The innovative role of sperm DNA fragmentation testing: understanding its impact on IVF outcomes and PGT-A

The innovative role of sperm DNA fragmentation testing: understanding its impact on IVF outcomes and PGT-A | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article The innovative role of sperm DNA fragmentation testing: understanding its impact on IVF outcomes and PGT-A Tsuyoshi Okubo, Tatsuya Kobayashi, Teruaki Hayashi, Noriyuki Onda, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7782850/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 23 Mar, 2026 Read the published version in Scientific Reports → Version 1 posted 23 You are reading this latest preprint version Abstract This retrospective study evaluated whether sperm DNA fragmentation (SDF), measured by DNA fragmentation index (DFI), predicts IVF outcomes in 124 couples undergoing IVF with PGT-A between 2015 and 2023. SDF was assessed using the Halosperm kit and categorized into low (≤ 15%), medium (15–30%), and high (≥ 30%) DFI groups. Fertilization was performed via IVF or ICSI, and embryos were analyzed with PGT-A using next-generation sequencing. High DFI was associated with advanced paternal age, reduced motility, lower fertilization rates (in ICSI), and fewer good-quality blastocysts. However, PGT-A outcomes showed no significant differences in euploidy, mosaicism, or aneuploidy rates across DFI groups. Similarly, implantation, pregnancy, and live birth rates following euploid embryo transfer were unaffected by DFI level. While high sperm DFI negatively impacts early embryonic development, it does not predict chromosomal integrity. SDF testing can help identify male infertility factors and guide IVF strategy, particularly regarding sperm selection. These findings suggest that early developmental impairment from sperm DNA damage may be mitigated by embryonic repair mechanisms at later stages. Biological sciences/Developmental biology Health sciences/Diseases Biological sciences/Genetics Health sciences/Medical research DNA fragmentation index embryology male infertility Preimplantation genetic testing for aneuploidy WHO criteria Figures Figure 1 1 INTRODUCTION Infertility is a serious problem for many couples, with causes that vary widely. Traditionally, infertility treatments have primarily focused on female factors 1 , while male factors, especially the sperm, have been relatively overlooked. In many fertility centers, semen analysis is limited solely to assessing parameters based on its concentration, motility, and morphology 2 . However, these evaluations are not standardized and often involve subjective elements 3 , raising concerns about diagnostic reliability 4 . Recent research has highlighted the crucial role of sperm quality in determining pregnancy outcomes 5 , 6 . Notably, beyond sperm count and motility, a key consideration is how sperm affect the fertilization of oocytes and subsequent embryo development 7 , 8 . It has been known that sperm play a crucial role from the early stages of fertilization to embryo formation and, ultimately, the development of the blastocyst. In addition to conventional semen analysis, methods such as morphological evaluation based on the Kruger criteria and the observation of sperm vacuoles under high magnification using the intracytoplasmic morphologically selected sperm injection method have been used as additional diagnostic tools for male infertility 9 , 10 . However, it has become clear that these examination methods alone are insufficient for fundamentally elucidating the causes of infertility attributable to male factors 11 . While semen analysis based on WHO criteria (Table 1 )—such as sperm concentration, motility, and normal morphology—provides a useful reference, meeting these standards does not necessarily rule out issues related to fertilization capacity or embryo development. The WHO criteria are only general indicators and do not fully reflect minute abnormalities in sperm quality or their effects on embryo development after fertilization 12 . Therefore, more refined analytical techniques are needed to assess the functional characteristics of sperm and their influence on embryo development 13 . Such a detailed evaluation will be key to more accurately identifying the causes of infertility attributable to sperm quality and to optimizing treatment strategies. Table 1. A Historical comparison of diagnostic criteria in WHO guidelines for semen examination from the 1st (1980) to the 6th (2021) edition. Semen characteristics WHO 1st (1980) WHO 2nd (1987) WHO 3rd (1992) WHO 4th (1999) WHO 5th (2010) WHO 6th (2021) Semen volume (mL) ND ≥2 ≥2 ≥2 ≥1.5 ≥1.4 Sperm count (10 6 /mL) 20-200 ≥20 ≥20 ≥20 ≥15 ≥16 Total sperm count (10 6 ) ND ≥40 ≥40 ≥40 ≥39 ≥39 Total motility (%) ≥60 ≥50 ≥50 ≥50 ≥40 ≥42 Progressive motility (%) ≥25 ≥25 ≥25 ≥25 ≥32 ≥30 Morphology (%) ≥80.5 ≥50 ≥30 ≥14 ≥4 ≥4 WHO: World Health Organization ND: Not Determined In reality, sperms are highly specialized cells that undergo various biological changes necessary for achieving fertilization. During spermatogenesis, sperm DNA undergoes structural modifications to achieve a highly organized, compact, and condensed form. This unique chromatin structure protects the paternal genome from external influences and ensures its accurate transmission to the oocyte. As a result, sperm DNA integrity has been suggested as a crucial factor for understanding the pathophysiology and etiology of male infertility. In recent years, SDF has emerged as an important parameter in semen analysis, with the DFI being of particular interest 14 . Studies have shown a negative correlation between DFI and sperm morphology and motility, with DFI increasing with male age 15 . Sperm DNA damage-induced dysfunction may impact IVF success rates, and previous research has reported that blastocysts derived from sperm with high DFI exhibit chromosomal abnormalities and morphological differences compared to those from sperm with low DFI. Furthermore, SDF has been suggested as a useful predictor of pregnancy success 16 . Since these findings suggest that additional semen analysis focused on SDF may serve as a critical assessment criterion in infertility treatment, we decided to validate its clinical impact on IVF outcomes. As previously mentioned, SDF is not only correlated with various factors in standard semen analysis but has also provided new insights into its association with blastocyst development and pregnancy outcomes in IVF treatment 17 . Several reports have indicated differences in the rates of good-quality blastocyst development, clinical pregnancy, and miscarriages among different DFI groups 18 , 19 . However, other studies have shown that blastocysts derived from sperm with high SDF do not exhibit significant differences in morphological grading or live birth outcomes 19 . This ongoing research continues to investigate and evaluate the relationship between sperm DNA damage and IVF success rates, but clear conclusions regarding causality and clinical significance have yet to be established 20 , 21 . This study aims to further investigate the correlation between SDF and IVF outcomes, providing new insights into the significance of SDF testing 22 . Previous reports have documented reduced fertilization rates and impaired blastocyst development in men with high SDF levels. In addition to these findings, we sought to present new perspectives based on embryonic aneuploidy analysis results. There have been no previous large-scale reports on the correlation between SDF and PGT-A results, making it particularly intriguing to explore how sperm DNA integrity influences the blastocyst formation process. PGT-A is a crucial technique in IVF for evaluating abnormalities in the number of chromosomes in blastocysts. A normal blastocyst typically has a positive diploid ploidy, 46 chromosomes in number. However, SDF may potentially influence chromosomal aneuploidy by causing excess or deficient chromosomes. Additionally, high DFI levels in semen have been linked to sperm aneuploidy and meiotic abnormalities, increasing the risk of embryo aneuploidy. In this study, we investigated the relationship between SDF and embryonic aneuploidy to provide a more detailed assessment of its impact on IVF success rates. Through this analysis, we aimed to offer new insights into male infertility factors that have not been previously explored in previous studies. Although the success of infertility treatment is influenced by a complex interplay of many factors, we examined the possibility that the DFI test, which provides a detailed assessment of male sperm health 23 , could play an essential role in optimizing treatment strategies in IVF. Through this study, we also aimed to verify the utility of the DFI test and to contribute to improving the success rate of infertility treatment by providing new insights into male infertility. 2 MATERIALS AND METHODS 2.1 Study population This study included 124 couples who underwent IVF and embryo aneuploidy analysis via PGT-A between May 2019 and April 2024, with male partners who opted for DFI testing during semen analysis. The study design was reviewed and approved by our institutional ethics committee (SYC2019-04). The research was conducted with patients who provided informed consent and agreed to participate. IVF cases involving oocytes or sperm not collected at our institution were excluded from the study (Fig. 1 ). ・Follicular monitoring and oocyte retrieval Starting on day 3 of the menstrual cycle, follicular monitoring was performed using ultrasound and hormone testing. Clomiphene citrate was administered at 0.5–1T/day, with additional FSH supplementation at 75 IU/day as needed. Once the dominant follicle reached a diameter of 16–18 mm and E2 levels reached 200 pg/mL, buserelin acetate (300 µg/mL) was administered intranasally, followed by oocyte retrieval 32–36 hours later. Oocytes were retrieved from the follicular fluid which was puncture-aspirated with a 22-gauge needle (Kitazato BioPharma, Japan). The collected oocytes were examined under an inverted microscope to assess their maturity based on the presence of first polar body extrusion. 2.2 Semen Examination and Analysis ・Sperm motility test (general semen test) The semen sample was collected through masturbation. The semen volume was measured using a sterile pipette when the semen was sufficiently liquefied. Subsequently, sperm concentration, total sperm count, and motility rate were analyzed using an automated sperm motility analysis system (SMAS, DETECT, Japan). ・Sperm morphology (Kruger sperm function test) Sperm morphology was evaluated using the Diff-Quik Staining Kit (Sysmex Corporation, Japan). More than 200 sperm were examined under a biological microscope (×400), with detailed observations of the head, midpiece, and tail. The percentage of sperm with normal morphology was then calculated. ・Sperm DNA fragmentation test (SDF test) DFI assessment was conducted using the Halosperm HT-HS10 kit (Halotech DNA, Spain). This kit enables the quantification of DNA fragmentation in the sperm nucleus. In normal sperm, a halo is visible around the head, formed by looped DNA strands. However, in fragmented sperm, the damaged DNA strands fail to form a halo 24 . The percentage of fragmented sperm was analyzed as the DFI rate, based on observations of over 300 sperm. The DFI rate was categorized into three groups: low (≤ 15%, n = 81), medium (15–30%, n = 34), and high (≥ 30%, n = 9). 