Jaime Gosalvez, Ph.D. (a), Raúl Sánchez Gutierrez, M.D., Ph.D. (b), Juan G. Alvarez, M.D., Ph.D. (c, d)
(a) Department of Biology, Universidad Autónoma de Madrid, Madrid, Spain
(b) Center of Translational Medicine (CEMT-BIOREN), Universidad de La Frontera, Temuco, Chile
(c) ANDROGEN Center, La Coruña, Spain
(d) Harvard Medical School, Boston, Massachusetts
Cryopreservation and sperm processing have been shown to result in sperm DNA fragmentation (SDF). However, the dynamics of SDF and type of damage, namely, single- (SSB) and double-strand breaks (DSB) have never been investigated after thawing of cryopreserved sperm. The main objective of this study was to report the extent and type of DNA damage produced after thawing of cryopreserved human sperm and its implications in assisted reproductive technologies. A total of 35 semen samples from fertile sperm donors were either cryopreserved directly or processed by density gradient centrifugation and incubated after thawing at 37°C for 5 and 30 minutes (short-term incubation) and 3, 6, and 24 hours (long-term incubation). Mean SDF values in spermatozoa from the 80% pellet at 0, 5, and 30 minutes incubation did not show any significant differences in SSB, DSB, or percent SDF although there was a trend towards an increase in all these parameters. However, significant differences in SSB and DSB were observed after 30 minutes of incubation. In 17 of 35 samples (48.5%), most of the DNA damage post-thawing at 3 and 6 hours was of the SSB type. In the remaining 18 samples (51.5%) there was a combination of SSB and newly produced DSB. There was significant variability between donors in sperm DNA longevity after thawing. In conventional in vitro fertilization (IVF) sperm are incubated for up to 19h at 37°C, sperm DNA damage may lead to D+3 embryo block and implantation failure; and reports indicate that embryo development is better after intracytoplasmic sperm injection than conventional IVF when using cryopreserved sperm. Our findings would discourage the use of cryopreserved sperm in conventional IVF— intracytoplasmic sperm injection should be recommended instead.
Although iatrogenic sperm DNA fragmentation (SDF) has been reported during sperm processing (1) and after cryopreservation (2), the dynamic behavior of DNA damage can be used as indicative of sperm DNA longevity and type of DNA damage, namely, single- (SSB) and double-strand breaks (DSB). These DNA lesions may occur during short- and long-term incubation after thawing of cryopreserved human sperm but have never been investigated. A previous report studied the differences in the dynamic behavior of SDF in fresh versus cryopreserved sperm samples after thawing but not the type of DNA damage (SSB vs. DSB) (3).
This is a prospective study directed to obtain baseline values in normozoospermic males and this is precisely why we performed the study using semen samples from sperm donors. Therefore, the aim of the study was to investigate the presence of these two types of DNA damage in a normal male population to generate baseline values to be compared later with other clinical situations associated to male factor infertility.
A total of 35 semen samples from fertile sperm donors were either cryopreserved directly or processed by density gradient centrifugation using a 40/80% Sperm Filter gradient (Cryos). All semen samples were from normozoospermic sperm donors with sperm concentrations ranging between 80 and 150 million/ml, with percent [a+b] motility between 60% and 90%, and percent normal forms above 4% using the strict Tygerberg morphology criteria (4).
Following centrifugation, spermatozoa from the 80% gradient pellet were aspirated and washed with SpermWash medium (Cryos). Aliquots of the semen and of the washed 80% pellet were mixed 1:1, v/v with the cryoprotectant, CryoProtec (Nidacon), transferred to 0.5 ml CBS straws, (CBS), thermosealed, exposed to liquid nitrogen vapors for 5 minutes and finally stored in liquid nitrogen. After cryopreservation, aliquots of the samples were thawed at 37°C for 5 minutes and immediately assessed for sperm DNA damage (0 minutes). Percent SDF and type of DNA damage (SSB and DSB) were assessed using the Halosperm test (Halotech- 3 DNA) and Two-tailed comet assay (5) (Figure 1), respectively. Thereafter, samples were incubated at 37°C for an additional 5 and 30 minutes (short- term incubation) and for 3, 6, and 24 hours (long-term incubation). The comparison of the dynamic loss of DNA quality was assessed using the nonparametric maximum likelihood Kaplan–Meier estimator. The log-rank test statistic (Mantel-Cox) which compares estimates of the hazard functions between the two groups at each time interval was used (SPSS v.17.0 for Windows; SPSS Inc.).
