Clinical Impact Of Sperm DNA Damage Biology Essay

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Tests of sperm DNA and chromatin integrity have been used increasingly in the evaluation of the infertile man with the hypothesis that these tests may better diagnose the infertility and predict reproductive outcomes. This paper discusses the etiology of sperm DNA damage, briefly reviews the tests of sperm DNA damage, and, evaluates the relationship between sperm DNA damage and reproductive outcomes. A systematic review of the literature allows us to conclude that sperm DNA damage is associated with lower natural, IUI and IVF pregnancy rates. Studies to date have not shown a clear association between sperm DNA and chromatin defects and pregnancy outcomes after ICSI; however, we cannot exclude the possibility that very high levels of DNA damage will impact on ICSI outcomes. In couples undergoing IVF or ICSI, there is evidence to show that sperm DNA damage is associated with an increased risk of pregnancy loss. A limitation of this systematic review and meta-analysis is that it does not address an important feature of the clinical studies on sperm DNA damage, the heterogeneity of the individual study characteristics. Although the clinical utility of tests of sperm DNA damage remains to be firmly established, the data suggest that there is clinical value in testing couples prior to assisted reproductive technologies - ARTs (IUI, IVF and ICSI) and in those couples with recurrent abortions. Additional, well-designed prospective studies are needed before testing becomes a routine part of patient care.

INTRODUCTION

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Standard semen parameters exhibit a high degree of biological variability, are only fair measures of fertility potential and poor predictors of reproductive outcomes (Guzick et al., 2001). As such, there is a need to develop new markers that might better discriminate infertile from fertile men, and help predict pregnancy outcome and adverse reproductive events. Tests of sperm chromatin and DNA integrity can help in the diagnosis of male infertility (Evenson et al., 1999, Kodama et al., 1997, Spano et al., 2000, Zini et al., 2001). Moreover, it has been reported that tests of sperm chromatin and DNA damage may help predict reproductive outcomes after assisted reproductive technologies (ARTs) but the value of these tests in the clinic remains to be more fully defined (Bungum et al., 2007).

ARTs have revolutionized the treatment of male infertility. With in vitro fertilization (IVF) with intra-cytoplasmic sperm injection (ICSI), men with severe oligozoospermia, obstructive and non-obstructive azoospermia can hope to father children. However, there are some questions regarding the safety of ARTs remains. These safety concerns are relevant because (1) ARTs can bypass the barriers to natural selection, (2) infertile men with severe male-factor possess substantially more sperm DNA damage than do fertile men, (3) pregnancy is possible regardless of the degree of sperm DNA damage and (4) experimentally, fetal and post-natal development is related to the degree of sperm DNA damage (Evenson et al., 1999, Fernandez-Gonzalez et al., 2008, Gandini et al., 2004, Kodama et al., 1997, Spano et al., 2000, Zini et al., 2001).

ETIOLOGY OF SPERM DNA DAMAGE

The etiology of sperm DNA damage is multi-factorial. Clinically, several conditions have been associated with sperm DNA and chromatin damage (e.g. chemotherapy, smoking, genital tract infection, varicocele) (Banks et al., 2005, Bungum et al., 2007, Erenpreiss et al., 2002, Fossa et al., 1997, O'Flaherty et al., 2008, Potts et al., 1999, Sailer et al., 1997, Saleh et al., 2003, Zini & Sigman, 2009). Broadly, these conditions can be categorized as primary or intrinsic defects in spermatogenesis (e.g. genetic or developmental abnormalities) and secondary or extrinsic noxious factors (e.g. gonadotoxins, hyperthermia, oxidants, endocrine disruption).

At the cellular level, a number of theories have been proposed to explain the DNA damage in human spermatozoa. Studies have suggested that protamine deficiency (with aberrant chromatin remodeling), reactive oxygen species (ROS), abortive apoptosis and alterations in topoisomerase II activity may be responsible for sperm DNA damage (Aitken & De Iuliis, 2007, Leduc et al., 2008, Sakkas et al., 2003, Aoki et al., 2006, Aoki et al., 2005, Cho et al., 2001, Tarozzi et al., 2007). Recently, De Iuliis et al, have proposed a two-step hypothesis to explain the generation of sperm DNA damage. Based on the model, oxidative stress acts on poorly protaminated cells (i.e. cells with incomplete replacement of histones by protamines) generated as a result of defective spermiogenesis (De Iuliis et al., 2009).

