Stesura Seveso Archivio Italiano di Urologia e Andrologia 2024; 96(4):12891 1 ORIGINAL PAPER In the context of assisted reproductive technology (ART), sperm preparation aims to isolate spermatozoa with opti- mal morphology and motility for successful fertilization (9). However, the cellular structure and function of sper- matozoa can be compromised during sperm preparation due to repeated centrifugation cycles, which induce ROS formation and result in the loss of seminal plasma antiox- idants (10). Studies have shown that ROS production in normal spermatozoa can increase 2-5 times after indirect swim-up preparation, with exposure to ROS from 1-2 hours leading to a 2-4-fold increase in DNA damage (11). To mitigate these effects, sperm selection methods such as the swim-up technique are employed to reduce the main sources of intracellular ROS, namely leukocytes and immature sperm (12). According to the WHO laboratory manual, the swim-up method is a simple method that allows motile sperm to swim into an overlaid medium, separating them from non-motile spermatozoa. This method enhances sperm motility, average velocity, nor- mal morphology and fertilization rates in vitro in mam- mals in compared to a lower fraction (13). However, sim- ple washing or swim-ups from the pellet have been asso- ciated with a sudden burst of ROS production, reduced motility, and impaired sperm-oocyte fusion in the zona- free hamster oocyte penetration test (14). Despite these challenges, the swim-up method has been shown to maintain better sperm DNA integrity compared to other procedures, such as density gradient centrifuga- tion, demonstrating reduced DNA fragmentation and vacuolization (15, 16). Antioxidant administration, including vitamins C, E, catalase and glutathione, has been suggested to protect sperm DNA integrity during preparation (17). Specifically, serum vitamin D levels have been correlated with improvements in sperm number, motility, morphol- ogy, and DNA fragmentation (18). Vitamin D plays a cru- cial role in calcium regulation within the male reproduc- tive system, and its activation in semen has been shown to enhance intracellular calcium levels, motility and acro- some reaction in spermatozoa (19). Additionally, vitamin D functions as a membrane antioxidant (20) and influ- ences gene expression related to cell proliferation, differ- Objective: This study aimed to identify the direct effect of vitamin D on sperm DNA integrity after swim-up preparation. Materials and methods: Normozoospermia samples were gath- ered from 12 men and assessed for their baseline characteris- tics, including DNA Fragmentation Index (DFI). Each sample was then prepared using the swim-up method. Half of the sam- ples were incubated with vitamin D, while the other half were incubated with a standard sperm-washing medium. Results: Vitamin D significantly reduced the DFI compared to the baseline (5.5 ± 3.4% versus 17.6 ± 4.2%; p < 0.001) and the swim-up-only group (5.5 ± 3.4% versus 12.0 ± 4.2%; p < 0.001). Microscopic examination reflected these results, showing a reduction in the number of small halos and no halos with an increased appearance of large to medium-sized halos. Conclusions: These results suggest that vitamin D incubation is valuable in protecting sperm from DNA damage that develops during sperm preparation. However, additional investigation is warranted to explore other preparation methods and to eluci- date the underlying mechanisms. KEY WORDS: Assisted reproductive technology; DNA damage; Male infertility; Swim up. Submitted 2 August 2024; Accepted 26 August 2024 INTRODUCTION Infertility is a significant global health issue, affecting approximately 1 in 7 couples, with contributions from both male and female factors equally distributed (1-3). Among the various factors contributing to male infertili- ty, damage to sperm DNA is a critical concern, impacting 20-40% of subfertile men. This DNA damage can arise from several sources, including imperfections in the apoptosis process during spermatogenesis (4), protami- nation during spermiogenesis (5), and oxidative stress from various endogenous and exogenous factors (6). Excessive production of reactive oxygen species (ROS) beyond the body’s antioxidant capacity can lead to sig- nificant damage to sperm function, causing lipid peroxi- dation, protein