Two-tailed comet assay in human spermatozoa showing normal spermatozoa and one mapping for single-strand breaks (SSB; Y-axis distribution) and double-strand breaks (DSB; X-axis distribution). (a) Original microscope capture. (b) Electronic filtered image enhancing the localization and distribution of DNA fragments.
Mean SDF values in spermatozoa from the 80% pellet at 0, 5, and 30 minutes of incubation did not show any significant differences in SSB, DSB, or percent SDF (χ2 = 1.01; df = 2; P = .063), although there was a trend towards an increase in all these parameters. However, significant differences in SSB and DSB were observed after 30 minutes of incubation (x2 =19, 2; df = 2; P<.0001). In 17 of 35 samples (48.5%), most of the DNA damage post-thawing at 3 and 6 hours was of the SSB type. In the remaining 18 samples (51.5%) there was a combination of SSB and newly produced DSB. There was significant variability between donors in sperm DNA longevity after thawing (x2 = 13.6, 2; P = .0001).
Sperm capacitation in vitro is a process that requires at least 2 hours of incubation at 37°C in the presence of 1.5 mM calcium, 25 mM bicarbonate, and high Human Serum Albumin (HSA) concentration (10 mg/ml) (6). Our results clearly show that significant DNA damage occurs already after 2 hours of incubation. In addition, it has been shown that sperm penetration of the oocyte occurs after 4 hours incubation at 37°C of sperm with the oocytes (7), which added to the sperm capacitation time would amount ≥ 6 hours of incubation at 37°C therefore, leading to high sperm DNA damage. Therefore, based on the results of our study, intracytoplasmic sperm injection (ICSI) should be considered when using cryopreserved sperm for its potential benefits since ICSI can be performed before waiting 30 minutes after thawing. It is true that swim-up and density gradient centrifugation are efficient techniques in eliminating spermatozoa containing double-strand DNA damage and also sperm with highly damaged (degraded) DNA) that is covered by SSB and DSB (8), our previous results showed that density gradient centrifugation is more efficient than swim-up in selecting spermatozoa that are free of SSB (9). In addition to DNA damage after thawing of cryopreserved liquefied semen samples, sperm subsets selected by density gradient centrifugation (including sperm from the gradient pellet) were also evaluated in this study for post-thawing sperm DNA damage and was precisely in these samples where significant SSB and DSB was observed. Therefore, the results of this study are novel in the sense that, for the first time, SSB and DSB have been shown to occur after short- and long-term incubation of cryopreserved sperm from the density gradient pellet.
Cryopreservation/thawing have been associated with reactive oxygen species (ROS) - induced sperm DNA damage (10-11). In addition, cryopreservation may result in the activation of sperm caspases and specific endonucleases leading to DSB (12). The increase in SSB is usually related to ROS-induced sperm DNA damage after thawing. It is noteworthy that DSB produced by endogenous enzymatic activity is not present during the first 30 minutes after thawing but it appears thereafter.
The results of this study show that significant DNA damage occurs after long-term incubation of cryopreserved sperm after thawing and that, therefore, conventional in vitro fertilization (IVF) should be discouraged when using cryopreserved sperm because it requires long term incubation (sperm capacitation in vitro plus penetration time after oocyte insemination). In addition, DSB have been associated with day-3 embryo block, low implantation rates, and miscarriage. Therefore, we feel that the significant sperm DNA damage that occurs after long-term incubation after thawing should be of high concern when performing conventional IVF. If cryopreserved sperm are microinjected by ICSI within 30 minutes after thawing, DNA damage would be minimized decreasing the probability of day-3 embryo block, implantation failure, and miscarriage.
In conclusion, significant SSB and DSB damage takes place after 30 minutes incubation at 37°C of cryopreserved sperm after thawing. In conventional IVF sperm are incubated with the oocytes for up to 19h at 37°C; sperm DNA damage, in particular DSB, may lead to D+3 embryo block, implantation failure, and spontaneous miscarriage; and reports indicate that embryo development is better after ICSI than conventional IVF when using cryopreserved sperm (13). Our findings would discourage the use of cryopreserved sperm in conventional IVF. ICSI should be recommended instead. These results suggest that new techniques of sperm cryopreservation, such as vitrification, should be evaluated for use in conventional IVF in order to improve DNA longevity.
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