was first slowly loaded into the inlet port.\nSubsequently, 750 µL of washing medium was added to the device,\nincluding 50 µL to prime the outlet port and 700 µL to cover\nthe membrane surface. The device was incubated for 30 minutes at 37°C. The\nsorted sperm was collected from the outlet port, with a final volume of up\nto 500 µL.\nSperm DNA fragmentation was assessed using the PhacoSperm® DNA\nFragmentation Kit, which is based on the sperm chromatin structure assay (SCSA).\nThis assay relies on the differential fluorescence of acridine orange (AO) when\nbound to double-stranded DNA (green emission) versus single-stranded DNA (red\nemission) under excitation by blue laser light. After acid-induced denaturation,\nfragmented DNA becomes single-stranded, while intact chromatin remains\ndouble-stranded. Flow cytometry was performed to determine the DNA fragmentation\nindex (DFI) by analyzing fluorescence signals from 5,000 spermatozoa per sample.\nA DFI greater than 15% was considered abnormal.\nControlled ovarian stimulation was initiated with recombinant\nfollicle-stimulating hormone (rFSH; Gonal-F ® , Merck\nSerono, Italy) on cycle day 2. The dose was adjusted according to individual\novarian reserve. A GnRH antagonist (Cetrotide ® , 250\nµg; Merck Serono, Germany) was administered from day 5 or 6 until the\novulation trigger. When at least two follicles reached ≥18 mm in\ndiameter, final oocyte maturation was induced using Ovitrelle® (250\nµg; Merck Serono).\nOocyte retrieval was performed 34-36 hours after hCG administration via\ntransvaginal ultrasound-guided aspiration under sedation. Following a 2-hour\nincubation period, cumulus cells were removed using hyaluronidase\n(HYASE-10X™, Vitrolife ® , Sweden). Sperm prepared by\neither density gradient centrifugation (DGC) or microfluidic sorting was\ninjected into mature oocytes using intracytoplasmic sperm injection (ICSI).\nFertilization was confirmed 17-20 hours later by the presence of two\npronuclei. Embryos were cultured in Continuous Single\nCulture ® -NX Complete medium (FUJIFILM Irvine\nScientific, USA) in a tri-gas incubator (5% CO₂, 5% O₂, 90% N₂) at 37°C.\nEmbryo quality was assessed on day 2 in accordance with the 2011 Istanbul\nconsensus. Good-quality embryos were defined as those containing 4-6 evenly\nsized blastomeres with less than 10% fragmentation. Moderate-quality embryos\nexhibited 10-25% fragmentation, while poor-quality embryos showed uneven\nblastomeres or greater than 25% fragmentation.\nEstrogen supplementation with estradiol valerate (6-8 mg/day) was initiated\non cycle day 2. Endometrial thickness was assessed by transvaginal\nultrasound on day 10. If the thickness was less than 8 mm, the dose was\nincreased to 12-16 mg/day and reassessed. Progesterone was initiated when\nthe endometrial thickness reached ≥8 mm, in the absence of\nintrauterine fluid, and when a trilaminar endometrial pattern was observed.\nEmbryo transfer cycles were canceled if the endometrial thickness remained\n<8 mm or exceeded 14 mm, if intrauterine fluid was present, or if the\nendometrial pattern appeared diffusely hyperechogenic.\nEmbryo transfer was performed on day 3, day 4, or day 5. Serum β-hCG\nlevels were measured 10 days after day-5 embryo transfer or 12 days after\nday-3 embryo transfer. Biochemical pregnancy was defined as a serum\nβ-hCG level greater than 25 IU/L. Clinical pregnancy was confirmed by\nthe presence of a gestational sac and fetal heartbeat at 4 weeks. Ongoing\npregnancy was confirmed by ultrasound at 12 weeks of gestation. Live birth\nwas defined as the complete expulsion or extraction of a fetus from the\nmother, showing any sign of life, regardless of gestational age.\nData analysis was performed using SPSS version 20.0 (IBM Corp., Armonk, NY, USA).\nContinuous variables were expressed as mean±standard deviation (SD),\nwhile categorical variables were presented as frequency and percentage. Group\ncomparisons for categorical variables were performed using the Chi-square test\nand the Fisher exact test in the case of expected frequency in any cell being\nless than 5. For continuous variables, the Student’s T-test was used to compare\nnormally distributed data, and the Mann-Whitney U test was applied for\nnon-normally distributed data. A two-tailed  p -value less than\n0.05 was considered statistically significant.\nPatient information was anonymized, kept confidential, and used solely for\nresearch purposes. The study was conducted only after obtaining written informed\nconsent from all participants. This comparative trial was approved by the\nleadership of the Center of IVF and Tissue engineering - Hanoi Medical\nUniversity Hospital, and received ethical approval from the Hanoi Medical\nUniversity Institutional Ethical Review Board (IBR-VN01.001 / IRB00003121 /\nFWA00004148)\nClinical Trial Registration:  This study was registered at\nClinicalTrials.gov with the identifier  NCT07004309 .