ANDROLOGY Evaluation of Sperm Parameters and DNA Integrity Following Different Incubation Times in PVP Medium Ali Nabi1,2 ,Farahnaz Entezari2,3, Seyed Mohsen Miresmaeili3, Serajoddin Vahidi2*, Keivan Lorian2, Fatemeh Anbari2 , Leila Motamedzadeh2 Purpose: Polyvinylpyrrolidone (PVP) is a chemical material used in intracytoplasmic sperm injection (ICSI) pro- gram. The aim of this study was to investigate the ideal time that sperm can be safely incubated in PVP with less structure and DNA damage. Method: Thirty-one Oligoasthenoteratospermia (OAT) samples were used. Sperm samples were prepared by dis- continuous density-gradients method and incubated in 10% PVP at different time intervals (0, 5, 10, 15, 20, and 30 min). The effect of PVP was assessed on sperm DNA fragmentation and viability via SCD assay and Eosin–ni- grosin staining respectively. Results: Data showed there was a significant increase in sperm DNA fragmentation at 10 min compared to 0 min. The viability rate also significantly reduced at 10 min compared to 0 min. Conclusion: As a result, sperm samples could be incubated with PVP for less than 10 min. While prolonged in- cubation may significantly damage the sperm DNA integrity and viability. Keywords: DNA fragmentation; intracytoplasmic sperm injections; polyvinylpyrrolidone (PVP); semen analyses; spermatozoa. 1Abortion research center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran. 2Andrology Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran. 3Department of Biology, Science and Arts University, Yazd, Iran. *Correspondence: Andrology Research Center, Yazd Reproductive Sciences Institute, Safayeh, Bou-Ali ave., Yazd, Iran. Postal code: 8916877391. Tel.: +983538247085; Fax: +983538247087; E-mail: vahidi.seraj@gmail.com. Received August 2021 & Accepted October 2021 INTRODUCTION Intracytoplasmic sperm injection (ICSI) is one of the methods of assistant reproductive technology (ART) used for the treatment of male infertility(1). During this method, sperm with motility and normal morphology is selected, and then they are immobilized under an in- verted microscope. Sperm immobilization is a neces- sary procedure that must be performed before sperm injection. Reducing the speed of sperm movement and stopping it are two processes used to immobilize sperm (2). Polyvinylpyrrolidone (PVP) is a water-soluble pol- ymer of N-vinyl-2-pyrrolidone(3). It is used for ICSI because increases the viscosity of sperm solution and reduces the sperm movement speed; therefore, sperm immobilization becomes facilitated. PVP is also used to control and manage sperm movement inside the injec- tion needle(4). Performing ICSI in oligoasthenoteratozoospermia (OAT) patients is often a challenge because it is not possible to find enough spermatozoa with acceptable morphology. Pasqualotto and Borges reported that the rate of fertilization was lower when ICSI is performed with sperm from men with OAT and non-obstructive azoospermia(5). Low sperm concentration with bad quality in these samples causes the time of sperm se- lection to be increased by the embryologist. Moreover, PVP has been shown to damage sperm membranes, mi- tochondrial membrane, and destroy axonal tubules and fibrous sheaths(6). Injected PVP with sperm remains in the oocyte for a long time and cannot be digested by lysozyme enzymes(7). The existence of PVP in oocyte delays the onset of calcium oscillation; therefore, the chromosomal abnormalities in embryos is increased(8,9). DNA integrity of sperm chromosomes is essential for natural fertilization and transmission of parental ge- netic information. Sperm DNA integrity plays an im- portant role in reproductive outcomes. Studies showed that high DNA fragmentation in sperm cells causes a lower development rate in embryos(10). Genetic dam- ages to gametes during the ICSI technique may affect the result of this process(11). Gomez and co-workers in 2007 showed that PVP can increase sperm DNA fragmentation. It was also shown that sperm immobi- lization before ICSI technique is one of the causes of ultra-structural sperm damage(12). These side effects of PVP are major concerns for infertility centers. There- fore, it is required to search for methods to reduce PVP side effects in the ART laboratory. The main aim of the present study was to investigate the appropriate and maximum time that sperm could stay