2.3 Semen Collection The density gradient centrifugation method, a common sperm sorting process, was performed by layering two layers of SepaSperm (Kitazato BioPharma, Japan), 80% and 40%, in a centrifuge tube, 1.0 mL each. Next, a sterile pipette was used to place 1.0 mL of liquefied semen sample in HTF medium onto the upper layer. The centrifuge tube was then centrifuged at 600G for 15 minutes. The supernatant was removed, and the remaining pellet was resuspended in 5 mL of washing HTF medium (Kitazato BioPharma, Japan) before undergoing a second centrifugation at 400G for 5 minutes. After removing the supernatant again, HTF medium was added to dilute and adjust sperm concentration. The sample was then stored in an incubator at 37°C until insemination. 2.4 IVF and Vitrification Mature oocytes underwent either conventional in-vitro fertilization (C-IVF) or intracytoplasmic sperm injection (ICSI), followed by individual culturing in 20 mL of Human Tubal Fluid (HTF) medium (Kitazato BioPharma, Japan) containing 0.4% human serum albumin. Fertilization was confirmed the day after insemination by identifying male and female pronuclei. Abnormally fertilized oocytes with more than three pronuclei and degenerated oocytes were excluded from the analysis. Fertilized embryos were cultured in one step medium (Naka medical, JAPAN) by time-lapse culture system on an Embryo Scope Flex (Vitrolife, Sweden). Blastocysts meeting the criteria for good-quality embryos were cryopreserved via vitrification using Cryotop (Kitazato BioPharma, Japan) 25 . The criteria for good-quality blastocysts were defined as the presence of at least 12 trophoectoderm (TE) cells at the outer perimeter of the blastocyst and an inner diameter of 170 µm on day 5 or 6 of culture. Blastocysts that did not meet these criteria by the end of day 6 were not cryopreserved. 2.5 TE Cell Biopsy and PGT-A Expanded blastocysts that met our clinic's PGT-A criteria (at least 12 TE cells on the outer perimeter and a blastocyst inner diameter of 170 µm or more) were eligible for PGT-A analysis. TE cells located away from the inner cell mass were isolated using the flicking method with a biopsy pipette, collecting 4–6 cells. The biopsied TE cells were washed in a 30 µL drop of phosphate-buffered saline (PBS) in at least three different locations and then transferred to a 2.5 µL drop of PBS solution containing 1% PVP in a polymerase chain reaction (PCR) tube. The samples were cryopreserved at -30°C. The frozen TE cells were then sent to a testing laboratory for next-generation sequencing (NGS) analysis to examine embryonic chromosomal aneuploidy. 2.6 EMBRYO TRANSFER The results of PGT-A analysis were classified into four categories: euploid, mosaic, aneuploid, and not detected. Only euploid embryos or low-frequency mosaic embryos were considered transferable, and informed consent was obtained from all patients prior to embryo transfer. All cryopreserved blastocysts were transferred during a natural ovulation cycle following the initial oocyte retrieval cycle, specifically 5 to 5.5 days after natural ovulation. If the endometrial thickness was less than 6 mm, embryo transfer for that cycle was canceled and postponed to a subsequent cycle. Additionally, all blastocysts underwent assisted hatching (AH) using laser technology to remove the zona pellucida, ensuring the embryo was in a completely hatched blastocyst state. Embryo transfer was performed as a single embryo transfer under transvaginal ultrasound guidance. 2.7 OUTCOMES The primary outcome was defined as live birth at > 22 weeks' gestation. Clinical pregnancy and ongoing pregnancy were defined as the presence of a gestational sac by the sixth week of pregnancy and the detection of fetal cardiac activity at ≥ 7 weeks' gestation, respectively. 2.8 STATISTICAL ANALYSIS Patient background data were summarized using mean and standard deviation for continuous variables. Comparisons with the control group were conducted using Dunnett's test. Fisher's exact test was used to compare IVF outcomes with the control group. All data were analyzed using JMP 11.2 software (SAS Institute, Cary, NC, USA), and statistical significance was determined at P < 0.05. 3 RESULTS Of the 455 couples whose DFI was measured at the time of the initial semen examination, 124 couples who underwent PGT-A were included in this study. The average age of the wives was 41.4 ± 2.9 years, while the husbands' average age was 42.1 ± 5.2 years. The mean DFI was 14.3 ± 10.4, and participants were categorized into three groups based on DFI levels: low (≤ 15%, n = 81), medium (15–30%, n = 34), and high (≥ 30%, n = 9). The proportion of patients in the high DFI group was 7.3% (9/124) (Table 2 ). Table 2 General semen findings and DFI in target patients (n = 124) Factors Mean ± SD Range Female Age (years) 41.4 ± 2.9 34–47 AMH (pmol/L) 1.89 ± 1.84 0.03–8.68 Male Age (years) 42.1 ± 5.2 29–59 Semen volume (mL) 2.7 ± 1.5 0.1–7.3 Concentration (10 6 /mL) 66.6 ± 66.5 1.3–380.0 Total sperm counts 185.3 ± 201.9 0.1-1194.8 Motility (%) 59.9 ± 19.1 8.9–89.6 Normal morphology (%) 2.8 ± 1.8 0-9.5 DFI (%) 14.3 ± 10.4 2.0-64.7 Data presented as mean ± standard deviation. AMH: Anti-Mullerian Hormone DFI: sperm DNA fragmentation index When comparing the correlation between DFI groups and various semen parameters with the control group (low DFI), both the medium and high DFI groups showed a significantly higher average male age and a tendency for decreased sperm motility. Regarding fertilization outcomes after insemination, normal fertilization rates in ICSI were significantly lower in the high DFI group compared to the control group. However, in C-IVF, there were no significant differences in normal or abnormal fertilization rates among the DFI groups. Additionally, the rate of good-quality blastocyst formation meeting our clinic’s cryopreservation criteria was significantly lower in the high DFI group than in the low and medium DFI groups (Table 3 ). Table 3 The relationship between DFI groups and semen analysis (n = 124) DFI P value Factors Low (≤ 15%) Medium (15–30%) High (≥ 30%) Low vs Medium Low vs High Percentage of the whole (%) 65.3 (81/124) 27.4 (34/124) 7.3 (9/124) Male age (years) 41.0 ± 4.8 43.6 ± 4.3 47.0 ± 7.9 0.0105 0.0014 Semen parameter Semen volume (mL) 2.8 ± 1.6 2.5 ± 1.6 2.6 ± 1.2 0.6331 0.9561 Concentration (10 6 /mL) 67.1 ± 65.2 61.5 ± 70.0 80.5 ± 71.0 0.8907 0.8113 Total sperm counts 187.0 ± 206.0 162.5 ± 178.3 253.6 ± 249.8 0.7717 0.5711 Motility (%) 64.7 ± 15.8 52.3 ± 21.3 44.3 ± 22.6 0.0020 0.0030 Normal morphology (%) 3.0 ± 2.0 2.5 ± 1.4 1.6 ± 1.4 0.3969 0.0519 Data presented as mean ± standard deviation. *Fisher’s exact test DFI: sperm DNA fragmentation index As for embryo aneuploidy based on PGT-A analysis, there were no significant differences in the proportions of euploid, mosaic, and aneuploid embryos among each DFI group. Based on PGT-A results, single embryo transfers were conducted in 103 cases where euploid or low-frequency mosaic embryos were observed. The implantation rates for the low, medium, and high DFI groups were 71.4% (50/70), 66.7% (18/27), and 100% (6/6), respectively. Pregnancy rates confirmed by gestational sac detection were 62.9% (44/70), 59.3% (16/27), and 66.7% (4/6), while live birth rates were 52.9% (37/70), 40.7% (11/27), and 50.0% (3/6). No significant differences were observed in pregnancy outcomes per embryo transfer among the DFI groups (Table 4 ). Table 4 The relationship between DFI groups and IVF results DFI P value Factors Low (≤ 15%) Medium (15–30%) High (≥ 30%) Low vs Medium Low vs High No. of oocytes (n) 6269 1827 586 ICSI outcome Normal fertilization (%) 79.6 (4403/5530) 80.4 (1318/1639) 75.9 (425/560) 0.4816 0.0381 Abnormal fertilization (%) 6.5 (359/5530) 5.5 (91/1639) 8.6 (48/560) 0.1683 0.0604 C-IVF outcome Normal fertilization (%) 80.4 (594/739) 78.7 (148/188) 88.5 (23/26) 0.6121 0.4396 Abnormal fertilization (%) 11.1 (82/739) 11.2 (21/188) 3.8 (1/26) 0.6672 0.3967 Blastocyst outcome No of cultured embryos (n) 4997 1466 448 - Cryopreserved blastocyst (%) 31.7 (1586/4997) 31.4 (460/1466) 27.2 (122/448) 0.7938 0.0489 Grade AA, AB and BA (%) 23.6 (375/1586) 22.0 (101/460) 17.2 (21/122) 0.4506 0.1048 Grade BB (%) 74.1 (1176/1586) 75.7 (348/460) 82.0 (100/122) 0.5149 0.0556 Grade BC and CB (%) 2.2 (35/1586) 2.4 (11/460) 0.8 (1/122) 0.8142 0.4834 PGT-A outcome No of biopsied embryos (n) 594 176 26 euploid 16.5 (98/594) 15.3 (27/176) 23.1 (6/26) 0.7146 0.5414 mosaic 9.8 (58/594) 10.8 (19/176) 11.5 (3/26) 0.6888 0.9688 aneuploid 69.7 (414/594) 69.3 (122/176) 65.4 (17/26) 0.9236 0.8027 not detected 4.0 (24/594) 4.5 (8/176) 0 (0/26) 0.9363 0.5989 No. of transferred blastocyst (n) 70 27 6 Pregnancy rate (%) 71.4 (50/70) 66.7 (18/27) 100 (6/6) 0.8323 0.2973 Clinical pregnancy rate (%) 62.9 (44/70) 59.3 (16/27) 66.7 (4/6) 0.7437 0.7985 Live birth rate (%) 52.9 (37/70) 40.7 (11/27) 50.0 (3/6) 0.2847 0.7707 Data presented as mean ± standard deviation. *Fisher’s exact test DFI: sperm DNA fragmentation index ICSI: Intracytoplasmic Sperm Injection C-IVF: Conventional In Vitro Fertilization PGT-A: Preimplantation Genetic Testing for Aneuploidy 4 DISCUSSION The findings of this study support previous research on DFI and reaffirm the impact of aging on DFI levels 26 . The observed increase in sperm DNA damage with advancing male age suggests that qualitative factors in sperm 27 —specifically, a decline in chromosomal stability—may be a contributing factor to infertility 28 . Among the male patients undergoing infertility treatment in this study 29 , the majority had low to medium DFI levels, while only about 7% exhibited high DFI (≥ 30%), making them a minority 30 . This highlights that although men with high DFI are relatively rare, they represent an important subgroup that warrants attention in infertility treatment 31 . Traditionally, infertility has often been attributed to female factors, but numerous studies have demonstrated that sperm quality is also crucial for IVF success 32 . In addition to fundamental semen parameters, such as sperm count and motility, sperm DNA integrity has been recognized as a key factor influencing IVF outcomes 33 . In particular, high DFI levels have been reported to be associated with reduced fertilization rates and impaired embryo developmental capacity. While there have been previous reports of cases that have been followed through to live births 34 , this study is the first to explore the impact of DFI on embryo aneuploidy risk. In the high DFI group, normal fertilization rates in ICSI were lower compared to the control group, and embryo development to the blastocyst stage showed a declining trend. This suggests a limitation in the qualitative assessment of sperm selected by humans during ICSI. Specifically, in cases with high DFI, sperm selection based solely on motility and morphology through visual assessment is insufficient to determine sperm DNA integrity 35 . Additionally, a negative correlation was observed between DFI and other semen parameters, indicating that DFI serves as a useful marker of sperm health 36 , 37 . This further supports the idea that aging in men contributes to increased DFI levels, ultimately leading to a decline in sperm quality 38 . By utilizing preimplantation genetic testing for aneuploidy (PGT-A), we investigated the relationship between embryo chromosomal aneuploidy and DFI. Our findings suggest that even in cases with high DFI, morphologically good-quality blastocysts do not necessarily carry an increased risk of aneuploidy. Therefore, when embryos are euploid chromosomes, embryo transfer should still be actively considered, even in cases of high DFI 39 . However, further research is needed to clarify how the ability to repair DNA during embryogenesis is affected in fertilized oocytes derived from high DFI patients 40 , 41 . Some studies suggest that sperm with high DFI may have a reduced ability to repair DNA damage during embryo development, which could potentially impact miscarriage rates 42 , 43 . Evaluating this aspect will be critical in future research 44 , 45 . Therefore, the assessment of sperm DNA fragmentation may play a crucial role in optimizing treatment strategies in IVF 46 . DFI test results can serve as important indicators of sperm health and embryo development. In cases where severe sperm DNA fragmentation (≥ 30%) is observed, it is essential to consider its potential impact on IVF outcomes and miscarriage risk when determining treatment plans 47 . Although the number of embryo transfer cases among high DFI patients in this study was limited, only three out of six implanted embryos resulted in live birth, highlighting the need for further validation with larger sample sizes 48 . As DFI testing becomes more widely adopted, it is expected to become a valuable diagnostic and treatment tool for male infertility. Several methods are available for detecting DFI, including TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) 49 , comet assay (single-cell gel electrophoresis-based detection), and AO test (fluorescence observation using acridine orange, a nucleic acid dye) 50 . However, these techniques generally require advanced equipment and can be costly. In this study, we quantified DFI using the sperm chromatin dispersion (SCD) test based on chromatin structure analysis of sperm nuclei with the Halo Sperm DNA kit 51 . This commercially available kit offers a relatively simple and cost-effective method for DFI analysis, making it a viable option for assessing male infertility. By employing DFI testing, improvements in the diagnostic accuracy of male infertility are anticipated. Based on the findings of this study and existing research, the importance of sperm selection in ICSI has become even more evident. Given the reported impact of sperm with severe DNA damage on fertilization rates and embryo development 52 , it is crucial to explore methods for selecting sperm with the least possible DNA damage 53 . The causes of sperm DNA damage are believed to include aging, oxidative stress, and lifestyle factors. However, many aspects remain unclear, and a definitive prevention or treatment strategy has yet to be established. Therefore, at this stage, reducing the DNA damage rate in sperm itself is challenging. Instead, improving the selection process for sperm used in fertilization could be a more practical and effective approach. In recent years, techniques have been developed to minimize sperm DNA damage while selecting sperm that maintain good genetic quality. These methods are considered promising for improving IVF outcomes. For example, several sperm selection methods have gained attention for reducing the proportion of DNA-fragmented sperm, including physiological hyaluronan-selected ICSI (PICSI), and magnetic-activated cell sorting (MACS). PICSI has the potential to select mature sperm with minimal DNA damage, enabling the selection of good-quality sperm 54 . MACS, on the other hand, is a technique that removes sperm that show characteristics of early apoptosis using magnetic separation, allowing for a more precise selection of sperm with intact DNA integrity 55 . In recent years, the MIGLIS (Menicon Life Science, Japan) method, which uses a microfluidic sperm selection device 56 , and the ZyMote (ZyMotefertility, USA) method, which is a non-centrifugal sperm sorting technique 57 , have also attracted attention. There are reports that sperm sorted by non-centrifugation can be retrieved with a much lower degree of DNA damage than those sorted by centrifugation 58 . Further research is needed to verify whether sperm selected through these methods can enhance favorable embryonic development and blastocyst formation 59 . In particular, a detailed evaluation of chromosomal aneuploidy risk and its impact on final birth rates would be particularly valuable. Additionally, investigating whether these selection techniques contribute to improvements in miscarriage rates and recurrent pregnancy loss could provide new insights into male infertility factors 60 . 5 CONCLUSION The clinical results of this study indicate that although high DFI patients represent a minority of cases overall, optimizing sperm selection methods to use sperm with minimal DNA damage may contribute to improved embryo quality and pregnancy success rates. Evaluating sperm DNA integrity and refining sperm purification processes to select good-quality sperm for insemination underscores the utility of DFI testing. This approach holds promise as an effective strategy for male infertility treatment. Declarations Funding Declaration Author Contribution Tsuyoshi Okubo：Concept and plan，Data collection，Substantial contribution to data analysis and interpretation，Drafting a paper and significant revision to its contentTatsuya Kobayashi：Substantial contribution to data analysis and interpretationNoriyuki Onda：Data collection and substantial contribution to data analysisTeruaki Hayashi：Data collection and substantial contribution to data analysisKenji Omi：Substantial contribution to data analysis and interpretationTomoya Segawa：Concept and plan, Substantial contribution to data analysis and interpretation Acknowledgement We thank K. Nakazato for proofreading the manuscript and assisting with its preparation and the staff of Shinbashi Yume Clinic. Data Availability The data underlying this article will be shared on reasonable request to the corresponding author. 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Sperm DNA damage caused by oxidative stress modifiable clinical, lifestyle and nutritional factors in male infertility. Reprod. Biomed. Online . 28 , 684–703 (2014). Meseguer, F. et al. Can Microfluidics Improve Sperm Quality A Prospective Functional Study. Biomedicines 12 , 1131 (2024). Kuwayama, M., Vajta, G., Ieda, S. & Kato, O. Comparison of open and closed methods for vitrification of human embryos and the elimination of potential contamination. Reprod. Biomed. Online . 11 , 608–614 (2005). Stone, B. A., Alex, A., Werlin, L. B. & Marrs, R. P. Age thresholds for changes in semen parameters in men. Fertil. Steril. 100 , 952–958 (2013). Rosiak-Gill, A. et al. Age-related changes in human sperm DNA integrity. Aging (Albany NY) . 13 , 5399–5411 (2019). Petersen, C. G. et al. The effects of male age on sperm DNA damage: an evaluation of 2,178 semen samples. JBRA Assist. Reprod. 22 , 323–330 (2018). Agarwal, A., Mulgund, A. M. & Chyatte, A. Hamada. M. R. A unique view on male infertility around the globe. Reprod Biol. Endocrinol 13 (2015). Evenson, D. P. Evaluation of sperm chromatin structure and DNA strand breaks is an important part of clinical male fertility assessment. Transl Androl. Urol. 6 , S495–S500 (2017). Vinnakota, C., Cree, L., Peek, J. & Morbeck, D. E. Incidence of high sperm DNA fragmentation in a targeted population of subfertile men. Syst. Biol. Reprod. Med. 65 , 451–457 (2019). Zidi-Jrah, I. et al. Relationship between sperm aneuploidy, sperm DNA integrity, chromatin packaging, traditional semen parameters, and recurrent pregnancy loss. Fertil. Steril. 105 , 58–64 (2016). Middelkamp, S. et al. Sperm DNA damage causes genomic instability in early embryonic development. Sci. Adv. 15 , eaaz7602 (2020). Mazzilli, R. et al. Effect of the male factor on the clinical outcome of intracytoplasmic sperm injection combined with preimplantation aneuploidy testing observational longitudinal cohort study of 1,219 consecutive cycles. Fertil. Steril. 108 , 961–972e963 (2017). Venkatesh, S. et al. Clinical significance of sperm DNA damage threshold value in the assessment of male infertility. Reprod. Sci. 18 , 1005–1013 (2011). Deenadayal Mettler, A. et al. Male age is associated with sperm DNA/chromatin integrity. Aging Male . 23 , 822–829 (2020). Christopher, L. R. B. et al. The diagnosis of male infertility an analysis of the evidence to support the development of global WHO guidance-challenges and future research opportunities. Hum. Reprod. Update . 23 , 660–680 (2017). Sedó, C. A. et al. Effect of sperm DNA fragmentation on embryo development: clinical and biological aspects. JBRA Assist. Reprod. 21 , 343–350 (2017). Ashwood-Smith, M. J. & Edwards, R. G. DNA repair by oocytes. Mol. Hum. Reprod. 2 , 46–51 (1996). Borini, A. et al. Sperm DNA fragmentation paternal effect on early post-implantation embryo development in ART. Hum. Reprod. 21 , 2876–2881 (2006). Dhawan, V. et al. Paternal factors and embryonic development: Role in recurrent pregnancy loss. Andrologia 51 , e13171 (2019). Evenson, D. P. et al. Utility of the sperm chromatin structure assay as a diagnostic and prognostic tool in the human fertility clinic. Hum. Reprod. 14 , 1039–1049 (1999). Tesarik, J., Greco, E. & Mendoza, C. Late, but not early, paternal effect on human embryo development is related to sperm DNA fragmentation. Hum. Reprod. 19 , 611–615 (2004). Busnelli, A. et al. Sperm DNA fragmentation and idiopathic recurrent pregnancy loss: Results from a multicenter case-control study. Andrology 11 , 1673–1681 (2023). De la Rochebrochard, E. & Thonneau, P. Paternal age and maternal age are risk factors for miscarriage; results of a multicentre European study. Hum. Reprod. 17 , 1649–1656 (2022). Cano-Extremera, M. et al. Superior Live Birth Rates, Reducing Sperm DNA Fragmentation (SDF), and Lowering Miscarriage Rates by Using Testicular Sperm Versus Ejaculates in Intracytoplasmic Sperm Injection (ICSI) Cycles from Couples with High SDF: A Systematic Review and Meta-Analysis. Biology 14 , 130 (2025). McQueen, D. B., Zhang, J. & Robins, J. C. Sperm DNA fragmentation and recurrent pregnancy loss: a systematic review and meta-analysis. Fertil. Steril. 112 , 54–60e53 (2019). Osman, A., Alsomait, H., Seshadri, S., El-Toukhy, T. & Khalaf, Y. The effect of sperm DNA fragmentation on live birth rate after IVF or ICSI: a systematic review and meta-analysis. Reprod. Biomed. Online . 30 , 120–127 (2014). Muratori, M. et al. DNA fragmentation in brighter sperm predicts male fertility independently from age and semen parameters. Fertil. Steril. 