oxidation, mitochondrial dysfunction, and DNA damage (7, 8). The effect of vitamin D in vitro supplementation on sperm deoxyribonucleic acid fragmentation Andri Rezano 1, 2, Ditto Rezkiawan 2, Vellyana Lie 2, Arya Srisadono 2, Rafly Mochamad Rivaldo 3, Amelia Reta Purba 3, Melia Juwita Adha 4, Tjahjo Djojo Tanojo 2, 5, Maria P.B.D. Pramesti 2, 6 1 Department of Biomedical Sciences, Faculty of Medicine, Universitas Padjadjaran, Sumedang, West Java, Indonesia; 2 Andrology Study Program, Faculty of Medicine, Universitas Airlangga, Surabaya, East Java, Indonesia; 3 Medical Study Program, Faculty of Medicine, Universitas Padjadjaran, Sumedang, West Java, Indonesia; 4 Holistic General Hospital, Purwakarta, West Java, Indonesia; 5 Andrology Clinic, Dr. Soetomo Academic Hospital, Surabaya, East Java, Indonesia; 6 Department of Biomedical Sciences, Faculty of Medicine, Universitas Airlangga, Surabaya, East Java, Indonesia. DOI: 10.4081/aiua.2024.12891 Summary Archivio Italiano di Urologia e Andrologia 2024; 96(4):12891 A. Rezano, D. Rezkiawan, V. Lie, et al. 2 entiation, apoptosis and oxidative stress through its receptors in the nucleus and post-acrosome of spermato- zoa (21-23). Although numerous studies have explored the role of vitamin D in the reproductive system, research specifical- ly examining its impact on reducing the DNA fragmenta- tion index (DFI) of human spermatozoa remains limited. The DFI is a crucial indicator of semen quality, reflecting the integrity and damage to sperm DNA, thereby detect- ing potential sperm damage (24). Blaseg et al. (2022) found no association between circulating vitamin D levels and human DFI. However, though the study was limited by using circulating vitamin D as a proxy for intratesticu- lar levels (25). Additionally, many studies have not con- sidered ART factors in their analyses (26, 27). Therefore, this study aims to investigate the effect of vita- min D in vitro administration on sperm DFI in normo- zoospermic samples following sperm preparation using the swim-up method, addressing a critical gap in current reproductive research. MATERIALS AND METHODS A total of 12 semen samples (n = 12) were included in this study, each with a volume of 3 mL. The inclusion cri- teria for selecting samples were men aged 26-35 years who agreed to participate and signed the informed con- sent, had abstained from ejaculation for 2-7 days, and were diagnosed with normozoospermia, characterized by a semen volume greater than 2 ml. Baseline (BL) charac- teristics assessed for each sample included patient age, body mass index, semen volume, sperm concentration, progressive motility, non-progressive motility, and DFI. Samples were collected using sterile, clean instruments maintained at the same temperature as the spermatozoa to prevent bacterial contamination, which can reduce sperm quality. All tools were sterilized, disinfected, wrapped in aluminium foil, and stored at 37°C until use. Following the WHO protocols (2021). Samples were col- lected via masturbation onto a sterile glass container and allowed to liquefy for 20-30 minutes. Semen volume, motility, and total sperm count were measured to ensure normal values before inclusion in the study. After the initial assessment, sperm samples were prepared using the swim-up method. The prepared samples were then divided into two groups: one undergoing swim-up preparation with vitamin D incubation (SD) and the other undergoing swim-up preparation only (SU). Unprepared samples (BL) were also included in the evaluation. Spermatozoa from all groups (BL, SD, and SU) were eval- uated for DNA fragmentation index. Sperm preparation Sperm preparation using the swim-up method was per- formed by mixing semen and Sperm Rinse™ medium (Vitrolife, USA) in a 1:1 ratio in a 5 ml tube. The mixture was then centrifuged at a speed of 400 G for 10 minutes. After centrifugation, the supernatant was discarded, and the pellet was resuspended with 2 ml of Sperm Rinse™ medium. The resuspended pellet was centrifuged again at a speed of 400 G for 5 minutes. The supernatant was dis- carded, and 2 ml of SpermRinse™ medium was gently added to the pellet along the tube wall. The tube was then placed at 45° angle and incubated at 37°C for 45 minutes. Finally, 1 ml of the upper medium fraction was carefully transferred to two new tubes, with 500 µl in each tube, for