\n\nA total of 119 couples completed the study and were included in the final analysis,\nwith 60 in the MC group and 59 in the DGC group.\nCompared to the DGC group, the MC group had significantly younger female\n(29.73±3.77  vs . 31.47±4.16 years,\n p= 0.018) and male participants (32.27±3.29\n vs . 34.81±4.59 years,  p= 0.001).\nMoreover, anti-Müllerian hormone (AMH) levels were significantly higher\nin the MC group than in the DGC group (5.63±4.09  vs .\n3.79±2.55 ng/mL,  p= 0.004), indicating a better ovarian\nreserve in this cohort ( Table 1 ).\nBaseline demographic and clinical characteristics of participants in the\nDGC and MC groups Values are presented as mean±standard deviation\nfor continuous variables and as number (percentage) for categorical\nvariables. Significant differences were observed in female age, male\nage, and AMH levels ( p <0.05). No statistically\nsignificant differences were found between groups in BMI, infertility\ntype, duration of infertility, AFC, or causes of infertility. Sample\nsize: DGC group (n=59), MC group (n=60). Abbreviations: DGC=Density\nGradient Centrifugation, MC=Microfluidic\nBoth DGC and MC methods significantly improved semen parameters, including\nprogressive motility, viability, and normal morphology (all\n p <0.0001). However, a key advantage of the microfluidic\ntechnique lies in its superior capacity to reduce the sperm DNA fragmentation\nindex (DFI) ( Table 2 ). Post-preparation\nDFI was significantly lower in the MC group (0.43±0.38%) compared to the\nDGC group (2.47±5.08%,  p =0.0024), particularly in\nsamples with high baseline DFI (≥15%), where the MC group achieved a\nmarked reduction ( p <0.0001). In samples with low baseline\nDFI (<15%), both methods were effective; however, the MC group still\ndemonstrated a significantly lower final DFI ( p =0.0001) ( Table 2 ).\nComparison of semen parameters before and after preparation using Density\nGradient Centrifugation (DGC) and Microfluidics (MC) Values are\npresented as mean±standard deviation. Within-group comparisons\n(p₁₂, p₃₄) and between-group comparisons (p₁₃, p₂₄) are shown. Both\nmethods significantly improved motility, viability, morphology, and DFI\n( p <0.0001). The MC group showed lower\npost-preparation DFI and higher motile sperm recovery rate.\nAbbreviations: DFI=DNA Fragmentation Index\nFurthermore, the MC group exhibited a significantly higher motile sperm recovery\nrate (44.41±3.95%  vs . 30.59±2.01%,\n p <0.0001), suggesting improved selection of functionally\ncompetent sperm. In contrast, total sperm recovery and post-preparation\nmorphology were comparable between the two groups ( Table 2 ).\nLinear regression analysis revealed a strong correlation between preand\npost-processing DFI in the MC group (r₁=0.999), indicating a proportional\nreduction in DFI across the full range of initial values ( Figure 1 ). Notably, samples with higher pre-processing DFI\nexperienced greater reductions, highlighting the clear advantage of microfluidic\ntechnology in improving DNA integrity, especially in cases with severe damage.\nBy contrast, the DGC group showed a weaker and more variable reduction\n(r₂=0.864), suggesting that DFI reduction was more dependent on other factors,\nmaking the outcomes less predictable ( Figure\n1 ).\nFigure 1 Correlation between initial DNA Fragmentation Index (DFI) and\nreduction in DFI after sperm processing using two different methods.\nThe graph illustrates the relationship between DFI before processing\nand the percentage reduction in DFI in two groups: the DGC group\n(red triangles) and the MC group (blue circles). Linear regression\nlines were fitted for each group.\nCorrelation between initial DNA Fragmentation Index (DFI) and\nreduction in DFI after sperm processing using two different methods.\nThe graph illustrates the relationship between DFI before processing\nand the percentage reduction in DFI in two groups: the DGC group\n(red triangles) and the MC group (blue circles). Linear regression\nlines were fitted for each group.\nThe number of oocytes retrieved was significantly higher in the Microfluidics\n(MC) group compared to the DGC group (19.63±8.80  vs .\n16.27±7.28,  p =0.025). However, no significant\ndifferences were observed between the groups in mature oocyte rate\n(68.89±19.36%  vs . 68.00±17.64%,\n p =0.792), fertilization rate (92.67±9.45%\n vs . 93.85±9.12%,  p =0.490), Day 2\nembryo formation rate (98.34±3.75%  vs .\n98.70±3.75%,  p =0.594), or good-quality Day 2 embryo rate\n(71.08±24.45%  vs . 