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Published-Ahead-of-Print September 5, 2007, DOI:10.2164/jandrol.107.003574
Journal of Andrology, Vol. 29, No. 1, January/February 2008
Copyright © American Society of Andrology
DOI: 10.2164/jandrol.107.003574

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Higher Sperm DNA Damage in Semen From Men With Spinal Cord Injuries Compared With Controls

NANCY L. BRACKETT, EMAD IBRAHIM, JASON A. GROTAS, TEODORO C. ABALLA AND CHARLES M. LYNNE

From the Miami Project to Cure Paralysis and the Department of Urology, University of Miami Miller School of Medicine, Miami, Florida.

Correspondence to: Dr Nancy L Brackett, Miami Project to Cure Paralysis, University of Miami Miller School of Medicine, Lois Pope Life Center, 1095 NW 14th Terrace, Miami, FL 33136 (e-mail: nbrackett{at}miami.edu).
Received for publication July 6, 2007; accepted for publication August 24, 2007.

   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
Semen from men with spinal cord injuries (SCI) and control subjects was investigated for sperm DNA damage using the sperm chromatin structure assay. Three experiments were performed. In experiment 1, the DNA fragmentation index (DFI) was compared in semen from SCI subjects and control subjects. In experiment 2, the % DFI was determined in repeated ejaculations to examine the effect of anejaculation on DFI. In experiment 3, the DFI was determined in neat vs processed semen to examine the effect of necrospermia or leukocytospermia on DFI. The results of experiment 1 showed a significantly higher mean (± SEM) DFI in the semen of SCI subjects (65.2% ± 6.6%; range, 42.3%–90.8%) compared with control subjects (15.4% ± 2.9%; range, 5.4%–33.5%; P < .001). In experiment 2, there was a high correlation between the DFIs obtained in the first semen specimens and the DFIs obtained 3 days later in semen of the same SCI subjects (rs = .94; P < .02). In experiment 3, the results showed no significant difference between mean DFI in aliquots of neat semen (79.3% ± 9.9%) vs matched aliquots of semen processed to remove dead sperm and leukocytes in SCI subjects (75.2% ± 16.1%). The DFI is higher in semen from men with SCI vs controls. The cause of this condition is unknown but does not seem to be due to prolonged anejaculation or to the proximate conditions of necrospermia or leukocytospermia. The relevance of these findings to fertility outcomes with SCI male partners remains to be determined.

     Key words: Infertility, DNA fragmentation, SCSA, SCI


The semen quality of men with spinal cord injuries (SCI) has been characterized in numerous studies. Findings have generally demonstrated normal sperm concentrations, low sperm motility, low sperm viability, variable sperm morphology, and abnormal seminal plasma constituents in the semen of men with SCI compared with that of healthy, noninjured men (DeForge et al, 2005; Brown et al, 2006; Kafetsoulis et al, 2006). Despite the abnormal semen quality in men with SCI, successful pregnancies with their sperm have occurred by intravaginal insemination, intrauterine insemination (IUI), and numerous forms of advanced assisted reproductive technology (Ohl et al, 2001; Biering-Sorensen et al, 2005; Engin-Uml et al, 2006; Kafetsoulis et al, 2006). The literature is divided regarding the comparability of pregnancy success rates with male partners with SCI vs other male-factor etiologies, with some studies showing similar success rates (Brackett et al, 1995; Shieh et al, 2003) and others showing lower success rates with male partners with SCI (Schatte et al, 2000).

The sperm DNA fragmentation index (DFI), as measured by the sperm chromatin structure assay (SCSA), determines the level of sperm DNA integrity in a semen sample. Semen samples containing more than 30% sperm with fragmented DNA have been associated with reduced pregnancy rates (Evenson and Wixon, 2006a). Increased DFIs have been shown in infertile men with normal semen analyses (Saleh et al, 2002). The SCSA has been proposed as an adjunct in the infertility clinic to identify couples with poor fertility prospects (Bungum et al, 2004; Boe-Hansen et al, 2006; Erenpreiss et al, 2006; Evenson and Wixon, 2006a; Bungum et al, 2007).

Sperm DNA fragmentation has been measured in male-factor infertility of various etiologies. For example, the DFI in sperm of men with varicocele was significantly higher than the DFI in sperm of men without varicocele (Saleh et al, 2003; Werthman et al, 2007). The purpose of the present study was to measure DNA fragmentation in sperm from men with SCI to further characterize the semen quality in this patient population.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 Conclusion
 References
 
Subjects

Subjects were volunteers in the Male Fertility Research Program of the Miami Project to Cure Paralysis at the University of Miami Miller School of Medicine. Subjects with SCI were in good general health and had been injured at least 1 year before the start of the study. Control subjects were age-matched, non-SCI, healthy, normospermic men with no history of infertility. No SCI subject or control subject had taken any medications known to affect semen quality within 6 months of the start of the study.

