Changes in DNA fragmentation during sperm preparation for intracytoplasmic sperm injection over time

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1 Changes in DNA fragmentation during sperm preparation for intracytoplasmic sperm injection over time Natalia Rougier, M.D., a,b Heydy Uriondo, M.Sc., a Sergio Papier, M.D., a Miguel Angel Checa, M.D., Ph.D., b Carlos Sueldo, M.D., a,c and Cristian Alvarez Sedo, M.Sc. a a CEGYR Reproductive Medicine, Buenos Aires, Argentina; b Department of Obstetrics and Gynecology, Parc de Salut Mar, Universitat Autonoma de Barcelona, Barcelona, Spain; and c University of California, San Francisco Fresno, Fresno, California Objective: To compare the DNA fragmentation of semen samples established by terminal deoxynucleotide transferase mediated dutp nick-end labeling (TUNEL) after incubation in polyvinylpyrrolidone (PVP) and hyaluronic acid (HA) for different time periods. Design: Comparative prospective study. Setting: Center for reproductive medicine. Patient(s): Twenty-seven semen samples from infertile patients. Intervention(s): None. Methods: Semen analysis and DNA fragmentation assays (TUNEL) were performed. Two groups were established: A) normal TUNEL (<20%); and B) Abnormal TUNEL (R20%). TUNEL was performed in neat (T0), postgradient (TG), 1-hour postgradient (TG1), and 2-hour postgradient (TG2) samples and in TG2 samples after 0.5, 1.0, and 1.5 hours of incubation in PVP or HA. Result(s): TUNEL levels were significantly reduced after gradient separation compared with neat values. In group A, TUNEL levels were significantly higher in the TG2 þ 1.5 hours in PVP and HA samples but did not reach abnormal levels. In group B, TUNEL levels were significantly higher in the TG2 þ 1 hour in PVP and HA samples. Conclusion(s): Sperm DNA fragmentation significantly decreased after centrifugation gradient, regardless of the initial levels of the sample. Samples with TUNEL R20% were more susceptible to a significant increase in DNA fragmentation over time, with similar increases being observed over time for samples that were incubated in HA or PVP. These data may be relevant for sperm preparation for intracytoplasmic sperm injection. (Fertil Steril Ò 2013;100: Ó2013 by American Society for Reproductive Medicine.) Key Words: Sperm DNA fragmentation, PVP, hyaluronic acid Discuss: You can discuss this article with its authors and with other ASRM members at fertstertforum.com/rougiern-sperm-dna-fragmentation-pvp-icsi/ Use your smartphone to scan this QR code and connect to the discussion forum for this article now.* * Download a free QR code scanner by searching for QR scanner in your smartphone s app store or app marketplace. In the past decade, the evaluation of sperm DNA fragmentation has become more important as a method to assess semen quality. Several authors have reported a negative correlation between high DNA fragmentation levels and assisted reproductive technology (ART) outcomes (1 4). DNA fragmentation is considered to be an important predictor of reproductive outcomes in IVF treatments compared with other sperm characteristics, such as progressive motility (5). Some reports have revealed that DNA fragmentation can undergo a significant increase with increased incubation times, and it has been Received November 28, 2012; revised February 18, 2013; accepted March 4, 2013; published online April 2, N.R. has nothing to disclose. H.U. has nothing to disclose. S.P. has nothing to disclose. M.A.C. has nothing to disclose. C.S. has nothing to disclose. C.A.S. has nothing to disclose. Reprint requests: Cristian Alvarez Sedo, M.Sc., CEGYR Reproductive Medicine, Viamonte 1432, Buenos Aires (1055), Argentina ( calvarez@cegyr.com). Fertility and Sterility Vol. 100, No. 1, July /$36.00 Copyright 2013 American Society for Reproductive Medicine, Published by Elsevier Inc. demonstrated that sperm from fertile men is more resistant to DNA fragmentation over time than sperm from infertile men. These reports have also demonstrated differences in DNA fragmentation dynamics based on the origin of the sample (i.e., fresh vs. frozen), showing that frozen samples presented greater DNA fragmentation in less time compared with fresh samples (6, 7). Intracytoplasmic sperm injection (ICSI) and intracytoplasmic morphologically selected sperm injection (IMSI) require the preselection of motile sperm, which can be accomplished with the use of a double-layer centrifugation VOL. 100 NO. 1 / JULY