TESTS OF SPERM DNA DAMAGE

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Several tests of sperm DNA and chromatin damage have been described (Chohan et al., 2006, Evenson et al., 1999, Zini & Sigman, 2009). These tests have been developed in the hope that they may (1) help in the diagnosis of male infertility, (2) predict reproductive outcomes in the context of assisted reproductive technologies (ARTs) and (3) provide some assurance regarding the integrity of the male gamete genome. Several factors must be considered when evaluating studies of sperm DNA and chromatin integrity. Firstly, the different assays measure different aspects of sperm DNA and chromatin (e.g. DNA fragmentation, level of protamination, DNA denaturation). Secondly, the assay conditions can greatly influence the accessibility of the dye or enzyme to the sites of damaged DNA and, therefore, impact on the final results (e.g. the concentration of reducing agents, such as, dithiotreitol [DTT] can alter the degree of sperm nuclear decondensation). Thirdly, current assays are limited because they do not selectively differentiate clinically important DNA fragmentation (e.g. degree or gene-specificity) from clinically insignificant damage (i.e. a normal threshold of DNA damage in a given cell has not been firmly established). Finally, sample preparation and handling prior to sperm DNA and chromatin integrity testing can impact on the final test results (e.g. centrifugation, prolonged incubation).

The COMET (single cell gel electrophoresis) and TUNEL (Terminal deoxynucleotidyl transferase-mediated dUTP Nick End-Labeling) assays are commonly utilized assays that detect DNA strand breaks directly. Some assays measure the susceptibility of DNA to denaturation â€" that is the formation of single stranded DNA from native double stranded DNA (e.g. SCSA-sperm chromatin structure assay) and depend on the premise that nicked DNA will denature more readily than intact DNA. Other assays rely on the differential binding of dyes or agents to single stranded and (Chohan et al., 2006) double stranded DNA (e.g. acridine orange) or to protamine-deficient sites (e.g. aniline blue or CMA3 test). Remarkably, the results of most sperm DNA or chromatin integrity assays correlate highly with each other â€" with the exception of the microscopic acridine orange test (Chohan et al, 2006). In order to provide clinically relevant information, an upper normal level (cutoff) of the percentage of cells with DNA fragmentation or chromatin defect has been set in most published studies although the method of defining the cutoff varies greatly (e.g. receiver operating characteristic curves, fertile populations, population mean). Samples with test results above the threshold or cutoff value are considered to have high levels of DNA damage (Zini & Sigman, 2009).

RELATIONSHIP BETWEEN SPERM DNA DAMAGE AND PREGNANCY

In order to assess the relationship between sperm chromatin/DNA damage and pregnancy outcomes a systematic review and meta-analysis of valid published studies was conducted. The strength of systematic reviews (with meta-analysis) is the improved precision of the summary estimates compared with the individual study estimates of the relationship between sperm DNA/chromatin defects and pregnancy outcomes. On the other hand, a weakness of meta-analyses (particularly on this topic) is the fact that it combines studies with highly variable study characteristics: data collection (prospective or retrospective), population characteristics (unselected, male factor), female inclusion/exclusion criteria, laboratory expertise in assessment of sperm DNA/chromatin damage, sperm DNA/chromatin test type and sperm DNA test cutoff.

Natural pregnancy and intra-uterine insemination (IUI) studies

Although small in number, the available studies suggest that sperm DNA and chromatin damage is related to lower rates of natural and IUI pregnancies. Studies have shown that sperm DNA damage is associated a prolonged time to pregnancy and a very low probability of achieving a natural pregnancy (Evenson et al., 1999, Giwercman et al., 2009, Loft et al., 2003, Spano et al., 2000). A systematic review and meta-analysis of published natural pregnancy studies demonstrates an important association between sperm DNA damage and failure to achieve a natural pregnancy (odds ratio - OR = 7.01, 95% CI 3.68, 13.36, P<0.001, Table 1). Sperm DNA damage has also been associated with lower IUI pregnancy rates (Bungum et al., 2007, Duran et al., 2002, Muriel et al., 2006). In the Bungum et al, 2007 study (the only evaluable IUI study because a 2x2 table cannot be constructed from the data in the Duran et al, 2002 and Muriel et al, 2006 studies) the diagnostic odds ratio (OR) = 9.9 (95% CI 2.37, 41.51, P<0.001) which demonstrates that sperm DNA damage is related to a significantly lower pregnancy rate at IUI.