Semen Collection

Semen specimens were obtained from SCI subjects by penile vibratory stimulation (PVS) or electroejaculation (EEJ). PVS was performed as previously described (Brackett, 1999) using the FERTI CARE personal vibrator (Multicept A/S, Albertslund, Denmark). EEJ was performed as previously described (Brackett et al, 2002) using the Seager model 14 EEJ unit (Dalzell USA Medical Systems, The Plains, Va). Control subjects produced specimens by masturbation following 3 to 5 days of abstinence from ejaculation.

Semen Analysis

Only antegrade fractions were used in this study; no retrograde fractions were included. Semen analysis was performed on all specimens according to World Health Organization (WHO) criteria (1999). Each specimen was first allowed to liquefy for 20 minutes at room temperature. Semen analysis was performed by placing 6 µL of semen onto a disposable Cell Vu semen analysis chamber (Millennium Sciences Inc, New York, NY). Sperm concentration was calculated as millions of sperm per mL of ejaculate. Total antegrade sperm count was calculated as (sperm concentration) x (total volume of antegrade ejaculate). Sperm motility was graded according to WHO methods: 1 = rapid linear, 2 = sluggish, 3 = nonprogessive, and 4 = immotile. The percent of motile sperm was calculated as the percentage of sperm with rapid linear plus sluggish motility.

DFI Determination

DFI was determined by the SCSA (SCSA Diagnostics, Brookings, SD), which was performed as previously described (Evenson et al, 2002). Technicians performing the SCSA were blinded to the treatment groups.

Experimental Design

     Experiment 1: DFI in SCI Subjects vs Control Subjects— The study consisted of three experiments. Experiment 1 compared the DFI in sperm of 10 SCI subjects and 12 control subjects. Table 1 shows the ages of control subjects and the ages, years postinjury, levels of injury, and methods of semen retrieval for SCI subjects. Semen specimens were obtained from each subject. Semen analysis was performed as described above. Semen specimens were then stored at –80°C until shipment in liquid nitrogen to SCSA Diagnostics for determination of DFI.


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  		Table 1. Demographic information for subjects in experiments 1 and 2
 

Group means for control subjects were compared with group means for SCI subjects using the t test. The following group means were compared: DFI, sperm concentration, sperm motility, leukocyte concentration, and age of subjects. DFI was correlated to sperm count, sperm motility, and leukocyte concentration using Pearson's correlation statistics. Values were considered significant at P < .05.

     Experiment 2: DFI in Repeated Ejaculations of SCI Subjects— Most men with SCI are anejaculatory, and it is possible that chronic anejaculation may contribute to DFI outcome. To examine this possibility, we compared semen specimens from 6 SCI subjects in experiment 1 (specimen 1) with semen specimens obtained from these same subjects in experiment 2 (specimen 2). Experiment 2 included subjects S5, S6, S7, S8, S9, and S10 (see Table 1 for ages, years postinjury, levels of injury, and methods of semen retrieval of these subjects). Specimen 1 was obtained from each subject after 2 to 4 weeks of anejaculation. Specimen 2 was obtained from each subject after 3 days of anejaculation. All specimens were stored at –80°C until ready for shipment in liquid nitrogen to SCSA Diagnostics for DFI determination.

     Experiment 3: DFI in Neat vs Processed Semen— Experiment 3 was performed to determine if necrospermia or leukocytospermia in men with SCI may contribute to their DFIs. Additionally, it is possible that semen processing can have an effect on DFI (Zini et al, 1999; Zini et al, 2000). Experiment 3 examined the effect of semen processing on DFI in SCI subjects and control subjects. For experiment 3, the DFI in unprocessed samples (with dead sperm and leukocytes) was compared with the DFI in processed samples (without dead sperm and leukocytes). The experiment was performed as follows.

Semen was obtained from 5 SCI subjects and 5 control subjects. Table 2 shows the ages of control subjects and the ages, years postinjury, levels of injury, and methods of semen retrieval for SCI subjects in experiment 3. Each semen specimen was divided into 2 aliquots. One aliquot received no treatment (neat semen), and the other aliquot was treated to remove dead sperm and leukocytes by processing the semen on an AllGrad 45/90 gradient (IVFonline LLC, Guelph, Canada) according to the manufacturer's instructions. Neat and processed aliquots were stored at –80°C until shipment in liquid nitrogen to SCSA Diagnostics for DFI determination.