104 , 582–590e584 (2015). Mohammadi, Z., Tavalaee, M., Gharagozloo, P., Drevet, J. R. & Nasr-Esfahani, M. H. Could high DNA stainability (HDS) be a valuable indicator of sperm nuclear integrity. Basic. Clin. Androl. 30 , 12 (2020). Tandara, M. et al. Sperm DNA integrity testing: big halo is a good predictor of embryo quality and pregnancy after conventional IVF. Andrology 2 , 678–686 (2014). Kuroda, S. et al. Early and late paternal effects of reactive oxygen species in semen on embryo development after intracytoplasmic sperm injection. Syst. Biol. Reprod. Med. 66 , 122–128 (2020). Bradley, C. K. et al. Intervention improves assisted conception intracytoplasmic sperm injection outcomes for patients with high levels of sperm DNA fragmentation a retrospective analysis. Andrology 4 , 903–910 (2016). Miller, D. et al. Physiological, hyaluronan-selected intracytoplasmic sperm injection for infertility treatment (HABSelect): a parallel, two-group, randomised trial. Lancet 393 , 416–422 (2019). Pacheco, A. et al. Magnetic-Activated Cell Sorting (MACS) A Useful Sperm-Selection Technique in Cases of High Levels of Sperm DNA Fragmentation. J. Clin. Med. 9 , 3976 (2020). Meitei, H. Y. et al. Centrifugation-Free, Sperm-Sorting Device Eliminates the Risks of Centrifugation in the Swim-Up Method While Maintaining Functional Competence and DNA Integrity of Selected Spermatozoa. Reprod. Sci. 28 , 134–143 (2021). Kocur, O. M. et al. Can a sperm selection technique improve embryo ploidy? Andrology 11 , 1605–1612 (2023). Albani, E. et al. Male age: negative impact on sperm DNA fragmentation. Aging (Albany NY) . 11 , 2749–2761 (2019). Johnson, D. S. et al. Preclinical validation of a microarray method for full molecular karyotyping of blastomeres in a 24-h protocol. Hum. Reprod. 25 , 1066–1075 (2010). Inversetti, A. et al. Recurrent pregnancy loss a male crucial factor-A systematic review and meta-analysis. Andrology 13 , 130–145 (2025). Additional Declarations No competing interests reported. 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Okubo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5UlEQVRIiWNgGAWjYHACZgbGBgY5CLtAgoGNkHoeqBZjCNcApiWBsJbEBogWmDgeLfYMzIeNeXfYpG+4kZ344IeBRR4f+wG2Bx9/4LOFLTmZ90xa7oYbuZsNewwkitl4EtgNZ+BzmPwb48O8bYdBWrZJA/2S2Ab0jjQPXr/wgLWkG9zI3f4bruUPAS3JQC0JQC3bmOFa8IbYAbZkw7ln0gxnnnm7WbIHpIUnsU2yJw23FvYG5sMSb3fYyPMdz9344UdFXeL89sPHJH7Y4NYCBwoH4ExgNBEF5IlUNwpGwSgYBSMQAAA/NEh42CCzDwAAAABJRU5ErkJggg==","orcid":"","institution":"Shinbashi Yume Clinic, Advanced medical research institute of fertility","correspondingAuthor":true,"prefix":"","firstName":"Tsuyoshi","middleName":"","lastName":"Okubo","suffix":""},{"id":542028277,"identity":"d9331bed-5768-4a84-911c-7f50b13112c5","order_by":1,"name":"Tatsuya Kobayashi","email":"","orcid":"","institution":"Haneda Clinic, Fujita Health University","correspondingAuthor":false,"prefix":"","firstName":"Tatsuya","middleName":"","lastName":"Kobayashi","suffix":""},{"id":542028278,"identity":"dab440eb-b1c5-423f-855e-cdcbb8e097e1","order_by":2,"name":"Teruaki Hayashi","email":"","orcid":"","institution":"Shinbashi Yume Clinic, Advanced medical research institute of fertility","correspondingAuthor":false,"prefix":"","firstName":"Teruaki","middleName":"","lastName":"Hayashi","suffix":""},{"id":542028279,"identity":"a5dcab84-54bd-4758-a1bf-49f6c1710af3","order_by":3,"name":"Noriyuki Onda","email":"","orcid":"","institution":"Shinbashi Yume Clinic, Advanced medical research institute of fertility","correspondingAuthor":false,"prefix":"","firstName":"Noriyuki","middleName":"","lastName":"Onda","suffix":""},{"id":542028280,"identity":"8bc3742c-d720-4e09-8372-a79f2ee50ed3","order_by":4,"name":"Kenji Omi","email":"","orcid":"","institution":"Shinbashi Yume Clinic, Advanced medical research institute of 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17:44:13","extension":"xml","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":132525,"visible":true,"origin":"","legend":"","description":"","filename":"c549b51bdea143a8bcc244ad34ab07481structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7782850/v1/bda3701ee5dfc7064a59e790.xml"},{"id":95860225,"identity":"1d462889-814b-4377-83cc-aff3a107cef7","added_by":"auto","created_at":"2025-11-13 17:44:13","extension":"html","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":144265,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7782850/v1/c444be3cea99a924825902a2.html"},{"id":95860217,"identity":"595f2d57-8536-4aa5-8519-a03dae379c93","added_by":"auto","created_at":"2025-11-13 17:44:13","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":121418,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental procedure flow diagram of DFI test and IVF results. DFI, DNA fragmentation index; IVF, in vitro fertilization; PGT-A, preimplantation genetic testing for aneuploidy.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7782850/v1/d30f82362eef16ad386f58b6.png"},{"id":105755554,"identity":"7cbe4968-a649-4e8f-be6a-82db52405f91","added_by":"auto","created_at":"2026-03-30 16:27:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":934628,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7782850/v1/1bd83fb6-2607-4a25-b536-59fbdcedca98.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The innovative role of sperm DNA fragmentation testing: understanding its impact on IVF outcomes and PGT-A","fulltext":[{"header":"1 INTRODUCTION","content":"\u003cp\u003eInfertility is a serious problem for many couples, with causes that vary widely. Traditionally, infertility treatments have primarily focused on female factors\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, while male factors, especially the sperm, have been relatively overlooked. In many fertility centers, semen analysis is limited solely to assessing parameters based on its concentration, motility, and morphology\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. However, these evaluations are not standardized and often involve subjective elements\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, raising concerns about diagnostic reliability\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Recent research has highlighted the crucial role of sperm quality in determining pregnancy outcomes\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Notably, beyond sperm count and motility, a key consideration is how sperm affect the fertilization of oocytes and subsequent embryo development\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIt has been known that sperm play a crucial role from the early stages of fertilization to embryo formation and, ultimately, the development of the blastocyst. In addition to conventional semen analysis, methods such as morphological evaluation based on the Kruger criteria and the observation of sperm vacuoles under high magnification using the intracytoplasmic morphologically selected sperm injection method have been used as additional diagnostic tools for male infertility\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. However, it has become clear that these examination methods alone are insufficient for fundamentally elucidating the causes of infertility attributable to male factors\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. While semen analysis based on WHO criteria (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e)\u0026mdash;such as sperm concentration, motility, and normal morphology\u0026mdash;provides a useful reference, meeting these standards does not necessarily rule out issues related to fertilization capacity or embryo development. The WHO criteria are only general indicators and do not fully reflect minute abnormalities in sperm quality or their effects on embryo development after fertilization\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Therefore, more refined analytical techniques are needed to assess the functional characteristics of sperm and their influence on embryo development\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Such a detailed evaluation will be key to more accurately identifying the causes of infertility attributable to sperm quality and to optimizing treatment strategies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e A Historical comparison of diagnostic criteria in WHO guidelines for semen examination from the 1st\u003csup\u003e\u0026nbsp;\u003c/sup\u003e(1980) to the 6th\u003csup\u003e\u0026nbsp;\u003c/sup\u003e(2021) edition.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"669\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 170px;\"\u003e\n \u003cp\u003eSemen characteristics\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003eWHO 1st (1980)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003eWHO 2nd (1987)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003eWHO 3rd (1992)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003eWHO 4th (1999)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003eWHO 5th (2010)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003eWHO 6th (2021)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003eSemen volume (mL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003e\u0026ge;2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003e\u0026ge;2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003e\u0026ge;2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003e\u0026ge;1.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003e\u0026ge;1.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003eSperm count (10\u003csup\u003e6\u003c/sup\u003e/mL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003e20-200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003e\u0026ge;20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003e\u0026ge;20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003e\u0026ge;20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003e\u0026ge;15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003e\u0026ge;16\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003eTotal sperm count (10\u003csup\u003e6\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003e\u0026ge;40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003e\u0026ge;40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003e\u0026ge;40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003e\u0026ge;39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003e\u0026ge;39\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003eTotal motility (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003e\u0026ge;60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003e\u0026ge;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003e\u0026ge;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003e\u0026ge;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003e\u0026ge;40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003e\u0026ge;42\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003eProgressive motility (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003e\u0026ge;25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003e\u0026ge;25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003e\u0026ge;25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003e\u0026ge;25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003e\u0026ge;32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003e\u0026ge;30\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003eMorphology (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003e\u0026ge;80.