DNA Integrity Tests in Clinical Practice: It Is Not a Simple Matter of Black and White (or Red and Green)

Transcription

1 Journal of Andrology, Vol. 27, No. 3, May/June 2006 Copyright E American Society of Andrology DNA Integrity Tests in Clinical Practice: It Is Not a Simple Matter of Black and White (or Red and Green) Androlog Summary ANTOINE A. MAKHLOUF AND CRAIG NIEDERBERGER From the Division of Andrology, University of Illinois, Chicago, Illinois. Note: Postings to Androlog have been lightly edited before publication. In vitro fertilization (IVF) presents a significant physical, emotional, and financial commitment on the part of the prospective parents. Couples often cling to hopes of successful IVF even when presented with adverse odds. Thus, any information that increases the accuracy of the estimate of success is of paramount importance. This is illustrated by a question from Mark Jutras, titled Extremely Abnormal SCSA : Have a guy seen for oligoasthenoteratozoospermia. He was found to be hypothalamic and was treated with hcg [human choriogonadotropin] 1500 SQ 3 times a week with normalization of T. Several months later there was some improvement in count but no normal sperm on any sample and all samples showing 50% 60% with 2 tails. This was also true before treatment with hcg. Specimen sent for SCSA [sperm chromatin structure assay] with call from [Donald] Evenson. Almost no signal in the normal range. Cannot even assign numbers to the value. His chromosomes were normal. They want to do IVF but in the current situation we have to tell them that we don t expect a pregnancy. Best plan I can come up with is since a repeat SCSA is indicated anyway, continue hcg and start Fertile One and recheck SCSA after 3 months of Fertile One. Will prep as for IVF/ICSI [intracytoplasmic sperm injection] and do the SCSA on that sample. My tech is skeptical that she can get anything through a column with current samples. If no go at that time thought I would send him to Cornell Urology for second opinion. Anyone else want him? Ideas and thoughts welcomed. Peter Schlegel was not surprised that the SCSA was abnormal given the gross morphological problems: The 2-tailed sperm are usually diploid. Sperm chromosome analysis (FISH [fluorescence in situ hybridization]) may be worthwhile to test this possibility before IVF/ DOI: /jandrol ICSI. Sounds like a failure of spermatid separation no surprise that DNA integrity is so poor. Jutras agreed with Schlegel s assessment and brought up an interesting anecdote on another patient with an abnormal SCSA: Brings up another interesting case we had. Couple with recurrent spontaneous abortion. Standard evaluation including genetics on both were normal. SCSA showed 2 distinct populations. About 50% of sperm had normal content of DNA and 50% were diploid. Best solution would have been IVF with PGD [preimplantation genetic diagnosis]. Since couple had one normal daughter they decided best solution was vasectomy. The reliance on SCSA results to predict clinical outcomes was questioned by this author (Craig Niederberger), who brought up the recent study by Payne et al (2005) on the predictive value of SCSA in assisted reproductive technology (ART): Regarding the patient with an abnormal SCSA: it s important to keep in mind that the original reported outcomes of this test have not yet been clinically verified by independent investigators. Payne and coauthors recently reported in an independent study that SCSA outcomes failed to identify a DFI [DNA fragmentation index] threshold above which pregnancy outcomes were negative after IVF or IVF/ICSI. In fact, almost half of the 19 couples with a DFI greater than 27% had clinical pregnancies. Interestingly, patients with low DFI (defined as less than 9%), what one would expect to have the highest quality sperm by this assay, were the least likely to become pregnant. Peter Schlegel concurred that more studies are required to further define the clinical relevance of DNA integrity testing: I agree with Dr Niederberger that additional information on SCSA and its relationship to pregnancy outcome is needed. Many studies have been done on this subject with highly variable findings, but essentially none that mirror the predictive value of SCSA for natural or IVF-ICSI cycles suggested by Evenson et al and Larson et al [Larson et al, 2000; Larson-Cook et al, 2003] in their original publications. Dr Schlegel then went on to give a nuanced view of the value of DNA integrity tests. While citing studies showing an association of paternal DNA damage with poor embryo development and recurrent miscarriage, he 316