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  		Table 2. Demographic information for subjects in experiment 3
 

Using a within-subjects design, the mean DFI in sperm from the neat semen of SCI subjects was compared to the mean DFI in sperm from the processed semen of those same subjects. Samples were similarly compared for control subjects. Comparisons were made by paired t tests.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 Conclusion
 References
 
Experiment 1: DFI in SCI Subjects vs Control Subjects

The mean DFI was significantly higher in SCI subjects compared with control subjects (Figure 1). Specifically, the mean (± SEM) DFI was fourfold higher in the SCI group (65.2% ± 6.6%) compared with the control group (15.4% ± 2.9%). There was no overlap in DFI between SCI and control subjects. For example, the lowest DFI for SCI subjects (range, 42.3%–90.8%) was higher than the highest DFI of control subjects (range, 5.4%–33.5%).


Figure 1
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  		Figure 1. The mean percent DFI in the semen of SCI subjects (65.2% ± 6.6%) was significantly higher than the mean percent DFI of control subjects (15.4% ± 2.9%; P < .001, t test).

 
There was a statistically significant inverse correlation between DFI and sperm motility in SCI subjects but not in control subjects (Figure 2). DFI was not correlated to leukocyte concentration or sperm count in SCI or control subjects (Figure 2).


Figure 2
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  		Figure 2. There was a statistically significant inverse correlation between percent DFI and sperm motility in SCI subjects but not in control subjects. Percent DFI was not correlated with leukocyte concentration or sperm count in SCI subjects or control subjects.

 
Similar to previous reports, the semen of SCI subjects had similar sperm concentrations, lower sperm motilities, and higher leukocyte concentrations than the semen of control subjects (Table 3).


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  		Table 3. Semen parameters of SCI subjects and control subjects in experiment 1*
 

Experiment 2: DFI in Repeated Ejaculations of SCI Subjects

Although the DFI in specimen 2 was slightly lower than the DFI in specimen 1 in all subjects in experiment 2, there was no statistically significant difference between the mean DFI of the 2 specimens (Table 4). The pairs were highly correlated (rs = .94; P < .02, Spearman's correlation), indicating a high degree of similarity between the DFIs obtained in specimen 1 and those obtained in specimen 2.


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  		Table 4. Percent DFI in specimen 1 vs specimen 2 of SCI subjects in experiment 2*
 

Experiment 3: DFI in Neat vs Processed Semen

When neat semen was compared with semen that had been processed to remove dead sperm and leukocytes, the results showed no significant difference between the mean DFI of neat vs processed specimens of SCI subjects: 79.3% ± 9.9% vs 75.2% ± 16.1% (Figure 3). Interestingly, the mean DFI was significantly higher in neat vs processed specimens of control subjects: 9.9% ± 1.3% vs 2.3% ± 1.4% (P < .02; Figure 3).


Figure 3
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  		Figure 3. There was no significant difference between mean percent DFI of neat (79.3% ± 9.9%) vs processed semen specimens of SCI subjects (75.2% ± 16.1%), but the mean percent DFI was significantly higher in neat (9.9% ± 1.3%) vs processed (2.3% ± 1.4%) specimens of control subjects.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
Conclusion
 References
 
The majority of SCI occur to young men in their parenting years (National Spinal Cord Injury Statistical Center, 2006). Most men with SCI are infertile due to a combination of erectile dysfunction, ejaculatory dysfunction, and semen abnormalities (Brown et al, 2006). The semen of men with SCI typically has normal sperm concentrations but low sperm motility and low sperm viability. Abnormal seminal plasma constituents have been proposed to contribute to this condition (Brackett et al, 1996; Brackett et al, 2000; Basu et al, 2004). In an effort to further characterize the semen quality of men with SCI, the present study examined the DFI in their semen by means of the SCSA.

The SCSA has been proposed as an objective measure of sperm viability and as a useful adjunct in the infertility clinic (Bungum et al, 2004; Boe-Hansen et al, 2006; Bungum et al, 2006; Erenpreiss et al, 2006). Conventional semen analysis falls short in discriminating infertile from fertile men (Zini and Libman, 2006) because it can be subject to significant interobserver variability and can fluctuate based on days of abstinence, illness, and method of collection (Tomlinson et al, 1999). These concerns may be especially true for men with SCI, most of whom are unable to ejaculate and must use assisted techniques such as PVS and EEJ. With the advances in assisted reproductive technologies, an increasing number of couples with male partners with SCI are seeking assistance to become biologic parents.