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003e\u0026ge;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003e\u0026ge;30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003e\u0026ge;14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003e\u0026ge;4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003e\u0026ge;4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eWHO: World Health Organization\u003c/p\u003e\n\u003cp\u003eND: Not Determined\u003c/p\u003e\n\u003cp\u003eIn reality, sperms are highly specialized cells that undergo various biological changes necessary for achieving fertilization. During spermatogenesis, sperm DNA undergoes structural modifications to achieve a highly organized, compact, and condensed form. This unique chromatin structure protects the paternal genome from external influences and ensures its accurate transmission to the oocyte.\u003c/p\u003e\n\u003cp\u003eAs a result, sperm DNA integrity has been suggested as a crucial factor for understanding the pathophysiology and etiology of male infertility. In recent years, SDF has emerged as an important parameter in semen analysis, with the DFI being of particular interest\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Studies have shown a negative correlation between DFI and sperm morphology and motility, with DFI increasing with male age\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Sperm DNA damage-induced dysfunction may impact IVF success rates, and previous research has reported that blastocysts derived from sperm with high DFI exhibit chromosomal abnormalities and morphological differences compared to those from sperm with low DFI. Furthermore, SDF has been suggested as a useful predictor of pregnancy success\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Since these findings suggest that additional semen analysis focused on SDF may serve as a critical assessment criterion in infertility treatment, we decided to validate its clinical impact on IVF outcomes.\u003c/p\u003e\n\u003cp\u003eAs previously mentioned, SDF is not only correlated with various factors in standard semen analysis but has also provided new insights into its association with blastocyst development and pregnancy outcomes in IVF treatment\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Several reports have indicated differences in the rates of good-quality blastocyst development, clinical pregnancy, and miscarriages among different DFI groups\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. However, other studies have shown that blastocysts derived from sperm with high SDF do not exhibit significant differences in morphological grading or live birth outcomes\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. This ongoing research continues to investigate and evaluate the relationship between sperm DNA damage and IVF success rates, but clear conclusions regarding causality and clinical significance have yet to be established\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThis study aims to further investigate the correlation between SDF and IVF outcomes, providing new insights into the significance of SDF testing\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Previous reports have documented reduced fertilization rates and impaired blastocyst development in men with high SDF levels. In addition to these findings, we sought to present new perspectives based on embryonic aneuploidy analysis results. There have been no previous large-scale reports on the correlation between SDF and PGT-A results, making it particularly intriguing to explore how sperm DNA integrity influences the blastocyst formation process.\u003c/p\u003e\n\u003cp\u003ePGT-A is a crucial technique in IVF for evaluating abnormalities in the number of chromosomes in blastocysts. A normal blastocyst typically has a positive diploid ploidy, 46 chromosomes in number. However, SDF may potentially influence chromosomal aneuploidy by causing excess or deficient chromosomes. Additionally, high DFI levels in semen have been linked to sperm aneuploidy and meiotic abnormalities, increasing the risk of embryo aneuploidy. In this study, we investigated the relationship between SDF and embryonic aneuploidy to provide a more detailed assessment of its impact on IVF success rates. Through this analysis, we aimed to offer new insights into male infertility factors that have not been previously explored in previous studies.\u003c/p\u003e\n\u003cp\u003eAlthough the success of infertility treatment is influenced by a complex interplay of many factors, we examined the possibility that the DFI test, which provides a detailed assessment of male sperm health\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, could play an essential role in optimizing treatment strategies in IVF. Through this study, we also aimed to verify the utility of the DFI test and to contribute to improving the success rate of infertility treatment by providing new insights into male infertility.\u003c/p\u003e"},{"header":"2 MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Study population\u003c/h2\u003e\u003cp\u003eThis study included 124 couples who underwent IVF and embryo aneuploidy analysis via PGT-A between May 2019 and April 2024, with male partners who opted for DFI testing during semen analysis. The study design was reviewed and approved by our institutional ethics committee (SYC2019-04). The research was conducted with patients who provided informed consent and agreed to participate. IVF cases involving oocytes or sperm not collected at our institution were excluded from the study (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e・Follicular monitoring and oocyte retrieval\u003c/p\u003e\u003cp\u003eStarting on day 3 of the menstrual cycle, follicular monitoring was performed using ultrasound and hormone testing. Clomiphene citrate was administered at 0.5\u0026ndash;1T/day, with additional FSH supplementation at 75 IU/day as needed. Once the dominant follicle reached a diameter of 16\u0026ndash;18 mm and E2 levels reached 200 pg/mL, buserelin acetate (300 \u0026micro;g/mL) was administered intranasally, followed by oocyte retrieval 32\u0026ndash;36 hours later. Oocytes were retrieved from the follicular fluid which was puncture-aspirated with a 22-gauge needle (Kitazato BioPharma, Japan). The collected oocytes were examined under an inverted microscope to assess their maturity based on the presence of first polar body extrusion.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Semen Examination and Analysis\u003c/h2\u003e\u003cp\u003e・Sperm motility test (general semen test)\u003c/p\u003e\u003cp\u003eThe semen sample was collected through masturbation. The semen volume was measured using a sterile pipette when the semen was sufficiently liquefied. Subsequently, sperm concentration, total sperm count, and motility rate were analyzed using an automated sperm motility analysis system (SMAS, DETECT, Japan).\u003c/p\u003e\u003cp\u003e・Sperm morphology (Kruger sperm function test)\u003c/p\u003e\u003cp\u003eSperm morphology was evaluated using the Diff-Quik Staining Kit (Sysmex Corporation, Japan). More than 200 sperm were examined under a biological microscope (\u0026times;400), with detailed observations of the head, midpiece, and tail. The percentage of sperm with normal morphology was then calculated.\u003c/p\u003e\u003cp\u003e・Sperm DNA fragmentation test (SDF test)\u003c/p\u003e\u003cp\u003eDFI assessment was conducted using the Halosperm HT-HS10 kit (Halotech DNA, Spain). This kit enables the quantification of DNA fragmentation in the sperm nucleus. In normal sperm, a halo is visible around the head, formed by looped DNA strands. However, in fragmented sperm, the damaged DNA strands fail to form a halo\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. The percentage of fragmented sperm was analyzed as the DFI rate, based on observations of over 300 sperm. The DFI rate was categorized into three groups: low (\u0026le;\u0026thinsp;15%, n\u0026thinsp;=\u0026thinsp;81), medium (15\u0026ndash;30%, n\u0026thinsp;=\u0026thinsp;34), and high (\u0026ge;\u0026thinsp;30%, n\u0026thinsp;=\u0026thinsp;9).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Semen Collection\u003c/h2\u003e\u003cp\u003eThe density gradient centrifugation method, a common sperm sorting process, was performed by layering two layers of SepaSperm (Kitazato BioPharma, Japan), 80% and 40%, in a centrifuge tube, 1.0 mL each. Next, a sterile pipette was used to place 1.0 mL of liquefied semen sample in HTF medium onto the upper layer. The centrifuge tube was then centrifuged at 600G for 15 minutes. The supernatant was removed, and the remaining pellet was resuspended in 5 mL of washing HTF medium (Kitazato BioPharma, Japan) before undergoing a second centrifugation at 400G for 5 minutes. After removing the supernatant again, HTF medium was added to dilute and adjust sperm concentration. The sample was then stored in an incubator at 37\u0026deg;C until insemination.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 IVF and Vitrification\u003c/h2\u003e\u003cp\u003eMature oocytes underwent either conventional in-vitro fertilization (C-IVF) or intracytoplasmic sperm injection (ICSI), followed by individual culturing in 20 mL of Human Tubal Fluid (HTF) medium (Kitazato BioPharma, Japan) containing 0.4% human serum albumin. Fertilization was confirmed the day after insemination by identifying male and female pronuclei. Abnormally fertilized oocytes with more than three pronuclei and degenerated oocytes were excluded from the analysis. Fertilized embryos were cultured in one step medium (Naka medical, JAPAN) by time-lapse culture system on an Embryo Scope Flex (Vitrolife, Sweden). Blastocysts meeting the criteria for good-quality embryos were cryopreserved via vitrification using Cryotop (Kitazato BioPharma, Japan) \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. The criteria for good-quality blastocysts were defined as the presence of at least 12 trophoectoderm (TE) cells at the outer perimeter of the blastocyst and an inner diameter of 170 \u0026micro;m on day 5 or 6 of culture. Blastocysts that did not meet these criteria by the end of day 6 were not cryopreserved.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 TE Cell Biopsy and PGT-A\u003c/h2\u003e\u003cp\u003eExpanded blastocysts that met our clinic's PGT-A criteria (at least 12 TE cells on the outer perimeter and a blastocyst inner diameter of 170 \u0026micro;m or more) were eligible for PGT-A analysis. TE cells located away from the inner cell mass were isolated using the flicking method with a biopsy pipette, collecting 4\u0026ndash;6 cells. The biopsied TE cells were washed in a 30 \u0026micro;L drop of phosphate-buffered saline (PBS) in at least three different locations and then transferred to a 2.5 \u0026micro;L drop of PBS solution containing 1% PVP in a polymerase chain reaction (PCR) tube. The samples were cryopreserved at -30\u0026deg;C. The frozen TE cells were then sent to a testing laboratory for next-generation sequencing (NGS) analysis to examine embryonic chromosomal aneuploidy.