In vitro fertilization (IVF) studies

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Over the past 10 years, more than twenty studies (11 evaluable) have examined the relationship between sperm DNA integrity and pregnancy rates after standard IVF. It is important to note that these studies are quite heterogeneous in terms of their design (prospective/retrospective), inclusion/exclusion criteria (e.g. female age, female infertility factors) and types of sperm DNA test employed (SCSA, TUNEL). With this limitation in mind, a systematic review and meta-analysis of evaluable IVF studies shows that sperm DNA damage is associated with lower IVF pregnancy rates; the combined OR is 1.70 (95% CI 1.30, 2.23, p<0.05) (Benchaib et al., 2007, Boe-Hansen et al., 2006, Borini et al., 2006, Bungum et al., 2007, Filatov et al., 1999, Frydman et al., 2008, Henkel et al., 2003, Host et al., 2000, Huang et al., 2005, Lin et al., 2008, Tarozzi et al., 2009). The characteristics of the 11 evaluable IVF studies included in the analysis are shown in Tables 2 and 3. Eleven otherwise valid studies must be excluded from the meta-analysis because (1) they include a mixed population (IVF and IVF/ICSI â€" see below) (Larson-Cook et al., 2003, Payne et al., 2005, Seli et al., 2004, Velez de la Calle et al., 2008, Virro et al., 2004) or (2) a 2x2 table could not be constructed (because a cutoff DNA damage level was not reported) (Bakos et al., 2008, Gu et al., 2009, Meseguer et al., 2008, Morris et al., 2002, Tomlinson et al., 2001, Tomsu et al., 2002). The results of this updated meta-analysis on IVF studies suggest that sperm DNA and chromatin damage has a modest influence on pregnancy rates at IVF and are in keeping with the results of a smaller meta-analysis (based on fewer studies) (Collins et al., 2008).

In vitro fertilization with intracytoplasmic sperm injection (IVF/ICSI) studies

Over 20 studies (14 evaluable) have evaluated the relationship between sperm DNA integrity and pregnancy rates after IVF/ICSI. As with IVF studies, these ICSI studies are quite heterogeneous in terms of design, inclusion/exclusion criteria and types of sperm DNA test employed. In keeping with a recent analysis (Collins et al., 2008), the results of this updated meta-analysis on ICSI studies indicate that sperm DNA damage is not related to ICSI pregnancy rates (combined OR of 1.15, 95% 0.90, 1.55, p=0.65) (Benchaib et al., 2007, Borini et al., 2006, Bungum et al., 2007, Check et al., 2005, Gandini et al., 2004, Hammadeh et al., 1996, Henkel et al., 2003, Host et al., 2000, Huang et al., 2005, Lin et al., 2008, Micinski et al., 2009, Tarozzi et al., 2009, Zini et al., 2005, Boe-Hansen et al., 2006). The characteristics of the 14 evaluable ICSI studies are shown in Tables 4 and 5. Nine otherwise valid studies were removed from the meta-analysis because (1) they included a mixed population (IVF and IVF/ICSI) (Larson-Cook et al., 2003, Payne et al., 2005, Seli et al., 2004, Velez de la Calle et al., 2008, Virro et al., 2004), (2) a 2x2 table could not be constructed (Bakos et al., 2008, Morris et al., 2002, Muriel et al., 2006) or (3) the assay type utilized in the study is not widely recognized (Virant-Klun et al., 2002). Although it may be surprising that studies indicate that sperm DNA and chromatin damage is not related to ICSI pregnancy, it is speculated that the careful selection of sperm and embryo during human ICSI mitigates the potential adverse effect(s) of sperm DNA damage on reproductive outcomes (Gandini et al., 2004). However, we cannot exclude the possibility that very high levels of DNA damage will impact on pregnancy outcome. Perhaps the most concerning aspect of these findings is the unknown long-term consequence (i.e. post-natal health) of a successful pregnancy with very high levels of DNA damage.