71.61±22.95%,\n p =0.903) ( Table\n3 ).\nComparison of laboratory outcomes between the DGC and MC groups Values\nare presented as mean±standard deviation. The MC group showed a\nsignificantly higher number of oocytes retrieved\n( p =0.025). No significant differences were observed in\nmature oocyte rate, fertilization rate, Day 2 embryo formation rate, or\ngood-quality Day 2 embryo rate.\nEmbryo transfer and pregnancy outcomes are shown in  Table 4 . Pregnancy rates following Day 5, Day 4, and\ncleavage-stage transfers were not significantly different. Total pregnancy rate\n(75.5%  vs . 72.2%,  p= 0.704), clinical pregnancy\nrate (67.4%  vs . 64.8%,  p= 0.786), ongoing\npregnancy rate (63.3%  vs . 59.3%,  p= 0.676), and\nlive birth rate (63.3%  vs . 59.3%,  p= 0.676)\nwere comparable between the DGC and MC groups ( Table 4 ).\nComparison of embryo transfer outcomes and pregnancy results between the\nDGC and Microfluidics (MC) groups Values are presented as number of\ncases (percentage). No significant differences were observed between\ngroups in overall pregnancy rates, clinical pregnancy, ongoing\npregnancy, or live birth rates.\nTable 5  presents pregnancy outcomes\naccording to baseline DFI levels in both the DGC and MC groups. Within each\ngroup, patients with high DFI (≥15%) showed slightly lower\npregnancy-related outcomes compared to those with low DFI (<15%); however,\nnone of the differences reached statistical significance.\nPregnancy outcomes in DGC and Microfluidics (MC) groups stratified by\nbaseline DNA fragmentation index (DFI)Values are presented as number of\ncases (percentage). Outcomes are compared between patients with low (DFI\n< 15%) and high (DFI ≥ 15%) DNA fragmentation in each group.\nNo statistically significant differences were observed in pregnancy,\nclinical pregnancy, ongoing pregnancy, or live birth rates between\nsubgroups.\nIn the DGC group, live birth rates were 64.9% for DFI <15% and 58.3% for DFI\n≥15% (p=0.683). Similarly, in the MC group, live birth rates were 62.5%\n vs . 50.0% for low  vs . high DFI\n( p =0.412). Comparisons between the high-DFI subgroups of\nboth groups also revealed no significant differences ( p =0.712).\nThese results suggest that both preparation methods may mitigate the adverse\nimpact of elevated DFI on pregnancy outcomes ( Table 5 ).\n\nThis randomized controlled trial aimed to compare the effectiveness of MC and DGC for\nsperm preparation in IVF cycles. The primary outcomes included live birth rates and\nsperm DNA fragmentation index (DFI), while secondary outcomes encompassed sperm\nquality, fertilization rates, embryo quality, and overall pregnancy outcomes. The\nfindings provide valuable insights into the relative advantages and limitations of\nthese two sperm preparation methods.\nIn this study, we successfully recruited 119 couples and randomly assigned them\ninto two groups using a random sequence. Despite efforts to minimize confounding\nfactors by excluding women with poor ovarian response according to the POSEIDON\ncriteria and ensuring that the causes of infertility were comparable between the\ntwo groups, we still observed some significant differences. Specifically, the\nintervention group (MC) had a younger average age for both female\n(29.73±3.77  vs . 31.47±4.16 years,\n p =0.018) and male partners (32.27±3.29\n vs . 34.81±4.59 years,  p =0.001),\nalong with a higher ovarian reserve (AMH 5.63±4.02  vs .\n3.79±2.55 ng/mL,  p =0.004) compared to the control group\n(DGC). These differences could potentially introduce a favorable bias for the MC\ngroup, as younger couples with higher ovarian reserves generally have better\nreproductive outcomes. However, previous studies have indicated that the age\nthresholds that significantly impact IVF success are 35 years for women and 40\nyears for men ( Vitagliano  et\nal. , 2023 ;  Gao  et\nal. , 2024 ;  Lu  et\nal. , 2023 ). Therefore, despite the age differences\nbetween the two groups, the fact that the average ages in both groups were below\nthese critical thresholds suggests that this disparity is unlikely to\nsignificantly influence the overall study outcomes.\nNumerous studies have confirmed that MC is more effective in reducing sperm DFI\ncompared to traditional DGC, especially in samples with high DFI ( Anbari  et al. , 2021 ;  Quinn  et al. , 2018 ;  Keskin  et al. , 2022 ). Our\nstudy not only supports this finding but also highlights the consistency of the\ntechnique: the reduction in DFI in the MC group was not only more pronounced but\nalso showed a nearly perfect linear correlation between preand post-processing\nDFI (r=0.999), indicating a stable reduction across the entire DFI spectrum\nregardless of baseline DNA damage.