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 EMBRYO TRANSFER\u003c/h2\u003e\u003cp\u003eThe results of PGT-A analysis were classified into four categories: euploid, mosaic, aneuploid, and not detected. Only euploid embryos or low-frequency mosaic embryos were considered transferable, and informed consent was obtained from all patients prior to embryo transfer. All cryopreserved blastocysts were transferred during a natural ovulation cycle following the initial oocyte retrieval cycle, specifically 5 to 5.5 days after natural ovulation. If the endometrial thickness was less than 6 mm, embryo transfer for that cycle was canceled and postponed to a subsequent cycle. Additionally, all blastocysts underwent assisted hatching (AH) using laser technology to remove the zona pellucida, ensuring the embryo was in a completely hatched blastocyst state. Embryo transfer was performed as a single embryo transfer under transvaginal ultrasound guidance.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 OUTCOMES\u003c/h2\u003e\u003cp\u003eThe primary outcome was defined as live birth at \u0026gt;\u0026thinsp;22 weeks' gestation. Clinical pregnancy and ongoing pregnancy were defined as the presence of a gestational sac by the sixth week of pregnancy and the detection of fetal cardiac activity at \u0026ge;\u0026thinsp;7 weeks' gestation, respectively.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 STATISTICAL ANALYSIS\u003c/h2\u003e\u003cp\u003ePatient background data were summarized using mean and standard deviation for continuous variables. Comparisons with the control group were conducted using Dunnett's test. Fisher's exact test was used to compare IVF outcomes with the control group. All data were analyzed using JMP 11.2 software (SAS Institute, Cary, NC, USA), and statistical significance was determined at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"3 RESULTS","content":"\u003cp\u003eOf the 455 couples whose DFI was measured at the time of the initial semen examination, 124 couples who underwent PGT-A were included in this study. The average age of the wives was 41.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9 years, while the husbands' average age was 42.1\u0026thinsp;\u0026plusmn;\u0026thinsp;5.2 years. The mean DFI was 14.3\u0026thinsp;\u0026plusmn;\u0026thinsp;10.4, and participants were categorized into three groups based on DFI levels: low (\u0026le;\u0026thinsp;15%, n\u0026thinsp;=\u0026thinsp;81), medium (15\u0026ndash;30%, n\u0026thinsp;=\u0026thinsp;34), and high (\u0026ge;\u0026thinsp;30%, n\u0026thinsp;=\u0026thinsp;9). The proportion of patients in the high DFI group was 7.3% (9/124) (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\u003eGeneral semen findings and DFI in target patients (n\u0026thinsp;=\u0026thinsp;124)\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\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\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFactors\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eRange\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFemale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\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\u003e41.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e34\u0026ndash;47\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAMH (pmol/L)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e1.89\u0026thinsp;\u0026plusmn;\u0026thinsp;1.84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.03\u0026ndash;8.68\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\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\u003e42.1\u0026thinsp;\u0026plusmn;\u0026thinsp;5.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e29\u0026ndash;59\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSemen volume (mL)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e2.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.1\u0026ndash;7.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eConcentration (10\u003csup\u003e6\u003c/sup\u003e/mL)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e66.6\u0026thinsp;\u0026plusmn;\u0026thinsp;66.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.3\u0026ndash;380.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTotal sperm counts\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e185.3\u0026thinsp;\u0026plusmn;\u0026thinsp;201.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.1-1194.8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMotility (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e59.9\u0026thinsp;\u0026plusmn;\u0026thinsp;19.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8.9\u0026ndash;89.6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNormal morphology (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e2.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0-9.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDFI (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e14.3\u0026thinsp;\u0026plusmn;\u0026thinsp;10.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.0-64.7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"3\"\u003eData presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation.\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"3\"\u003eAMH: Anti-Mullerian Hormone\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"3\"\u003eDFI: sperm DNA fragmentation index\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eWhen comparing the correlation between DFI groups and various semen parameters with the control group (low DFI), both the medium and high DFI groups showed a significantly higher average male age and a tendency for decreased sperm motility.\u003c/p\u003e\u003cp\u003eRegarding fertilization outcomes after insemination, normal fertilization rates in ICSI were significantly lower in the high DFI group compared to the control group. However, in C-IVF, there were no significant differences in normal or abnormal fertilization rates among the DFI groups. Additionally, the rate of good-quality blastocyst formation meeting our clinic\u0026rsquo;s cryopreservation criteria was significantly lower in the high DFI group than in the low and medium DFI groups (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eThe relationship between DFI groups and semen analysis (n\u0026thinsp;=\u0026thinsp;124)\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\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\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eDFI\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003eP value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFactors\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLow\u003c/p\u003e\u003cp\u003e(\u0026le;\u0026thinsp;15%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMedium\u003c/p\u003e\u003cp\u003e(15\u0026ndash;30%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHigh\u003c/p\u003e\u003cp\u003e(\u0026ge;\u0026thinsp;30%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eLow vs Medium\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eLow vs High\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePercentage of the whole (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e65.3 (81/124)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e27.4 (34/124)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e7.3 (9/124)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMale age (years)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e41.0\u0026thinsp;\u0026plusmn;\u0026thinsp;4.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e43.6\u0026thinsp;\u0026plusmn;\u0026thinsp;4.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e47.0\u0026thinsp;\u0026plusmn;\u0026thinsp;7.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e0.0105\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e0.0014\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSemen parameter\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSemen volume (mL)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.6331\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.9561\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eConcentration (10\u003csup\u003e6\u003c/sup\u003e/mL)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e67.1\u0026thinsp;\u0026plusmn;\u0026thinsp;65.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e61.5\u0026thinsp;\u0026plusmn;\u0026thinsp;70.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e80.5\u0026thinsp;\u0026plusmn;\u0026thinsp;71.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.8907\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.8113\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTotal sperm counts\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e187.0\u0026thinsp;\u0026plusmn;\u0026thinsp;206.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e162.5\u0026thinsp;\u0026plusmn;\u0026thinsp;178.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e253.6\u0026thinsp;\u0026plusmn;\u0026thinsp;249.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.7717\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.5711\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMotility (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e64.7\u0026thinsp;\u0026plusmn;\u0026thinsp;15.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e52.3\u0026thinsp;\u0026plusmn;\u0026thinsp;21.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e44.3\u0026thinsp;\u0026plusmn;\u0026thinsp;22.