in the PVP media without possible damage such as DNA fragmentation and viability. MATERIALS AND METHODS Study participants In this study, the semen samples were obtained from patients referring to Yazd reproductive sciences insti- tute. The age range of the male participants was 20-45 years. Male partners with diseases such as varicocele, cancer, urinary tract infection, diabetes, or orchitis were Urology Journal/Vol 19 No. 3/ May-June 2022/ pp. 232-237. [DOI:10.22037/uj.v18i.6936] Vol 19 No 3 May-June 2022 100 excluded. Samples were prepared by masturbation in sterile specimen container after 2-5 days of sexual abstinence. Then, thirty -one moderate OAT samples were incubated in 37 ˚C for 20 min for liquefaction. The routine seminal parameters were performed ac- cording to the World Health Organization criteria(13). This study was approved by the Ethics Committee of Yazd Reproductive Sciences Institute (IR.SSU.RSI. REC.1398.030). Sperm preparation Semen samples were prepared by the discontinuous density – gradients method. A 40/80 gradient (Percep- tion, SAGE, USA) was prepared in a 15ml conical tube and then a layer of 1 ml semen is placed on top of the density media. The first centrifuge (Eppendorf, North America) was performed with 300×g for 20 min. Then the supernatant was removed and the sediment was kept. The sediment was washed with 5ml Ham’s F10 (Biochrome, Berlin, Germany) media for two times at 300×g for 5 min and then sediment resuspend to assess for sperm concentration, motility, viability, normal morphology and DNA integrity(14). PVP preparation In this study, 10% PVP solution (10% PVP Solution with HAS (5mg/mL), Irvine Scientific, USA) accord- ing to the laboratory routine was used. The pre-warmed PVP was placed in a petri dish (Falcon, USA) as 20 µl droplets. After that, sterile mineral oil (Life Global, Belgium) was poured on drops to cover the surface of all drops. Study design Each sample was divided into 6 equal parts after wash- ing by a discontinuous density-gradients method. Then, 20 µl of samples were placed in 10% PVP droplets on a hot plate with a temperature of 37◦C at 0, 5, 10, 15, 20 and 30 min and, each part was evaluated at specific times for sperm viability and DNA integrity. Sperm parameters 10 µl of sample was placed on the Makler chamber slide for sperm motility and concentration assessment. The number of sperm in 10 vertical squares and 10 hori- zontal squares was counted and the average of the two was taken. For motility assessment according to WHO Sperm parameters Before preparation (N=31) After preparation (N=31) P-value Sperm count (×106) 10.14 ± 3.29 18.00 ± 7.40 .0001* Progressive motile sperm (%) 17.68 ± 7.26 29.14 ± 8.72 .0001* Non-progressive motile sperm (%) 8.18 ± 3.09 11.73 ± 4.10 .0001* Immotile sperm (%) 74.05 ± 9.33 58.95 ± 10.79 .0001* Sperm morphology (%) 1.95 ± 0.78 5.77 ± 2.67 .0001* Sperm viability (%) 41.59 ± 7.33 58.91 ± 10.68 .0001* Table 1. Sperm parameters before and after discontinuous density – gradients method The data were presented as mean ± SD. Data were calculated using Paired t-test. * P-Value <0.05 was significant. Figure 1. Different halo patterns were appeared around sperm cells. A: Large and B: medium-sized halos were considered as sperm cells with no DNA fragmentation. C: Small halo or D: No halos were considered as sperm cells with DNA fragmentation. Effect of PVP on sperm DNA integrity-Nabi et al. Vol 19 No 3 May-June 2022 233 Andrology 234 2010 recommendation, the percentages of progressive motile, non-progressive motile and non-motile sperma- tozoa were recorded(13). For sperm morphology, Papan- icolaou staining, OG-6 (Merck, Germany) was used. In this staining, the head becomes blue and the midpiece and tail of the sperm becomes red(13). This parameter was assessed by 1000× magnification light microscope. Eosin–nigrosin staining was used to assess sperm vi- ability. The sperm viability was assessed by light mi- croscopy at ×1000 magnification. Viable spermatozoa remained colorless; while, dead spermatozoa stained pink or red(13). The results were recorded before and af- ter the discontinuous density-gradients method and also at different times that sperm was incubated with PVP media. Sperm chromatin dispersion (SCD) test SCD test was used to assess DNA integrity. This test was performed according to Fernandez et al(15). In brief, the slides were covered by 0.65% standard agarose (Merck, Germany). 