2 ORIGINAL ARTICLE: ANDROLOGY gradient. Motile spermatozoa obtained in the pellet are suspended in culture medium and stored for at least 2 3 hours. After this time (while oocytes are prepared for injection), motile spermatozoa are placed in polyvinylpyrrolidone (PVP) for hours to slow their movement and perform sperm tail breakage to allow their selection for ICSI or IMSI. Hyaluronic acid (HA) treatment is an alternative to PVP medium before ICSI and/or IMSI. According to recent publications, HA achieves the same results as PVP and reduces the levels of DNA fragmentation in selected spermatozoa (8, 9). The aim of the present article was to compare the DNA fragmentation of semen samples measured by terminal deoxynucleotide transferase mediated dutp nick-end labeling (TUNEL) after incubation in PVP and HA for different lengths of time. MATERIALS AND METHODS Population Twenty-seven patients who were undergoing ART were selected for this study. Men with normal hormonal profiles (FSH, LH, PRL, and T), semen samples of >1 ml, and sperm concentrations > /ml were eligible for inclusion. Men with urogenital infection, varicocele, or cryptorchidism were excluded. All procedures were performed at CEGYR Reproductive Medicine in Buenos Aires, Argentina, from January to June Semen samples were donated for research with informed written consent by couples undergoing assisted reproduction procedures at CEGYR. This study was approved by the Internal Review Board and Ethics Committee of CEGYR. There is no conflict of interest at the present study. Semen Samples Preparation Samples were collected by masturbation after sexual abstinence for 2 4 days. After semen liquefaction (30 60 minutes), basic semen analysis was performed (motility, concentration, and survival) and motile sperm were selected with the use of a double-layer gradient (45% 90%) (Isolate; Irvine Scientific) according to the procedures established by the World Health Organization (2010) (10). Isolated motile sperm was adjusted to a final concentration of DNA fragmentation (TUNEL) was assessed in neat (T0), postgradient (TG), 1-hour postgradient (TG1), and 2-hour postgradient (TG2) samples at room temperature in Modified Human Tubal Fluid (Irvine Scientific). After this time (TG2), samples were divided into two equal volume fractions: the first fraction were incubated in PVP (Irvine Scientific) and the second in HA (Sperm Slow; Origio Medicult) under oil (Irvine Scientific) over an inverted microscope equipped with a heating plate (37 C). Aliquots were taken at 0.5, 1.0, and 1.5 hours (TG2-30, TG2-60, and TG2-90, respectively) for assays. For PVP fractions, sperm were carefully aspirated from the peripheral region. Similarly in HA, incubation was carried out according to the supplier, and sperm were selected from HA and clean medium. Evaluation of DNA Fragmentation (TUNEL) All fractions were fixed in 2% formaldehyde (Sigma) in 1 phosphate-buffered saline solution (PBS; ph 7.4; Gibco) for at least 1 hour. Each sample was placed into one well of a multiwell (4-mm diameter) Teflon-printed slide (Electron Microscopy Sciences) and allowed to settle. After 2 3 hours,each well was washed with 1 PBS (three times, 5 minutes each), and the cells were permeabilized with cold methanol (Merck). Before incubation with the TUNEL solution, each well was washed again with 1 PBS. For each sample, one extra well was incubated with DNAse (1 U/mL; Sigma) for 30 minutes at 37 Cas a positive control, and in another well the TUNEL enzyme solution was omitted as a negative control. Then all samples were incubated in TUNEL solution (Roche,) for 1 hour at 37 C. All samples were finally washed with 1 PBS (three times, 5 minutes each), and mounted in Vectashield H-1000 medium (Vector Laboratories). A total of 500 spermatozoa were counted by fluorescence microscopy for each fraction. TUNEL staining was evaluated by fluorescence microscopy using a green filter (fluorescein isothiocyanate, 488 nm). Figure 1 shows light and dark field images of negative ( enzyme of TUNEL solution omitted; A, A 0,A 00 ) and positive (incubated in DNAse such that all spermatozoa have green head fluorescence; B, B 0,B 00 ) control samples. Only complete spermatozoa (Fig. 1C and C 0, arrows) were considered for percentage estimates; heads without tails were not considered (Fig. 1C, 1C 0, and 1C 00 arrowhead). Statistics The Student t test was used for between-group comparisons, and the Mann-Whitney U-test was used to assess homogeneity. Differences were considered to be significant when P<.05. MedCalc 12.4 software (Belgium) was used for the statistical analysis. RESULTS To permit a more accurate analysis and interpretation of the results, the population was separated into two groups: samples with T0 TUNEL <20% (group A) and those with T0 TUNEL R20% (group B). The sperm characteristics of both populations are described in Table 1. Because volume and concentration were used as inclusion criteria, no significant difference was found