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e0.0020\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e0.0030\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNormal morphology (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.3969\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.0519\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"7\"\u003eData presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. *Fisher\u0026rsquo;s exact test\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"7\"\u003eDFI: sperm DNA fragmentation index\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eAs for embryo aneuploidy based on PGT-A analysis, there were no significant differences in the proportions of euploid, mosaic, and aneuploid embryos among each DFI group. Based on PGT-A results, single embryo transfers were conducted in 103 cases where euploid or low-frequency mosaic embryos were observed. The implantation rates for the low, medium, and high DFI groups were 71.4% (50/70), 66.7% (18/27), and 100% (6/6), respectively. Pregnancy rates confirmed by gestational sac detection were 62.9% (44/70), 59.3% (16/27), and 66.7% (4/6), while live birth rates were 52.9% (37/70), 40.7% (11/27), and 50.0% (3/6). No significant differences were observed in pregnancy outcomes per embryo transfer among the DFI groups (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eThe relationship between DFI groups and IVF results\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"8\"\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\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eDFI\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c8\" namest=\"c6\"\u003e\u003cp\u003eP value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFactors\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLow\u003c/p\u003e\u003cp\u003e(\u0026le;\u0026thinsp;15%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMedium\u003c/p\u003e\u003cp\u003e(15\u0026ndash;30%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHigh\u003c/p\u003e\u003cp\u003e(\u0026ge;\u0026thinsp;30%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eLow vs Medium\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eLow vs High\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c8\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNo. of oocytes (n)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6269\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1827\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e586\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c8\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eICSI outcome\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c8\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNormal fertilization (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e79.6 (4403/5530)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e80.4 (1318/1639)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e75.9 (425/560)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.4816\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e0.0381\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c8\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAbnormal fertilization (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6.5 (359/5530)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5.5 (91/1639)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e8.6 (48/560)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.1683\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.0604\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c8\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eC-IVF outcome\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c8\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNormal fertilization (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e80.4 (594/739)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e78.7 (148/188)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e88.5 (23/26)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.6121\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.4396\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c8\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAbnormal fertilization (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e11.1 (82/739)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e11.2 (21/188)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.8 (1/26)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.6672\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.3967\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c8\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBlastocyst outcome\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c8\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNo of cultured embryos (n)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4997\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1466\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e448\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c8\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCryopreserved blastocyst (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e31.7 (1586/4997)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e31.4 (460/1466)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e27.2 (122/448)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.7938\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e0.0489\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c8\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGrade AA, AB and BA (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e23.6 (375/1586)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e22.0 (101/460)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e17.2 (21/122)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.4506\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.1048\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c8\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGrade BB (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e74.1 (1176/1586)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e75.7 (348/460)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e82.0 (100/122)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.5149\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.0556\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c8\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGrade BC and CB (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.2 (35/1586)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.4 (11/460)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.8 (1/122)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.8142\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.4834\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c8\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePGT-A outcome\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c8\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNo of biopsied embryos (n)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e594\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e176\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c8\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eeuploid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e16.5 (98/594)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e15.3 (27/176)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e23.1 (6/26)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.7146\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.5414\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c8\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003emosaic\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e9.8 (58/594)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10.8 (19/176)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e11.5 (3/26)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.6888\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.9688\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c8\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eaneuploid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e69.7 (414/594)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e69.3 (122/176)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e65.4 (17/26)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.9236\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.8027\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c8\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003enot detected\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4.0 (24/594)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4.5 (8/176)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0 (0/26)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.9363\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.5989\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c8\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNo. of transferred blastocyst (n)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c8\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePregnancy rate (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e71.4 (50/70)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e66.7 (18/27)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e100 (6/6)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.8323\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.2973\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c8\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eClinical pregnancy rate (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e62.9 (44/70)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e59.3 (16/27)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e66.7 (4/6)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.7437\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.7985\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c8\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLive birth rate (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e52.9 (37/70)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e40.7 (11/27)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e50.0 (3/6)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.2847\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.7707\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c8\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"8\"\u003eData presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. *Fisher\u0026rsquo;s exact test\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"8\"\u003eDFI: sperm DNA fragmentation index\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"8\"\u003eICSI: Intracytoplasmic Sperm Injection\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"8\"\u003eC-IVF: Conventional In Vitro Fertilization\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"8\"\u003ePGT-A: Preimplantation Genetic Testing for Aneuploidy\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"4 DISCUSSION","content":"\u003cp\u003eThe findings of this study support previous research on DFI and reaffirm the impact of aging on DFI levels\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. The observed increase in sperm DNA damage with advancing male age suggests that qualitative factors in sperm\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e\u0026mdash;specifically, a decline in chromosomal stability\u0026mdash;may be a contributing factor to infertility\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Among the male patients undergoing infertility treatment in this study\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, the majority had low to medium DFI levels, while only about 7% exhibited high DFI (\u0026ge;\u0026thinsp;30%), making them a minority\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. This highlights that although men with high DFI are relatively rare, they represent an important subgroup that warrants attention in infertility treatment\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eTraditionally, infertility has often been attributed to female factors, but numerous studies have demonstrated that sperm quality is also crucial for IVF success\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. In addition to fundamental semen parameters, such as sperm count and motility, sperm DNA integrity has been recognized as a key factor influencing IVF outcomes\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. In particular, high DFI levels have been reported to be associated with reduced fertilization rates and impaired embryo developmental capacity. While there have been previous reports of cases that have been followed through to live births\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, this study is the first to explore the impact of DFI on embryo aneuploidy risk.