further analysis (13). Vitamin D incubation A solution of active vitamin D, 1.25(OH)2D3 (Calcitriol, molecular weight 416.64), was prepared in 100% ethanol with a stock concentration of 10 μg/ml (24 μM) and stored in a -20°C. To analyse the effect of in vitro vitamin D administration on DNA fragmentation, 500 uL post- preparation spermatozoa were incubated with 1 nM 1.25(OH)2D3 at 37°C, for 45 minutes (19). The working solution concentration was prepared in stages starting from 1 mM, 10 μM, 10 nM, and finally 1 nM. The con- trol group without vitamin D was incubated with SpermRinse™ medium under the same conditions. DNA fragmentation index DNA Fragmentation was assessed using the DNA Fragmentation Kit (SpermFunc®, BRED-002, BRED Life Science Technology Inc.), following the manufacturer’s pro- tocol. Observations were made using a light microscope at 400X magnification to distinguish between unfrag- mented DNA (big and medium halos) and fragmented DNA (small halos, no halos, and damaged spermatozoa) in 500 spermatozoa cells. Normal sperm DNA presented FIgure 1. Determination of DNA fragmentation based on halo size (28). 1) The diameter of the core as the determinant of DNA fragmentation. 2) Large halo and 3) medium halo show spermatozoa without DNA fragmentation. 4) Small halo and 5) no halo indicating spermatozoa DNA fragmentation. Archivio Italiano di Urologia e Andrologia 2024; 96(4):12891 3 Vitamin D and sperm deoxyribonucleic acid fragmentation as medium (halo thickness smaller than the length but greater than one-third of the core minor diameter) to big halos (halo thickness equal to or greater than the length of the core minor diameter). In contrast, damaged/frag- mented sperm DNA presented as no halos or small halos (halo thickness equal to or less than one-third of the core minor diameter) (Figure 1). The percentage of spermato- zoa with DNA fragmentation was calculated as the DNA Fragmentation Index (DFI). Statistics Data were recorded and analysed to compare the effect of vitamin D incubation on sperm DNA integrity between the vitamin D-treated group (SD), the control group (SU), and the unprepared samples (BL). This comparison aimed to determine whether vitamin D supplementation during sperm preparation could enhance DNA integrity in sper- matozoa, thus potentially improving outcomes in ART. Statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS) version 26.0 for Windows. The Shapiro-Wilk test was used to determine whether the data were normally distributed (p > 0.05) or not normally distributed (p < 0.05). Differences in DFI val- ues between groups were tested using paired t-test, and results were expressed as mean ± standard deviation. A p- value of less than 0.05 was considered statistically signifi- cant. Ethical approval The study was approved by the Health and Humanities Research Ethics Committee, Faculty of Medicine, Universitas Airlangga (Code: 242/EC/KEPK/FKUA/2023). Samples were collected between August-October 2023. RESULTS Patient characteristics A total of 12 volunteers participated during the study peri- od. All DFI data between groups were found to be normal- ly distributed (p > 0.05). The mean age of volunteers was 30.9 years (30.9 ± 2.6), reflecting the reproductive age of men. The body mass index (BMI) was 27.4 kg/m2 (27.4 ± 4.1), classified as level 1 obesity; the mean baseline DFI value was 17.6 ± 4.2%, which falls within the normal range. Baseline characteristics are summarized in Table 1. DFI between groups A paired t-test was conducted to compare the DFI within each sample group (Table 2). The SU group exhibited a significantly reduced DFI (12.0 ± 4.2%) compared to the BL characteristics (17.6 ± 4.2%). Furthermore, the DFI was significantly decreased in the SD group (5.5 ± 3.4%) compared to both the SU group (12.0 ± 4.2%) and the initial BL (17.6 ± 4.2%), with mean reductions of 6.5% and 12.1% respectively. These findings suggest that vita- min D incubation during sperm preparation resulted in a significant improvement in sperm DNA integrity com- pared to the control groups. Evaluation of spermatozoa DNA fragmentation examination In each group, variations in the appearance of a large halos, medium halos, small halos and no halos were observed in semen samples before and after preparation. Specifically, the presence of small halos and no halos was