30 µl of sperm suspension was mixed with 70 µl low melting agarose (Roche, Germa- ny). 50 µl of the prepared mixture was placed on pre- coated slides and then allowed to become solid. They were put at 4 °C for 4 min. Then, Denaturation solution (0.08 N HCl) (Merck, Germany) was used to immerse slides for 7 min at RT in dark. The slides were placed into lysing solution 1 (0.4 M Tris, 2-Mercaptoethanol, 1% SDS, and 50 mM EDTA, pH 7.5) for 20 min at RT. After that, the slides were transferred in the lysing solu- tion 2 (0.4 M Tris, 2 M NaCl, and 1% SDS, pH 7.5) for 15 min at RT and washed in Trisborate- EDTA buffer (0.09 M Tris-borate and 0.002 M EDTA, pH 7.5 and 12 min at RT. To dehydrate samples, 70%, 90% and 100% ethanol were used (2 min each) and then they became dry at RT. The Wright stain solution (Sigma- Aldrich, USA) which was mixed with phosphate buffer solution (PBS) (1: 1) was used to stain samples. After staining for 10 min, samples were evaluated for size of the halos by light microscope. Different halo patterns appeared around sperm cells. Large or medium-sized halos ap- peared around sperm cells with no DNA fragmentation (16). Small halo or no halo, sign that DNA fragmentation occurred in sperm cells (Figure 1). Two hundred sper- matozoa were evaluated for DNA fragmentation and the percentage of spermatozoa was recorded. Statistical analysis We used SPSS 20 (SPSS, Inc., IL, USA) for statistical analysis. Normalization of data was determined with the Kolmogorov-Smirnov normality test. Paired t-test was used to compare sperm parameters before and after the discontinuous density-gradients method. Repeated measure One-way ANOVA was done between groups and multiple comparison was followed by paired t-test. Significance level was corrected using Bonferroni cor- rection and it was considered as 0.0125. RESULTS Sperm parameters before and after discontinuous den- sity gradients method are summarized in Table 1. All parameters except count were significantly different before and after preparation by this method. The DNA fragmentation rate after the preparation of sperm by discontinuous density-gradients method (0 min) was not significantly increased compared to the 5 min after incubation with PVP (P = .296). In 10 min (P<.001), 15 min, 20 min and 30 min (P < .0001) after Figure 2. Effect of different incubation times in PVP on the sperm DNA damage. P values were significant between all of the groups except between 0 and 5. ***= p < 0.001, **** = p < 0.0001. Data were calculated using repeated measure ANOVA. Effect of PVP on sperm DNA integrity-Nabi et al. Vol 19 No 3 May-June 2022 100 incubation with PVP, the DNA fragmentation was sig- nificantly increased compared to the 0 min (Figure 2). The sperm viability rate in 0 min compared to 5 min after PVP incubation was not significantly decreased (p = 0.42). However, the percentage of sperm viability decreased significantly in 10 (P = .041), 15, 20 and 30 min (P < .0001) after PVP incubation compared to the 0 min after processing (Figure 3). Moreover, the DNA fragmentation rate in 5 min compared to 10 min was not significantly increased after incubation with PVP (P = .169). According to Table 2, the DNA fragmentation and the rate of viability were significantly increased be- tween other times after incubation with PVP (Table 2). DISCUSSION 10% PVP is conventionally applied in ICSI process to reduce the speed of sperm movement. Although, PVP facilities the ICSI process, it can inhibit the deconden- sation of the sperm head and also increase DNA dam- age, which can affect the outcomes of fertilization and embryonic development(15). In the present study, the best time that sperm could be incubated with PVP with less damage was 5min, while the exposure times high- er than ten minutes had negative effects on DNA and viability of sperm. Performing of ICSI procedure with spermatozoa from OAT patients is still a challenge. Enough spermatozoa is not available and subsequently lower fertilization and pregnancy rates is observed after ICSI(17). In these cases, time frame for the search of spermatozoa is long due to morphological defects and low concentration of sperm. A cohort study showed that the extended sperm search for both ejaculated and TESE decreased the fertilization and pregnancy