in these parameters. However, significant differences were observed in age, morphology (Kruger), and motility (P<.05). The average TUNEL levels decreased significantly after centrifugation through a double-layer gradient (T0 vs. TG), regardless of the initial TUNEL status of the sample. T0 vs. TG was, respectively, % vs % in group A and % vs % in group B (Fig. 2, *P<.05). This feature can be clearly distinguished in Figure 3A and B. After the gradient centrifugation, motile sperm were incubated at room temperature for 2 hours in culture medium. During this period, the TUNEL levels within each group remained stable (group A: % vs %; group B: % vs %; Figs. 2, 3C, and 3D). In patients with TUNEL <20%, we 70 VOL. 100 NO. 1 / JULY 2013

3 Fertility and Sterility FIGURE 1 Terminal deoxynucleotide transferase mediated dutp nick-end labeling (TUNEL) assay. The negative control was performed by omitting the enzyme solution [phase contrast (A 0 ), under green fluorescence (A), and dark field þ fluorescence]. A DNAse-treated sample was used as the positive control [green fluorescence, 100% of positive cells (B), phase contrast (B 0 ), and dark field þ fluorescence (B 00 )]. Only complete spermatozoa (C, arrow) were considered in the evaluation of TUNEL; sperm heads without tails were not counted for this purpose (C, arrowhead). observed a significant increase of DNA fragmentation at TG2-90 (Figs. 2, 3G, and 3G 0 ;**P<.05), but no differences were observed in the PVP ( % to %) and HA (10.2 3% to %) treated samples, and DNA fragmentation levels were not considered to be altered (<20%) in either case. Moreover, in patients with TUNEL R20%, we observed a significant increase in DNA fragmentation at TG2-60 (Figs. 2, 3F, and 3F 0 ; ***P<.05), but no differences were observed between the PVP ( %1 to %) and HA ( % to %) treated samples. Sperm motility between TG and TG2 varied from % to %. DISCUSSION Our results show that DNA damage increases more rapidly over sample preparation time in semen samples with R20% DNA fragmentation than in samples with <20% DNA fragmentation, but incubation in PVP and HA had similar effects. During sperm preparation for ART procedures (ICSI or IMSI), it is important to consider sperm DNA fragmentation before the treatment. High values can significantly affect the reproductive outcomes. The most common sperm selection methods used in ART are the swim-up and double-layer gradient centrifugation methods (11). The swim-up technique is based on the ascending capacity of the sperm, i.e., their progressive motility, and the gradient technique is based on the migration of mature motile sperm against centrifugal force. The swim-up technique is useful in sperm samples with good concentration and motility. In samples with high DNA fragmentation, the double-layer gradient technique has been proven to significantly reduce DNA damage compared with the swim-up method. In TABLE 1 Sperm parameter values between groups A and B. Parameter A: TUNEL <20% B: TUNEL R20% P value n Age (y) ( ) ( ).04 Strict sperm morphology (%) ( ) ( ).02 Volume (ml) ( ) ( ).2 Sperm concentration (10 6 /ml) ( ) ( ).4 Progressive motility (A þ B) ( ) ( ).01 Note: Values are presented as mean SD (95% CI). TUNEL ¼ terminal deoxynucleotide transferase mediated dutp nick-end labeling. VOL. 100 NO. 1 / JULY

4 ORIGINAL ARTICLE: ANDROLOGY FIGURE 2 DNA fragmentation dynamics. The levels of DNA fragmentation decreased significantly after obtaining a centrifugation gradient in all samples (*). In patients with terminal deoxynucleotide transferase mediated dutp nick-end labeling (TUNEL) <20% (A), we observed a significant increase of DNA fragmentation after TG2-90 (***) (10.2 3% to %). In patients with TUNEL R20% (B), we observed a significant increase in DNA fragmentation beginning at TG2-60 (**) ( to ). HA ¼ hyaluronic acid; PVP ¼ polyvinylpyrrolidone. addition, gradient centrifugation could also improve some sperm parameters, such as survival time, morphology, and others (12, 13). Therefore, we chose to perform the assay with the double-layer gradient technique. Our results confirm that the use of gradient centrifugation significantly reduces DNA fragmentation levels. This experiment reproduces the events that occur before ICSI or IMSI, with the exception of sperm immobilization by tail breakage. Our aim was to evaluate when a significant increase of sperm DNA fragmentation takes place after gradient centrifugation (TG), to improve and/or adjust the processing time used for this type of sample. In normal samples (TUNEL <20%), it was observed that TUNEL levels increased significantly after TG2-90, which represents a total of 3.5 hours after gradient. No samples reached pathologic levels during the indicated