Sperm cryopreservation is widely used for both research and reproduction purposes, but its effect on sperm DNA damage remains controversial. Sperm DNA fragmentation (SDF) has become an important biomarker to assess male infertility. In particular, the differentiation between single- and double-stranded DNA fragmentation (ssSDF and dsSDF) has clinical implications for male infertility where ssSDF is associated with reduced fertility, whereas dsSDF is associated with increased risk of miscarriage. In this study, semen samples from 30 human males have been analysed in both fresh and cryopreserved using the alkaline and neutral Comet assays. Results show an increase of about 10% of ssSDF, assessed by the alkaline Comet assay, regardless of the male fertility status. Neutral Comet analysis of dsSDF does not show any statistical increase when comparing fresh and cryopreserved samples in any of the patient groups. Results support previous reports that oxidative stress is the major effector in DNA damage during sample cryopreservation, as, on one hand, ssSDF has previously been related to oxidative damage and, on the other hand, we have not found any effect on dsSDF. Therefore, there might be a slight risk of decreased fertility after using a freezed sample, but no evidence for increased miscarriage risk from cryopreserved spermatozoa should be expected.
The sperm DNA damage analysis has become a complementary biomarker in determining male infertility, which is mainly diagnosed through macroscopic and microscopic semen parameters, determination of chromosomal aneuploidies, meiotic studies, hormonal analysis and karyotype (Egozcue et al., 1997; Benet et al., 2005; Martin, 2006; Carrell, 2008; Templado et al., 2011). Sperm DNA fragmentation (SDF) has been developed as a marker of sperm DNA quality, and many studies have shown an increase in SDF in infertile patients compared with fertile donors, and have established clinical threshold values for infertility using different techniques (Sergerie et al., 2005; Evenson & Wixon, 2008; Velez de la Calle et al., 2008; Sharma et al., 2010; Simon et al., 2011; Ribas-Maynou et al., 2012b). Moreover, a distinction of different groups of infertile patients such as varicocoele patients, recurrent miscarriage patients or chromosomal rearrangement carriers can be performed by using methods with higher sensitivity for SDF analysis such as the Comet assay (Ribas-Maynou et al., 2013). The aetiology of SDF has also been widely discussed, locating the DNA damage at different levels (Aitken & De Iuliis, 2010; Sakkas & Alvarez, 2010): (i) at the testicular level, where there can occur apoptosis during spermatogenesis, DNA breaks during spermiogenesis as a result of nuclease activity, radiotherapy and chemotherapy or environmental toxicants (Maione et al., 1997; Sailer et al., 1997; Sotolongo et al., 2005; Rubes et al., 2007; O'Flaherty et al., 2008); (ii) at the epididymis level, where the DNA damage would be mainly caused by oxidative stress and (iii) at vas deferens level, where the oxidative stress is increasing with respect to the epididymis (Agarwal et al., 2008; Makker et al., 2009; Aitken & Koppers, 2011).
The effect of the sperm DNA damage on the embryo has been less studied owing to a lack of physiological studies. However, some authors report that fertilization with a DNA-damaged spermatozoon might lead to DNA errors at different levels of embryogenesis (Aitken & De Iuliis, 2007; Lewis & Simon, 2010) or a slower embryo development (Gawecka et al., 2013). Moreover, if the DNA breaks carried by the sperm cell are not repaired, the embryo might be miscarried (Ribas-Maynou et al., 2012b) or the child affected by different childhood diseases (Cooke et al., 2003; Aitken et al., 2009).