Our study found that the mean DFI was significantly higher in SCI subjects compared with healthy non-SCI control subjects. The cause of increased sperm DNA damage in men with SCI is not clear. In our study, repeated ejaculation in 6 SCI subjects yielded similar DFI levels, making chronic anejaculation unlikely to be the cause. Another possible explanation for the higher DFIs in men with SCI is the high concentrations of dead sperm and leukocytes typically found in their ejaculates. Necrospermia (ie, the term for high concentrations of dead sperm in the semen) may contribute to elevated DFI (Guerin et al, 2005). Similarly, leukocytospermia, the term for elevated concentrations of leukocytes in the semen, may contribute to elevated DFI (Alvarez et al, 2002). Most men with SCI have necrospermia (Brackett et al, 1998) and leukocytospermia (Aird et al, 1999; Basu et al, 2002; Trabulsi et al, 2002). Our study showed similar DFIs in neat vs processed semen of SCI subjects, indicating that necrospermia or leukocytospermia are unlikely to be the immediate cause; however, it is possible that toxic substances released by degenerating sperm cells or leukocytes may have exerted negative effects on sperm DNA prior to semen processing. Whereas sperm processing did not elevate percent DFI in SCI subjects, the data showed a significantly decreased percent DFI in control subjects, which is in agreement with Larson et al (1999, 2000).

Several hypotheses have been formulated to account for the elevated DNA fragmentation in non-SCI male-factor infertility patients. An increased sperm histone: protamine ratio and an excess of nuclear histones result in poorer chromatin compaction and a subsequent increased susceptibility to external stress (Barone et al, 1994; Agarwal and Said, 2003; Erenpreiss et al, 2006; Oliva, 2006; Zhang et al, 2006; Zini and Libman, 2006). Others have speculated that increased DNA damage results from sperm that have experienced abortive apoptosis during spermatogenesis (Sakkas et al, 2003). Multiple studies have also suggested that DNA damage can be induced by oxidative stress (Sawyer et al, 2003; Lewis and Aitken, 2005; Aitken and Baker, 2006).

Elevated levels of cytokines and free radicals have been found in the seminal plasma of men with SCI (Padron et al, 1997; Basu et al, 2004). The seminal plasma of men with SCI was found to be toxic to normal sperm. For example, when seminal plasma of men with SCI was mixed with sperm from non-SCI normospermic men, a rapid and profound impairment to normal sperm motility occurred (Brackett et al, 1996). Furthermore, sperm unexposed to the seminal plasma (ie, aspirated from the vas deferens) had significantly higher motility than sperm in the ejaculate of men with SCI (Brackett et al, 2000).

Studies have shown an association between DFI and pregnancy outcomes. For example, it has been reported that if a man has a DFI of greater than 30%, IUI should probably not be considered and the couple should move to routine in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) (Evenson and Wixon, 2006b). Another study of 637 couples undergoing 998 cycles found that the group with DFIs greater than 30% had significantly better results with ICSI than with IVF (Bungum et al, 2006). Increased DNA damage has also been seen in infertile patients with normal semen parameters (Saleh et al, 2002).

Our study was not designed to examine DFIs in male partners with SCI who achieved pregnancy vs those who did not achieve pregnancy in their female partners. We therefore cannot make predictions about pregnancy outcomes with sperm from male partners with SCI. There is abundant literature reporting live births from couples with male partners with SCI by the methods of intravaginal insemination, IUI, and numerous forms of advanced assisted reproductive technology (Ohl et al, 2001; Biering-Sorensen et al, 2005; Engin-Uml et al, 2006; Kafetsoulis et al, 2006). Of the men in our study, only 1 patient (patient S9) attempted to achieve pregnancy with his wife. The couple failed 1 IUI cycle and 2 ICSI cycles. Pregnancy, culminating in a live birth of twins, was achieved on the third cycle of ICSI. SCSA studies suggest that a DFI greater than 30% is the threshold that places a man at a statistical risk for a longer time to pregnancy and increased IVF cycles.

Based on the results of our study and the literature to date, it is impossible to know for sure if men with SCI with DFIs above 30% can achieve pregnancy resulting in live birth, but it seems that this possibility exists.


   Conclusion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 Conclusion
References
 
Semen of men with SCI had a significantly higher percent of sperm with DNA damage than semen of non-SCI control subjects. Elevated DFI was apparently not attributable to prolonged anejaculation or to the proximate conditions of necrospermia or leukocytospermia. The cause of elevated DFI in sperm of men with SCI is unknown. The relevance of these findings to fertility outcomes using sperm from SCI male partners remains to be determined.


   Acknowledgments
 
The authors thank SCSA Diagnostics and Don Evenson, PhD, HCLD, for technical advice and for performing the SCSA.


   Footnotes
 
Support was provided by the State of Florida Department of Health and the Miami Project to Cure Paralysis.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
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