rates from 44% to 23%(18). The study of the effects of sperm incubation in PVP is one of the topics that have been considered by researchers in the last decade. Previous studies have been conducted on the effects of sperm incubation in PVP drops by evaluating sperm param- eters such as motility, viability, and morphology. By understanding the important role of sperm DNA health and quality infertility, many research groups have made great efforts to find a relationship between sperm cell health and sperm DNA integrity(19). Sperm DNA integrity is an important parameter of sperm quality that plays a fundamental role in ART out- comes. It is proved that increasing DNA damage is as- sociated with poor embryo development and decreased implantation and pregnancy outcomes(20). Some assays have been developed to evaluate sperm DNA status. SCD is an assay that evaluates the property of fragmentation DNA with denaturation solution un- der certain conditions. However, the SCD assay is less complex, cost effective, and not time-consuming which presents similar sensitivity for the assessment of DNA fragmentation compared to other assays(21,22). In this study, the effect of different time intervals on DNA fragmentation and viability of sperm cells, which were incubated in PVP, was investigated. The results demonstrated that after ten min of sperm incubation in PVP, DNA fragmentation increased and sperm viabili- ty reduced significantly. Sterler et al. showed that PVP had negative effects on plasma membrane, acrosomal membrane, and sperm mitochondrial membrane on the samples which were exposed to 10% PVP solution for 30 min(23). In an animal study conducted by Kato and Nagao in 2009, the effect of PVP at different times, 0, 15, 30 and 60 min on the capacity and acrosomal reac- Figure 3. The effect of PVP on sperm viability in different times. P values were significant between all of the groups except between 0 and 5. ** = p < 0.05, ****= p < 0.0001. Data were calculated using repeated measure ANOVA. Effect of PVP on sperm DNA integrity-Nabi et al. Vol 19 No 3 May-June 2022 235 Andrology 236 tion of cow spermatozoa was assessed. They found that the highest rate of fertilization and embryo growth oc- curred in the first 15 min and by passing time most de- structive effects of PVP on sperm were observed(24). An- other study showed that nuclear and chromatin damage was noticed in sperms exposed to PVP(25). Although, the best way is to avoid the use of exogenous substances in ART procedures, selecting sperm without a reduction in the speed of their movements is not practical. Howev- er, a physiological alternative to PVP is suggested. For instance, materials such as sodium hyaluronate slow sperm movements and prevent sperm from sticking to the dish or pipette. However, sperm motility in a media containing hyaluronate is faster than in a media contain- ing PVP, since no difference was observed in post-ICSI zygote growth in both of these media(26). This study suggested that sperm addition to PVP in ART procedure for more than 10 min caused possible damages, such as DNA fragmentation and sperm viabil- ity reduction, that ultimately affect fertility outcomes. Therefore, PVP could be potentially dangerous for the sperm nucleus during ICSI treatments, and it may be better to reduce the length of the treatment period of PVP solution. CONCLUSIONS Taken together, following sperm incubation in PVP at different times (0, 5, 10, 15, 20, and 30 min), we pre- sented incubation times in PVP medium for >10 min, causes significantly increased fragmentation of sperm DNA. Therefore, ICSI should be performed within 10 min of sperm incubation in PVP. CONFLICT OF INTEREST There is no conflict of interest in this study. REFERENCES 1. Esteves SC, Roque M, Bradley CK, Garrido N. Reproductive outcomes of testicular versus ejaculated sperm for intracytoplasmic sperm injection among men with high levels of DNA fragmentation in semen: systematic review and meta-analysis. Fertil Steril. 2017;108:456- 67. e1. 2. Hussain T, Ranjha NM, Shahzad Y. Swelling and controlled release of tramadol hydrochloride from a pH-sensitive hydrogel. Des Monomers Polym. 2011;14:233-49. 3. Ding D, Wang Q, Li X, et al. Effects of different polyvinylpyrrolidone concentrations on intracytoplasmic sperm injection. Zygote. 2020;28:148-53. 