Mixed or combined IVF and IVF/ICSI studies

Seven mixed IVF and IVF-ICSI studies (5 evaluable) have assessed the relationship between sperm DNA integrity and pregnancy. Again, these combined studies are quite heterogeneous in terms of design, inclusion/exclusion criteria and types of sperm DNA test employed. A systematic review and meta-analysis of the 5 mixed IVF and IVF-ICSI studies shows that sperm DNA damage is associated with pregnancy rates in this context (combined OR of 1.63, 95% 1.03, 2.59, p<0.05) (Larson-Cook et al., 2003, Meseguer et al., 2008, Payne et al., 2005, Seli et al., 2004, Virro et al., 2004). Two otherwise valid studies were removed from the meta-analysis because a 2x2 table could not be constructed (Tavalaee et al., 2009, Velez de la Calle et al., 2008). The results of this meta-analysis on mixed IVF and IVF-ICSI studies are in keeping with the meta-analysis of IVF studies (see above) and support the premise that sperm DNA damage has a measurable but modest impact on pregnancy rates at IVF.

Pregnancy loss after IVF and IVF-ICSI

Eleven (11) studies have reported on sperm DNA damage and its association with pregnancy loss after IVF and IVF/ICSI (see Table 4). These pregnancy loss studies are quite heterogeneous in terms of design, inclusion/exclusion criteria and types of sperm DNA test employed. A meta-analysis of the evaluable studies demonstrates a combined OR of 2.48 (95% CI; 1.52, 4.04, p<0.0001) indicating an important association between sperm DNA damage and the rate of pregnancy loss after IVF and ICSI (Zini et al., 2008). The characteristics of the 11 evaluable studies (from 7 reports) are shown in Table 6. There was no difference in the OR according to the type of ART (IVF or ICSI). These data provide an additional mechanism or cause for pregnancy loss after IVF and IVF/ICSI. It may be speculated that the pregnancy loss (at IVF or ICSI) is a result of impaired embryo/blastocyst development associated with sperm DNA damage (Seli et al., 2004, Zini et al., 2005).

CLINICAL VALUE OF TESTS OF SPERM DNA AND CHROMATIN DAMAGE

Four common clinical scenarios are highlighted so as to illustrate the clinical value of sperm DNA and chromatin integrity tests. The recommendations for sperm DNA testing are based on (1) a systematic review and meta-analysis of the relevant studies, (2) the characteristics of sperm DNA testing (e.g. sensitivity, positivity rate) and (3) on disease prevalence (e.g. pregnancy, pregnancy loss).

1. Screening test for first pregnancy planners

The data from 3 studies show that sperm DNA damage is associated with a significantly reduced natural pregnancy rate (combined OR 7.01, 95% CI 3.68, 13.36, p<0.0001). Remarkably, the 3 studies reported very similar associations between sperm DNA damage and natural pregnancy rate (with ORs of 6.54, 6.82 and 7.59). An analysis of the 3 studies reveals a median pregnancy rate of 53%, with a median positive predictive value (PPV) of 83% and a median negative predictive (NPV) of 58% associated with sperm DNA testing (Evenson et al., 1999, Giwercman et al., 2009, Spano et al., 2000). As such, the analysis predicts that in populations with an overall pregnancy rate of 53% (at 6 to 12 months of follow-up), the pregnancy rate is 17% when there is a positive test for sperm DNA damage and at 58% when the test result is normal. Therefore, testing for sperm DNA damage can discriminate between pregnancy rates of 17% and 58%. However, because the prevalence of a positive test in this context (first pregnancy planners) is low (<10%) and 17% of couples with a positive test will achieve a pregnancy, indiscriminate sperm DNA testing in this context is not advocated. Clinicians may want to test first pregnancy planners but clinicians should also understand the predictive value and limitations (e.g. sensitivity, specificity) of the sperm DNA test in this context and discuss these issues with the patients.