\u003c/p\u003e\u003cp\u003eIn the high DFI group, normal fertilization rates in ICSI were lower compared to the control group, and embryo development to the blastocyst stage showed a declining trend. This suggests a limitation in the qualitative assessment of sperm selected by humans during ICSI. Specifically, in cases with high DFI, sperm selection based solely on motility and morphology through visual assessment is insufficient to determine sperm DNA integrity\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Additionally, a negative correlation was observed between DFI and other semen parameters, indicating that DFI serves as a useful marker of sperm health\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. This further supports the idea that aging in men contributes to increased DFI levels, ultimately leading to a decline in sperm quality\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eBy utilizing preimplantation genetic testing for aneuploidy (PGT-A), we investigated the relationship between embryo chromosomal aneuploidy and DFI. Our findings suggest that even in cases with high DFI, morphologically good-quality blastocysts do not necessarily carry an increased risk of aneuploidy. Therefore, when embryos are euploid chromosomes, embryo transfer should still be actively considered, even in cases of high DFI\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. However, further research is needed to clarify how the ability to repair DNA during embryogenesis is affected in fertilized oocytes derived from high DFI patients\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Some studies suggest that sperm with high DFI may have a reduced ability to repair DNA damage during embryo development, which could potentially impact miscarriage rates\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Evaluating this aspect will be critical in future research\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eTherefore, the assessment of sperm DNA fragmentation may play a crucial role in optimizing treatment strategies in IVF\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. DFI test results can serve as important indicators of sperm health and embryo development. In cases where severe sperm DNA fragmentation (\u0026ge;\u0026thinsp;30%) is observed, it is essential to consider its potential impact on IVF outcomes and miscarriage risk when determining treatment plans\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Although the number of embryo transfer cases among high DFI patients in this study was limited, only three out of six implanted embryos resulted in live birth, highlighting the need for further validation with larger sample sizes\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. As DFI testing becomes more widely adopted, it is expected to become a valuable diagnostic and treatment tool for male infertility.\u003c/p\u003e\u003cp\u003eSeveral methods are available for detecting DFI, including TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e, comet assay (single-cell gel electrophoresis-based detection), and AO test (fluorescence observation using acridine orange, a nucleic acid dye) \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. However, these techniques generally require advanced equipment and can be costly. In this study, we quantified DFI using the sperm chromatin dispersion (SCD) test based on chromatin structure analysis of sperm nuclei with the Halo Sperm DNA kit\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. This commercially available kit offers a relatively simple and cost-effective method for DFI analysis, making it a viable option for assessing male infertility. By employing DFI testing, improvements in the diagnostic accuracy of male infertility are anticipated.\u003c/p\u003e\u003cp\u003eBased on the findings of this study and existing research, the importance of sperm selection in ICSI has become even more evident. Given the reported impact of sperm with severe DNA damage on fertilization rates and embryo development\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e, it is crucial to explore methods for selecting sperm with the least possible DNA damage\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. The causes of sperm DNA damage are believed to include aging, oxidative stress, and lifestyle factors. However, many aspects remain unclear, and a definitive prevention or treatment strategy has yet to be established. Therefore, at this stage, reducing the DNA damage rate in sperm itself is challenging. Instead, improving the selection process for sperm used in fertilization could be a more practical and effective approach. In recent years, techniques have been developed to minimize sperm DNA damage while selecting sperm that maintain good genetic quality. These methods are considered promising for improving IVF outcomes.\u003c/p\u003e\u003cp\u003eFor example, several sperm selection methods have gained attention for reducing the proportion of DNA-fragmented sperm, including physiological hyaluronan-selected ICSI (PICSI), and magnetic-activated cell sorting (MACS). PICSI has the potential to select mature sperm with minimal DNA damage, enabling the selection of good-quality sperm\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. MACS, on the other hand, is a technique that removes sperm that show characteristics of early apoptosis using magnetic separation, allowing for a more precise selection of sperm with intact DNA integrity\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. In recent years, the MIGLIS (Menicon Life Science, Japan) method, which uses a microfluidic sperm selection device\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e, and the ZyMote (ZyMotefertility, USA) method, which is a non-centrifugal sperm sorting technique\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e, have also attracted attention. There are reports that sperm sorted by non-centrifugation can be retrieved with a much lower degree of DNA damage than those sorted by centrifugation\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eFurther research is needed to verify whether sperm selected through these methods can enhance favorable embryonic development and blastocyst formation\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. In particular, a detailed evaluation of chromosomal aneuploidy risk and its impact on final birth rates would be particularly valuable. Additionally, investigating whether these selection techniques contribute to improvements in miscarriage rates and recurrent pregnancy loss could provide new insights into male infertility factors\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e"},{"header":"5 CONCLUSION","content":"\u003cp\u003eThe clinical results of this study indicate that although high DFI patients represent a minority of cases overall, optimizing sperm selection methods to use sperm with minimal DNA damage may contribute to improved embryo quality and pregnancy success rates. Evaluating sperm DNA integrity and refining sperm purification processes to select good-quality sperm for insemination underscores the utility of DFI testing. This approach holds promise as an effective strategy for male infertility treatment.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eDeclaration\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eTsuyoshi Okubo：Concept and plan，Data collection，Substantial contribution to data analysis and interpretation，Drafting a paper and significant revision to its contentTatsuya Kobayashi：Substantial contribution to data analysis and interpretationNoriyuki Onda：Data collection and substantial contribution to data analysisTeruaki Hayashi：Data collection and substantial contribution to data analysisKenji Omi：Substantial contribution to data analysis and interpretationTomoya Segawa：Concept and plan, Substantial contribution to data analysis and interpretation\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe thank K. Nakazato for proofreading the manuscript and assisting with its preparation and the staff of Shinbashi Yume Clinic.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data underlying this article will be shared on reasonable request to the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePractice Committee of the American Society for Reproductive Medicine. Diagnostic evaluation of the infertile female: a committee opinion. \u003cem\u003eFertil. Steril.\u003c/em\u003e \u003cb\u003e103\u003c/b\u003e, e44\u0026ndash;50 (2015).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e; ESHRE Guideline Group on RPL et al. ESHRE guideline: recurrent pregnancy loss: an update in 2022. \u003cem\u003eHum. Reprod. Open.\u003c/em\u003e (2023). (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eElisabetta, B. et al. 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Reprod.\u003c/em\u003e \u003cb\u003e25\u003c/b\u003e, 1066\u0026ndash;1075 (2010).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eInversetti, A. et al. Recurrent pregnancy loss a male crucial factor-A systematic review and meta-analysis. \u003cem\u003eAndrology\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e, 130\u0026ndash;145 (2025).\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":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"DNA fragmentation index, embryology, male infertility, Preimplantation genetic testing for aneuploidy, WHO criteria","lastPublishedDoi":"10.21203/rs.3.rs-7782850/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7782850/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis retrospective study evaluated whether sperm DNA fragmentation (SDF), measured by DNA fragmentation index (DFI), predicts IVF outcomes in 124 couples undergoing IVF with PGT-A between 2015 and 2023. SDF was assessed using the Halosperm kit and categorized into low (\u0026le;\u0026thinsp;15%), medium (15\u0026ndash;30%), and high (\u0026ge;\u0026thinsp;30%) DFI groups. Fertilization was performed via IVF or ICSI, and embryos were analyzed with PGT-A using next-generation sequencing. High DFI was associated with advanced paternal age, reduced motility, lower fertilization rates (in ICSI), and fewer good-quality blastocysts. However, PGT-A outcomes showed no significant differences in euploidy, mosaicism, or aneuploidy rates across DFI groups. Similarly, implantation, pregnancy, and live birth rates following euploid embryo transfer were unaffected by DFI level. While high sperm DFI negatively impacts early embryonic development, it does not predict chromosomal integrity. SDF testing can help identify male infertility factors and guide IVF strategy, particularly regarding sperm selection. These findings suggest that early developmental impairment from sperm DNA damage may be mitigated by embryonic repair mechanisms at later stages.\u003c/p\u003e","manuscriptTitle":"The innovative role of sperm DNA fragmentation testing: understanding its impact on IVF outcomes and PGT-A","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-13 17:44:09","doi":"10.21203/rs.3.rs-7782850/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision 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