\nIn contrast, DGC showed less consistent DFI reduction, particularly in samples\nwith high DFI. The lower correlation coefficient (r=0.864) suggests variability\nin DFI reduction efficacy. This may be due to steps in the DGC process, such as\ncentrifugation and exposure to resin particles, which can cause mechanical and\noxidative stress on sperm. Several studies have also indicated that DGC may not\nbe suitable for all sperm samples, especially those with poor quality (OAT),\nfrom older men, or with high oxidative stress-commonly seen in men who smoke,\nare exposed to toxins, or have unhealthy lifestyles ( Conti  et al. , 2024 ;  Muratori  et al. , 2019 ). Although our study\nexcluded OA samples and limited male age to under 40 to minimize confounding,\nfactors such as smoking and lifestyle are difficult to control completely and\nmay affect DGC efficiency. Therefore, for semen samples with DFI ≥15% or\nin men with a history of smoking or unhealthy habits, it may be advisable to\nconsider Microfluidics as the initial sperm selection method for ICSI.\nMicrofluidics mimics the physiological environment of the female reproductive\ntract, allowing sperm to actively migrate through microchannels without\ncentrifugation or exposure to chemicals. This significantly reduces mechanical\nand oxidative stress-two major causes of sperm DNA damage. The technology also\nallows for effective selection of highly motile, morphologically normal sperm\nwhile eliminating dead or DNA-damaged sperm. These benefits are particularly\nevident in samples with high DFI ( Agarwal\n et al. , 2020 ).\nSeveral recent studies have reported that MC may improve IVF outcomes in certain\nselected patient populations, such as increasing fertilization rates in\nrecurrent IVF failure cases ( Yildiz &\nYuksel, 2019 ), enhancing blastocyst formation rates ( Ozaltin  et al. , 2023 ), or\nimproving blastocyst quality and euploidy rates in patients with\nastheno-teratozoospermia ( Guler  et\nal. , 2021 ). However, a recent meta-analysis found no\nstatistically significant difference in clinical pregnancy rates when comparing\nMC with conventional sperm preparation methods. This suggests that while MC may\nenhance sperm quality, it does not necessarily translate into improved clinical\nIVF outcomes ( Ferreira Aderaldo  et\nal. , 2023 ).\nIn our study, we intentionally excluded low-quality and low-count sperm samples\ndue to concerns about sperm recovery for ICSI-this exclusion criterion\neffectively defined a selected patient population. When comparing clinical\noutcomes between the two groups, no significant differences were observed in\nfertilization rates, embryo formation, day-2 embryo quality, pregnancy rates,\nclinical pregnancy, ongoing pregnancy, or live birth rates. The only notable\ndifference was a higher number of oocytes retrieved in the MC group, which is\nconsistent with the younger age and better ovarian reserve of the women in that\ngroup.\nEven when further stratifying outcomes based on baseline DFI levels (≥15%\n vs . <15%), no significant differences were found in\npost-transfer success rates. This indicates that although MC is effective at\nreducing DFI-especially in high-DFI samples-it is not sufficient on its own to\nsignificantly improve IVF outcomes. Moreover, in both DGC and Microfluidics\ngroups, clinical pregnancy rates tended to be lower in patients with DFI\n≥15%, suggesting that baseline DFI may remain an independent prognostic\nfactor affecting IVF outcomes, regardless of the sperm preparation method\nused.\nThis study has some methodological limitations that should be acknowledged.\nFirst, the sample size was not calculated a priori, as the study was designed as\nan exploratory trial. Second, it was conducted at a single center, which may\nlimit the generalizability of the findings to other clinical settings. While\nembryologists performing ICSI and embryo assessment were blinded to group\nallocation, sperm preparation was not blinded, which could introduce a degree of\nperformance bias. Moreover, the intervention group had slightly more favorable\nbaseline characteristics, such as younger female age and higher AMH levels,\nwhich may have influenced outcomes. Lastly, neonatal outcomes were not\nevaluated, as the study focused primarily on live birth as the final\nendpoint.\n\nMicrofluidics is an effective method for reducing sperm DNA fragmentation,\nparticularly in samples with high DFI. However, this improvement does not\nnecessarily guarantee better clinical outcomes in IVF. Baseline sperm DNA\nfragmentation index (DFI) may still act as an independent prognostic factor\ninfluencing IVF success, regardless of the sperm preparation method used.","source_license":"CC-BY-4.0","license_restricted":false}