4. Kato Y, Nagao Y. Effect of polyvinylpyrrolidone on sperm function and early embryonic development following intracytoplasmic sperm injection in human assisted reproduction. Reprod Med Biol. 2012;11:165-76. 5. Pasqualotto FF, Borges E, Jr. Re: sperm defect severity rather than sperm source is associated with lower fertilization rates after intracytoplasmic sperm injection. Int Braz J Urol. 2008;34:231-2. 6. Strehler E, Baccetti B, Sterzik K, et al. Detrimental effects of polyvinylpyrrolidone on the ultrastructure of spermatozoa (Notulae seminologicae 13). Hum Reprod. 1998;13:120-3. 7. Jean M, Mirallié S, Boudineau M, Tatin C, Barrière P. Intracytoplasmic sperm injection with polyvinylpyrrolidone: a potential risk. Fertil Steril. 2001;76:419-20. 8. Nyboe Andersen A, Goossens V, Ferraretti A, et al. Assisted reproductive technology in Europe, 2004: results generated from European registers by ESHRE. Hum Reprod. 2008;23:756-71. 9. Ding D, Wang Q, Li X, et al. Effects of different polyvinylpyrrolidone concentrations on intracytoplasmic sperm injection. Zygote. 20201-6. 10. Mohammad HN-E, Mohammad S, Shahnaz R, et al. Effect of sperm DNA damage and sperm protamine deficiency on fertilization and embryo development post-ICSI. Reprod Biomed Online. 2005;11:198-205. 11. Matsuura R, Takeuchi T, Yoshida A. Preparation and incubation conditions affect the DNA integrity of ejaculated human spermatozoa. Asian J Androl. 2010;12:753. 12. Gomez-Torres MJ, Ten J, Girela JL, Romero J, Bernabeu R, De Juan J. Sperm immobilized before intracytoplasmic sperm injection undergo ultrastructural damage and acrosomal disruption. Fertil Steri. 2007;88:702-4. 13. Organization WH. WHO Laboratory Manual for the Examination and Processing of Human Semen. fifth ed: World Health Organization; 2010. 14. Jayaraman V, Upadhya D, Narayan PK, Adiga SK. Sperm processing by swim-up and density gradient is effective in elimination of sperm with DNA damage. J Assist Reprod Genet. 2012;29:557-63. 15. Fernández JL, Muriel L, Rivero MT, Goyanes V, Vazquez R, Alvarez JG. The sperm chromatin dispersion test: a simple method for the determination of sperm DNA fragmentation. J Androl. 2003;24:59-66. 16. Anbari F, Khalili MA, Agha-Rahimi A, Maleki B, Nabi A, Esfandiari N. Does sperm DNA fragmentation have negative impact on embryo morphology and morphokinetics in IVF programme? Andrologia. 2020e13798. 17. Nordhoff V, Fricke RK, Schüring AN, Zitzmann M, Kliesch S. Treatment strategies for severe oligoasthenoteratozoospermia (OAT) (<0.1 million/mL) patients. Andrology. 2015;3:856-63. 18. Palermo GD, Neri QV, Schlegel PN, Rosenwaks Z. Intracytoplasmic sperm injection (ICSI) in extreme cases of male infertility. PLoS One. 2014;9:e113671. 19. Zhang X-D, Chen M-Y, Gao Y, Han W, Liu D-Y, Huang G-N. The effects of different sperm preparation methods and incubation time on the sperm DNA fragmentation. Hum Fertil. 2011;14:187-91. 20. Liffner S, Pehrson I, Garcia-Calvo L, et al. Diagnostics of DNA fragmentation in human spermatozoa: Are sperm chromatin structure analysis and sperm chromatin dispersion tests (SCD-HaloSpermG2((R)) ) comparable? Effect of PVP on sperm DNA integrity-Nabi et al. Vol 19 No 3 May-June 2022 100 Andrologia. 2019;51:e13316. 21. Anbari F, Halvaei I, Nabi A, Ghazali S, Khalili MA, Johansson L. The quality of sperm preparation medium affects the motility, viability, and DNA integrity of human spermatozoa. J Hum Reprod Sci. 2016;9:254- 8. 22. Nabi A, Khalili MA, Halvaei I, Roodbari F. Prolonged incubation of processed human spermatozoa will increase DNA fragmentation. Andrologia. 2014;46:374-9. 23. Strehler E, Capitani S, Collodel G, et al. Submicroscopic mathematical evaluation of spermatozoa in assisted reproduction. I. Intracytoplasmic sperm injection. (Notulae seminologicae 6). J Submicrosc Cytol Pathol. 1995;27:573-86. 24. Kato Y, Nagao Y. Effect of PVP on sperm capacitation status and embryonic development in cattle. Theriogenology. 2009;72:624-35. 25. Tarozzi N, Nadalini M, Borini A. Effect on sperm DNA quality following sperm selection for ART: new insights. Genetic Damage in Human Spermatozoa: Springer; 2019:169-87. 26. BM A, MA A, KG A, AS O, SA B. Efficiency of hyaluronic acid binding ability to improve sperm selection in intracytoplasmic sperm injection (ICSI). Egypt J Hosp Med. 2016;65:536-46. Effect of PVP on sperm DNA integrity-Nabi et al. Vol 19 No 3 May-June 2022 237