2. Couples with mild male-factor infertility - IUI candidates

Data from one valid IUI study shows that sperm DNA damage is related to a significantly reduced IUI pregnancy rate (OR 9.9, 95% CI, 2.37, 41.51, p<0.0001). In the Bungum et al, (2007) study, the overall IUI pregnancy rate is 20%, the PPV is 97% and NPV is 24% (Bungum et al., 2007). Therefore, in populations with an IUI pregnancy rate of 20%, a positive test for sperm DNA damage predicts the pregnancy rate to be 3% and a normal test result predicts the pregnancy rate to be 24%. Therefore, testing for sperm DNA damage prior to IUI can differentiate between pregnancy rates of 3% and 24%. According to the Bungum et al, study, couples with high levels of sperm DNA damage should proceed to IVF and/or ICSI rather than IUI. However, it is important to note that the sensitivity and prevalence of a positive test in this context (couples with mild male-factor infertility) are low (<20%) and these recommendations are derived from only one reliable study (Bungum et al., 2007). As such, additional IUI studies are needed before routine testing is recommended prior to initiating IUI treatments.

3. Couples with severe male-factor infertility - IVF or ICSI candidates

Data from more than 20 studies (11 evaluable) demonstrate that sperm DNA damage is associated with a modest but significant reduction in the IVF pregnancy rate (combined OR of 1.70, 95% CI 1.30, 2.23, p<0.05). Further analysis of the 11 evaluable IVF studies (with a median pregnancy rate of 33%) reveals a median PPV of 77% and median NPV of 34% (see Table 3). In clinical terms, this means that in populations with an overall IVF pregnancy rate of 33%, a positive test for sperm DNA damage predicts the IVF pregnancy rate to be 23% and if the test is negative, 34%. As such, couples with sperm DNA damage may choose to proceed to ICSI, where pregnancy rates are independent of test results (combined OR of 1.15, 95% 0.90, 1.55, p=0.65, see Table 4). However, the clinical value of an 11% difference in pregnancy rates (34% vs. 23%) is modest and it may be hard to justify routine testing in this setting. However, clinicians may want to test select couples (e.g. with failed IVF) so as to better counsel these couples in future ART cycles.

Testing couples with severe male-factor infertility may also be valuable because sperm DNA damage is associated with a significantly higher rate of pregnancy loss after IVF or ICSI (combined OR of 2.48, 95% CI; 1.52, 4.04, p<0.0001). Data derived form these studies (PPV and NPV) indicate that in populations with an overall rate of pregnancy loss of 18%, the rate of pregnancy loss is estimated at 37% when the test is positive and 10% when it is negative. The difference between a pregnancy loss rate of 37% and 10% may be valuable to patients and clinicians. Although the effect of DNA damage on pregnancy loss should be discussed with patients prior to undergoing ART, many couples will proceed with these treatments regardless of sperm DNA test results and the impact on pregnancy loss.

4. Couples with pregnancy loss after IVF or IVF/ICSI

The prevalence of a positive test, sensitivity and specificity of sperm DNA testing in the context of pregnancy loss after IVF and ICSI are and 25%, 40% and 85%, respectively. This indicates that sperm DNA damage is a minor cause of pregnancy loss after IVF and ICSI (based on the low prevalence and low sensitivity). However, if the test is positive, it suggests that the sperm DNA damage (or male-factor) may be the cause of the pregnancy loss (based on the high specificity). In this setting it may be advisable to evaluate or re-evaluate the male and correct any potential male factor (e.g. varicocele) that may contribute to the DNA damage.

SUMMARY

Tests of sperm DNA and chromatin integrity are being used in the evaluation of the infertile man with the hypothesis that these tests may better diagnose the infertility and predict reproductive outcomes. To date, the clinical studies on sperm DNA and chromatin defects allow us to conclude that sperm DNA damage is associated with lower natural, IUI and IVF pregnancy rates, but not with ICSI pregnancy rates. Moreover, sperm DNA damage is associated with an increased risk of pregnancy loss in those couples undergoing IVF or ICSI. Although the clinical utility of tests of sperm DNA/chromatin damage remains to be firmly established, the data suggest that there is clinical value in testing couples with recurrent abortions or prior to initiating ART cycles.