time; therefore, in sperm samples with normal TUNEL, it would be acceptable to wait at least 3.5 hours before the injection. In contrast, samples with abnormal initial TUNEL values (R20%) exhibited a significant increase after TG2-60, or 3 hours after gradient. Consequently, it would be inappropriate to process these samples for a period longer than 3 hours. The sperm in group B had worse quality, but the increase in DNA damage occurred only one-half-hour sooner in group B than in group A. However, this does not indicate that greater differences would be observed after longer time periods (8 24 hours). It has been reported that DNA fragmentation may be associated with sperm apoptosis events (14 19). There is a relationship between increased reactive oxygen species (ROS) and apoptosis with consequent sperm DNA damage. Oxidative stress occurs when the amount of ROS exceeds the antioxidant capacity of the seminal plasma. Sperm are particularly susceptible to damage induced by ROS. These molecules cause injury by attacking the sperm plasma membranes, which contain large amounts of polyunsaturated fatty acids. Additionally, sperm cytoplasm contains low concentrations of reducing enzymes and apparently no mechanisms to repair DNA damage (20). DNA damage and ROS production can be clearly distinguished in the sperm of patients who are R40 45 years old, and these events can also induce a gradual decrease in sperm motility. In the present study, patients from group B were significantly older and had significantly diminished sperm motility compared with the patients from group A (Table 1). These factors could explain why the patients in group B have an increased susceptibility to DNA damage. No significant differences were observed between the samples that were incubated in PVP or in HA, even though it has been reported that the use of HA, such as PICSI or Sperm Slow, improves nonfragmented sperm selection. Additionally, HA has been proposed to replace PVP as a sperm immobilizer for ICSI or IMSI (8, 21). In our experience, however, both media have similar effects. The amount of DNA fragmentation at a time close to 4 hours after TG did not exceed 6% in group A or group B. This finding is contradictory to results reported by other authors, who observed an increase of >30% in the same time period in infertile patients (7). However, those authors did not clarify if the study was performed in neat or processed 72 VOL. 100 NO. 1 / JULY 2013

5 Fertility and Sterility FIGURE 3 Sperm DNA fragmentation dynamics at various time points. T0 (A), TG (B), TG1 (C), TG2 (D), TG2-30 (E), TG2-60 (F), and TG2-90 in polyvinylpyrrolidone (G) and TG2 30 (E 0 ), TG2 60 (F 0 ), and TG2 90 (G 0 ) in hyaluronic acid. Images of green fluorescence/dark field and phase contrast microscopy (insets) were captured. VOL. 100 NO. 1 / JULY

6 ORIGINAL ARTICLE: ANDROLOGY samples. Those studies used the sperm chromatin dispersion (SCD) test to assay DNA fragmentation, but SCD and TUNEL results have been reported to have a high correlation (R ¼ 0.9) in both fertile and infertile men (22). There is no doubt that an increase in DNA damage occurs over time, but in our experience, the levels of damage do not increase more than 25% in 4 hours. It has been reported that DNA damage may be higher over 8 24 hours, which we did not evaluate in our experiment. Moreover, those incubation periods are far beyond the usual sperm-processing time involved in ART. It has been reported that levels of DNA damage could vary depending on the motile sperm selection method (swim-up or gradient), incubation temperature (room temperature or 37 C), atmospheric conditions (5% CO 2 ), and use of sperm freezing (23). We think that our findings could be complemented by future experiments using the same conditions but comparing the results of sample incubation at 37 C (at all steps) versus room temperature and by experiments to establish the differences in the results of the gradient and swim-up procedures. Finally, excessive time spent on sperm preparation influences DNA damage levels; therefore, in patients with DNA fragmentation R20%, it is especially important to minimize the processing time before ICSI or IMSI. Sperm in this subgroup seem to deteriorate more rapidly over time. Finally, follow-up studies should be performed to confirm that our results are correlated with ART outcomes, especially live birth rates. REFERENCES 1. Lewis SE, Agbaje I, Alvarez J. Sperm DNA tests as useful adjuncts to semen analysis. Syst Biol Reprod Med 2008;54: Speyer BE, Pizzey AR, Ranieri M, Joshi R, Delhanty JDA, Serhal P. Fall in implantation rates following ICSI with sperm with high DNA fragmentation. 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