noticeably reduced in the post-preparation samples of the SD group (Figure 2). FIgure 2. Evaluation of spermatozoa’s DNA fragmentation in the BL, SU and SD groups. Image caption: a. Large halos; b. Medium halos; c. Small halos; d. No halos. Table 1. Age and semen profile of the study subjects. Subject characteristics Mean ± SEM (n = 12) Patient age (in years) 30.9 ± 2.6 Body Mass Index (kg/m2) 27.4 ± 4.1 Semen volume (ml) 3.4 ± 1.0 Sperm concentration (million/ml) 48.7 ± 25.1 Progressive motility (%) 52.4 ± 13.4 Non-progressive motility (%) 6.8 ± 3.7 DNA fragmentation index (%) 17.6 ± 4.2 Table 2. Paired t-test results from each treatment group on DFI. Variable Group 1 Group 2 P-value (Mean ± SD) (Mean ± SD) DFI BL (17.6 ± 4.2) SU (12.0 ± 4.2) < 0.001 SU (12.0 ± 4.2) SD (5.5 ± 3.4) < 0.001 BL (17.6 ± 4.2) SD (5.5 ± 3.4) < 0.001 Archivio Italiano di Urologia e Andrologia 2024; 96(4):12891 A. Rezano, D. Rezkiawan, V. Lie, et al. 4 DISCUSSION The decline in sperm quality and function due to increas- ing reproductive disorders globally is a significant con- cern in reproductive health. A recent study highlighting the impact of vitamin D deficiency on mouse fertility and subsequent improvements in semen parameters has shed new light on the role of vitamin D in male reproductive function (29). Numerous studies have emphasized the role of vitamin D in male reproductive health, particular- ly in enhancing sperm quality and motility (30). Infertility affects approximately 15-20% of couples of reproductive age, with male factors contributing to around 50% of cases (31, 32). In our study, the average age of the sample population was 30.9 ± 2.6 years, con- sistent with previous research (33). Additionally, the average BMI of the participants fell within the category of level 1 obesity. Obesity is known to increase the risk of sperm DNA damage in infertile men due to its association with increased oxidative stress (34, 35). Sperm DNA integrity is critical to successful fertilization and embryogenesis (36). However, previous studies have been limited in evaluating the functional status of sperma- tozoa (37), indicating the need for further research. In our study, despite having normal semen parameters, semen samples exhibited a DFI of 17.6 ± 4.2%. This finding aligns with previous research conducted by Halim et al., where the DFI before semen processing was 16.12% (13.48- 19.04) (38). Oxidative stress levels that are insufficient to induce cell death can still disrupt sperm function, high- lighting the importance of addressing DNA fragmentation in infertility (39). The extent of DNA fragmentation’s impact on fertilization depends on both the level of DNA damage and the DNA repair capacity of the oocyte (40). Our study revealed that supplementation of vitamin D, calcitriol [1.25(OH)2D3] at a concentration of 1 nM after sperm preparation, led to a significant reduction in sperm DNA fragmentation by 6.5%. Notably, there was a syner- gistic effect between sperm preparation and vitamin D administration in decreasing DNA fragmentation com- pared to baseline levels. These findings differ from a study by Moghadam et al. (2019), which reported no repair of DNA damage with vitamin D administration after swim-up (41). This discrepancy may be attributed to differences in the DNA damage examination methods used, with the sperm chromatin dispersion method prov- ing more effective than the TUNEL method in diagnosing sperm DNA damage in unexplained infertility (40). The protective mechanism of vitamin D on sperm DNA integrity can be explained in several ways. Firstly, the hydrophobic part of vitamin D binds to fatty acid residues on the spermatozoa membrane, thereby protecting mem- brane integrity (20). Additionally, vitamin D has been shown to enhance the integrity of spermatozoa membranes during cryopreservation by reducing intracellular ROS lev- els (41). VDR expression in testes and spermatozoa, along with cellular uptake of circulating vitamin D, play crucial roles in regulating spermatozoa motility and acrosome function (19). Moreover, administration of 20,000 nM of vitamin D has been associated with increased expression of heat shock protein 70 (HSP70), a marker of oxidative stress and lipid peroxidation, indicating a dose-response rela- tionship of vitamin D as an antioxidant (42). While our study supports