Gamete cryopreservation is widely used for a variety of purposes, such as fertility preservation previous to chemotherapy treatment, donor or conjugal sperm cryopreservation or research (Sanger et al., 1992; Anger et al., 2003; Jensen et al., 2011; Di Santo et al., 2012). Because of that, it is important to understand the effects of cryopreservation to preserve the better quality of the thawed sample. It has been shown that cryopreservation reduces sperm motility and sperm vitality (Thomson et al., 2010; Lee et al., 2012; Satirapod et al., 2012). Recent studies have been focused on the effect of cryopreservation on sperm DNA damage, showing that the main effector of DNA damage during the process of freezing and thawing a semen sample are the reactive oxygen species (Lasso et al., 1994; Thomson et al., 2009; Said et al., 2010). However, the effect of cryopreservation on sperm DNA integrity remains controversial with some reports showing an effect (Spano et al., 1999; Donnelly et al., 2001; de Paula et al., 2006; Thomson et al., 2009; Zribi et al., 2010), whereas others report none (Host et al., 1999; Duru et al., 2001; Isachenko et al., 2004). These controversial data may be resolved by controlling for additional factors that affect sperm DNA integrity during freeze/thawing, such as the previous state of the sample (Donnelly et al., 2001; Kalthur et al., 2008; Ahmad et al., 2010), the technique used for cryopreservation or the cryoprotectant applied (Di Santo et al., 2012).
Different techniques have been used to assess sperm DNA damage in cryopreservation, such as TUNEL (Duru et al., 2001; de Paula et al., 2006; Thomson et al., 2009; Zribi et al., 2010), SCSA (Spano et al., 1999; Gandini et al., 2006), SCD (Gosálvez et al., 2010) and the Comet assay (Donnelly et al., 2001; Kalthur et al., 2008; Ahmad et al., 2010). However, to our knowledge there have been no cryopreservation studies differentiating single-stranded sperm DNA fragmentation (ssSDF) and double-stranded sperm DNA fragmentation (dsSDF) on the same semen sample, using both fertile and subfertile patients. This differentiation could be helpful to understand the mechanisms through which DNA fragmentation is produced in cryopreservation. In this sense, it has been proposed that ssSDF can be related to oxidative stress DNA damage and would be extensively distributed throughout the genome, whereas dsSDF is associated with some kind of enzymatic activity having acting in a non-extensive manner (Sotolongo et al., 2005; Ribas-Maynou et al., 2012a,b). The sperm Comet assay allows researchers to distinguish between these two types of DNA damage, depending on whether it is performed with a previous alkaline denaturation or with neutral conditions respectively (Enciso et al., 2009; Ribas-Maynou et al., 2012a). The Comet assay has a higher sensitivity than the SCD test because of the electrophoresis component of the former (Ribas-Maynou et al., 2013). The SCD test has a similar sensitivity as two other common SDF assays, the TUNEL assay and SCSA (Chohan et al., 2006; Garcia-Peiró et al., 2011).
The main aim of the present work is to evaluate the effect of cryopreservation on semen samples attending single-stranded or double-stranded sperm DNA fragmentation using the Comet assay methodology. A secondary objective of this work was to analyse the effect of cryopreservation in different groups, taking into account their clinical status.
Materials and methods
Semen samples from 44 human males were obtained by masturbation after an abstinence period of 3–7 days. Samples were divided into three clinical groups: fertile donors (n = 10), recurrent miscarriage patients without a female factor (RPL) (n = 8) and a group of subfertile patients (n = 26) which includes altered semenogram samples from subfertile couples (7 asthenozoospermic, 4 teratozoospermic, 9 asthenoteratozoospermic, 1 oligoasthenoteratozoospermic and 5 asthenoteratozoospermic with varicocoele) who had unprotected intercourse without a pregnancy during 12 months. The age of all donors ranged from 18 to 38 years and there were no differences among different groups. Informed consent was obtained for all donors and the appropriate ethics committee approved the study.
After allowing the sample to liquefy for 30 minutes, semen parameters according to WHO 2010 guidelines were analysed by using SCA software (Sperm Class Analyzer; Microptic, Barcelona, Spain). Sperm count (106 spermatozoa/mL), motility (% A+B) and morphology (% normal forms) for the samples were (mean ± standard deviation): 124.35 ± 58.42, 50.95 ± 9.63 and 8.11 ± 2.89, respectively, for fertile donors; 122.14 ± 128.02, 46.5 ± 17.46 and 4.14 ± 2.12, respectively, for recurrent miscarriage patients and 61.93 ± 63.33, 24.54 ± 13.00 and 2.86 ± 3.35, respectively, for subfertile patients.