the role of antioxidants in maintaining sperm chromatin integrity during sperm preparation, definitive conclusions cannot be drawn due to certain limitations. This study did not directly examine intraspermatozoal ROS levels and endogenous antioxi- dants. Future research should address these factors and analyse abnormal semen samples to further elucidate the role of vitamin D in infertility. Additionally, investiga- tions into other variables, such as ROS levels, antioxi- dants, lipid peroxidation, and acrosome reactions, as well as their impacts on fertilization outcomes, pregnancy, and embryo development, are warranted. Comparative studies between swim-up and other sperm selection pro- cedures, such as density gradient centrifugation, are also needed to evaluate their efficacy and drawbacks in reduc- ing DNA damage with vitamin D administration. CONCLUSIONS This study demonstrated that DFI significantly decreased following sperm preparation using the swim-up method. Additionally, the DFI further decreased significantly in the group treated with vitamin D compared to both the pre-preparation and post-preparation without vitamin D administration groups. These findings highlight the protective role of vitamin D against DNA damage incurred during sperm preparation. However, further studies are necessary to elucidate the underlying mechanism of this protective effect and to compare the efficacy of vitamin D supplementation with other sperm preparation methods. Future research should also consider examining different variables such as ROS levels, antioxidants, lipid peroxidation, acrosome reactions, and their impacts on fertilization outcomes, pregnancy, and embryo development. ACKNOWLEDGMENTS The authors express sincere gratitude to the staff at the Department of Biomedical Sciences Faculty of Medicine Universitas Airlangga and Andrology Clinic, Dr. Soetomo Academic Hospital, for their invaluable assistance and sup- port throughout this study. REFERENCES 1. Agarwal A, Baskaran S, Parekh N, et al. Male infertility. Lancet 2021; 397:319-33. 2. Turner KA, Rambhatla A, Schon S, et al. Male Infertility is a Women’s Health Issue-Research and Clinical Evaluation of Male Infertility Is Needed. Cells 2020; 9:990. 3. World Health Organization. 2020. Infertility. Available at: https://www.who.int/news-room/fact-sheets/detail/infertility, accessed May 2024. 4. Asadi A, Ghahremani R, Abdolmaleki A, Rajaei F. 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Open Access Maced J Med Sci 2021; 9:626-30. 40. Agarwal A, Farkouh A, Saleh R, et al. Technical Aspects and Clinical Limitations of Sperm DNA Fragmentation Testing in Male Infertility: A Global Survey, Current Guidelines, and Expert Recommendations. World J Mens Health 2024; 42:202-15. Archivio Italiano di Urologia e Andrologia 2024; 96(4):12891 A. Rezano, D. Rezkiawan, V. Lie, et al. 6 41. Moghadam MT, Fard YA, Saki G, Nikbakht R. Effect of vitamin D on apoptotic marker, reactive oxygen species and human sperm parameters during the process of cryopreservation. Iran J Basic Med Sci 2019; 22:1036. 42. Moghadam MT, Hamidian O, Mansouri E, Nikbakht R. Effects of vitamin D3 on the level of heat shock protein 70 and oxidative stress in human sperm: a pilot study. Middle East Fertil Soc J 2020; 25:1-8. Correspondence Andri Rezano, MD-Andrologist, PhD, Associate Professor andri.rezano@unpad.ac.id Department of Biomedical Sciences, Faculty of Medicine Universitas Padjadjaran Jl. Ir. Soekarno KM. 21 Jatinangor, Sumedang 45363, West Java, Indonesia Ditto Rezkiawan ditto.rezkiawan-2021@fk.unair.ac.id Vellyana Lie vellyana.lie-2022@fk.unair.ac.id Arya Srisadono arya.srisadono-2022@fk.unair.ac.id Tjahjo Djojo Tanojo tjahjodjojo@gmail.com Maria PBD Pramesti pramestidyan@gmail.com Andrology Study Program, Faculty of Medicine, Universitas Airlangga, Surabaya 60132, East Java, Indonesia Mochamad Rivaldo rafly22001@mail.unpad.ac.id Medical Study Program, Faculty of Medicine, Universitas Padjadjaran, Sumedang, 45363, West Java, Indonesia Amelia Reta Purba amelia23007@mail.unpad.ac.id Medical Study Program, Faculty of Medicine, Universitas Padjadjaran, Sumedang 45363, West Java, Indonesia Melia Juwita Adha meliarezano@gmail.com Holistic General Hospital, Purwakarta 41115, West Java, Indonesia Conflict of interest: The authors declare no potential conflict of interest.