The cryopreservation technique used in this work has previously been published in Ribas-Maynou et al. (2012a). The total semen sample was mixed in equal proportions with test-yolk buffer (14% glycerol, 30% egg yolk, 1.98% glucose and 1.72% sodium citrate, pH 7.5) and, after homogenizing, each sample was divided into cryotubes and frozen in isopropanol at −80 °C overnight, which allows a cooling ramp of −1 °C/min. The following day, samples were transferred to liquid nitrogen until they were thawed to start the analysis of DNA fragmentation.
Thawing and sample preparation
Samples were thawed at room temperature. Then, three washes were performed using PBS without Ca2+ or Mg2+, centrifuging at 600 g for 5 min. Finally, the sperm concentration was adjusted at 1 × 106 spermatozoa/mL to assess sperm DNA damage.
The SDF analysis was performed twice using the alkaline and neutral Comet assays: once starting within the first hour after obtaining the fresh sample and again starting immediately after thawing a cryopreserved fraction of the same sample.
The Comet assay was performed in alkaline or neutral conditions to analyse single-stranded DNA fragmentation and double-stranded DNA fragmentation, respectively, as previously described (Ribas-Maynou et al., 2012a). Briefly, 15 μL of semen sample was mixed with 25 μL of LMP agarose, and allowed to solidify with a coverslip on two slides. After coverslips were removed, slides were incubated in two lysis solutions for half an hour each. Then slide designated for alkaline Comet was denatured in an alkaline buffer for 2.5 min and electrophoresed for 4 min, and slide designated for neutral Comet was electrophoresed for 12.5 min and washed in a NaCl buffer. Finally, both slides were washed in neutralization buffer and ethanol series. After allowing to dry horizontally, staining was performed using DAPI SlowFade® Gold antifade (Invitrogen, Eugene, OR, USA) and 400 spermatozoa were classified as fragmented or non-fragmented following the criteria reported before (Ribas-Maynou et al., 2012a) using a fluorescence microscope (Olympus AX70, Hamburg, Germany). Results were expressed as a percentage of the fragmented spermatozoa (%SDF).
Statistical analysis was performed with SPSS v20 software (Statistics Package for the Social Sciences software, Inc., Chicago, IL, USA). As the two fresh and cryopreserved groups are related samples, the comparisons between them were performed using the Wilcoxon test for paired samples. The significance level was established at 95% of the confidence interval to be considered statistically significant.
Cryopreservation and SDF
The data were classified attending the clinical status of the donors into three groups: fertile donors, recurrent miscarriage without female factor patients and general subfertile patients including altered semenogram patients as described in Material and Methods. The SDF analysed with alkaline and neutral Comet assay regarding these three groups before and after cryopreservation is shown in Table 1.
Table 1. Percentage of spermatozoa showing DNA fragmentation assessed by the Comet assay before and after cryopreservation (mean ± standard deviation)
|Total samples (n = 44)||40.13 ± 17.67||49.80 ± 17.64||67.36 ± 19.57||68.88 ± 17.83|
|Fertile donors (n = 10)||21.05 ± 10.63||33.63 ± 12.34||63.70 ± 28.52||65.57 ± 24.80|
|RPL patients (n = 8)||34.97 ± 18.51||38.55 ± 17.23||84.60 ± 16.11||84.88 ± 14.74|
|Subfertile patients (n = 26)||49.05 ± 12.79||59.48 ± 12.43||63.46 ± 13.29||65.23 ± 12.81|
The alkaline Comet results showed statistical differences between fresh and cryopreserved spermatozoa in all three groups (Table 1). These differences were greater for the fertile donors (p = 0.005) and subfertile males group (0.000) than for males from couples with recurrent pregnancy loss (p = 0.045). Overall, there was approximately a 10% increase in ssDNA damage in cryopreserved spermatozoa as measured by the alkaline Comet assay (p < 0.001). Interestingly, the neutral Comet assay did not show any statistical difference between fresh and cryopreserved samples in any of the groups (p > 0.05).
SDF and cryopreservation
Semen cryopreservation has become widely used technique in reproduction, applied to both assisted reproduction techniques and research. The human sperm cryopreservation has been studied in many publications, with different results between them. Some studies have been focused on the effect of cryopreservation to seminal parameters such as sperm motility, vitality and morphology, showing a decrease on these parameters (Thomson et al., 2010; Di Santo et al., 2012; Lee et al., 2012; Satirapod et al., 2012). However, the growing interest on SDF assessment requires studies to approach the actual DNA damage on the cryopreserved spermatozoa. In this sense, opposite results have been described on literature, some showing DNA damage after cryopreservation (Spano et al., 1999; Donnelly et al., 2001; de Paula et al., 2006; Thomson et al., 2009; Zribi et al., 2010), and some showing no effect of cryopreservation (Host et al., 1999; Duru et al., 2001; Isachenko et al., 2004). Nevertheless, cryopreservation studies have been performed with different techniques and, because of the controversy on this topic (Garcia-Peiró et al., 2011), it might be necessary to perform the analysis at the same time with different techniques, or using the most sensitive ones, such as Comet assay (Ribas-Maynou et al., 2013). For that, in this work we performed the analysis through the alkaline and neutral Comet assays. Comet results showed a statistical increase on SDF (Table 1), agreeing with some previous studies using this technique (Donnelly et al., 2001; de Paula et al., 2006; Thomson et al., 2009). In this sense, a remarkable result obtained is that the percentage of spermatozoa with single-stranded DNA fragmentation is increased by a 10% after cryopreservation (Table 1). This would mean that a semen sample would have roughly 10% more fragmented spermatozoa, which would have worse DNA integrity, and therefore, they would be less likely to end up with a pregnancy. Regarding neutral Comet, no differences have been observed between before and after cryopreservation (Table 1), showing no effect on double-stranded DNA integrity. To our knowledge there have not been results using this technique related to cryopreservation, but taking into account that ssSDF has recently been related to oxidative damage (Enciso et al., 2009; Ribas-Maynou et al., 2012a), these results would fit to the consideration that oxidative stress would be the main effector of DNA damage during cryopreservation (Mazzilli et al., 1995; Thomson et al., 2009). Moreover, this increase only on ssSDF might have a clinical effect on pregnancy achievement, but the lack of increase on dsSDF would not produce an increase on the miscarriage risk (Ribas-Maynou et al., 2012b). In relation to that, when different clinical statuses were analysed, all fertile donors, recurrent miscarriage patients and subfertile patients showed a statistical increase on alkaline Comet after cryopreservation, but none of them showed an increase on neutral Comet (Table 1). Therefore, as different cryopreservation protocols can have different effects on DNA integrity, a comparative study of the cryopreservation techniques and a standardization of the best one would be necessary to solve the different effects found in the literature (Donnelly et al., 2001; Kalthur et al., 2008; Ahmad et al., 2010).
The effect of cryopreservation on alkaline Comet assay showed an increase of 10% of ssSDF, whereas the neutral Comet assay showed no effect after thawing. Therefore, these results show that a mean of 10% of the cryopreserved sperm cells present worse single-stranded DNA integrity than before cryopreservation. This suggests that cryopreservation may affect the pregnancy capacity of the sperm cell without increasing the associated miscarriage risk.
This work has been supported by Fondo de Investigación Sanitária, Instituto de Salud Carlos III (PI11/00630), and Generalitat de Catalunya (2009 SGR 1107), and J. Ribas-Maynou has a grant from Generalitat de Catalunya (FI-DGR 2011). We would like to thank Dr. Steve Ward for his exhaustive revision and useful comments on the final manuscript.
J.R.-M. contributed in experimental procedures, statistical analysis, table elaboration and document writing. A.F.-E. contributed in experimental procedures. A.G.-P. contributed in experimental design, results discussion and statistical analysis. E.P., C.A. and M.J.A. contributed in recruitment of patients, samples collection, storage and semen parameters analysis. J.N. and J.B. contributed in experimental design and direction and coordination of the work.