2 Makhlouf and Niederberger N DNA Integrity Tests 317 cautioned against using absolute SCSA cutoff points to predict pregnancy outcomes: Unfortunately, the study design of some of the additional reports has been poor. Payne et al [2005] essentially tested the hypothesis that DFI was the only variable affecting IVF outcome. No one would expect this to be true and, ignoring other known factors that affect IVF outcome (eg, female age), may well have explained the apparently contradictory results of this study. So, we are left with a field that has conflicting data. I do believe SCSA (and other tests of DNA integrity) have some value in identifying male factors involved with fertility. For example, patients with recurrent miscarriage have a substantive increase in DNA breaks than couples undergoing IVF for other indications or fertile donors (Carrell et al, 2003). In addition, the observation of increased DNA breaks in sperm from couples with poor embryo development suggests a possible role of this test in identifying a male factor (Tesarik et al, 2004). Interestingly, DNA breaks (abnormal SCSA results) do not appear to predict fertilization failure. At present, I see tests like DNA integrity evaluations (TUNEL [terminal uridine nick end labeling], SCSA, Comet) as well as sperm aneuploidy testing as allowing us new insight into male factor contributions to infertility that we ve never considered before. However, as Dr Niederberger appropriately points out, there is no absolute cut-point for SCSA (as originally suggested) that will preclude success for natural pregnancy nor IVF/ICSI. On the other hand, I expect we will find that the man with 70% DFI (DNA fragmentation index) has a lower chance of contributing to a pregnancy than other couples in the same situation. Certainly we (and many other centers) have reported pregnancies with sperm obtained from samples with high DFI levels. However, having fragmented and unstable DNA cannot be good for sperm. Confounding factors that complicate the interpretation of DNA integrity testing were neatly summarized and discussed by Juan Alvarez. He first pointed out that different tests measure distinct aspects of DNA: I recently published a letter to the editor in Human Reproduction in which I address the issue of the predictive value of the SCSA test in ART (Alvarez, 2005). First, I point out that we have to differentiate between tests that measure real DNA fragmentation vs tests that measure susceptibility to DNA denaturation. In the former group we have tests like TUNEL, in situ nick translation and COMET under neutral ph conditions. In the latter group we have tests like the SCSA, COMET under alkaline or acidic conditions, SCD [sperm chromatin dispersion], chromomycin, etc. Dr Alvarez also discussed the confounder of heterogeneity in the sperm population. He questioned whether the abnormal sperm as detected by SCSA are a distinct subpopulation of otherwise healthy sperm or whether they are the worst representatives of a globally damaged sperm population (ie, the tip of the iceberg ; Evenson et al, 1999, 2002): Another factor that should be considered is whether DNA damage affects the whole sperm population in a given semen sample or only a fraction of it. For example, DNA fragmentation induced by apoptosis usually affects a fraction of the spermatozoa whereas DNA fragmentation induced by oxidative stress and radiation therapy leads to double-stranded DNA fragmentation and nucleotide damage of the 8-OH-2- deoxyguanosine (oxo8dg) type in most spermatozoa. DNA fragmentation induced by the hydroxyl radical results in the formation of oxo8dg in a first stage followed by double-stranded DNA fragmentation thereafter (Cui et al, 2000). While DNA damage of the first type could be repaired to some extent by the oocyte, double-stranded DNA damage is virtually irreversible and incompatible with the development of a viable pregnancy. Since DNA fragmentation values in ejaculated spermatozoa above 10%, as assessed by TUNEL (Benchaib et al, 2003), or above 30%, as assessed by the SCSA test (Evenson et al, 1999), are associated with low pregnancy rates, one would think that the remaining 90% or 70% of the spermatozoa, respectively, could fertilize the egg and result in a viable pregnancy. However, in addition to double-stranded breaks, a significant proportion of these spermatozoa could have DNA nucleotide modifications of the oxo8dg type. Therefore, the probability that a spermatozoon with normal DNA would fertilize the egg would be much lower than that expected from a DNA fragmentation value of 10% or 30%, respectively. That is, in addition to the measurable 10% and 30% of spermatozoa with DNA fragmentation, the remaining 90% and 70% of spermatozoa would have some type of DNA damage that is not compatible with the development of a viable pregnancy. This concept has been designated as the iceberg effect (Evenson et al 1999). Another confounder is that not all DNA damage is lethal. Dr Alvarez reminded us that the majority of DNA is noncoding and that the oocyte is capable of DNA repair. Another factor is whether DNA damage affects exons vs introns. Since more than 90% of DNA is comprised of non protein-coding regions or introns, DNA damage is most likely to affect these noncoding regions of DNA and, therefore, may not affect embryo development. This could explain, at least in part, why relatively high levels of sperm DNA fragmentation can result in viable pregnancies. Another important factor is the capability of the oocyte to repair sperm DNA damage. DNA damage in the fertilizing spermatozoon may be repaired by the oocyte. This is most likely to occur in those cases where female age is,35 years. Dr Alvarez then concluded that these confounding factors place inherent limitations on the predictive value of DNA integrity tests. He proposed combining DNA integrity tests with measures of reactive oxygen species damage to get a clearer picture of sperm DNA quality: Therefore, based on the above, it can be concluded that the predictive value of sperm DNA fragmentation tests

3 318 Journal of Andrology N May ÙJune 2006 will always have DNA region and oocyte-derived uncertainty factors and, thus, cannot have a 100% negative predictive value, as was originally suggested by the iceberg effect model proposed by Evenson et al [1999]. The concomitant determination of DNA fragmentation and nucleotide modifications of the oxo8dg type in sperm DNA may significantly improve the predictive value of DNA fragmentation tests in ART, especially when using tests like TUNEL, in situ nick translation, or COMET under neutral ph conditions that measure real DNA damage. These tests should have a higher predictive value than those that measure susceptibility to DNA denaturation like the SCSA. This is supported by a recent report by Greco et al, in which they showed that microinjection of spermatozoa with TUNEL test values.15% resulted in a pregnancy rate of 5.6% while microinjection of spermatozoa with TUNEL values equal or,6% in a second ICSI attempt in these same couples resulted in a pregnancy rate of 44.4% (Greco et al, 2005). Peter Schlegel further elaborated on the issue of sperm population heterogeneity. He argued that since the predictive value of SCSA is better when applied to unprocessed sperm, and not the fraction used in ART, then SCSA must be measuring some global insult that affects the entire sperm population: Dr Alvarez has nicely summarized some of the existent data on DNA integrity and IVF outcomes. Several points deserve emphasis and consideration of how the data were constructed in these studies. In the original studies reported by Evenson et al [Evenson et al, 1999; Larson et al, 2000; Larson-Cook et al, 2003], DNA integrity was evaluated by SCSA in the neat (unprocessed) semen specimen. This result helped predict IVF outcome, whereas the processed sperm SCSA test was less predictive. So, the actual sperm used for IVF/ICSI did not have many DNA breaks. The tendency of the sperm sample as a whole to form DNA breaks with acid treatment presumably predicted a defect? small DNA defects? excess ROS [reactive oxygen species]? other factors? that may affect IVF outcome, including (based on Tesarik et al [2004] and Carrell et al [2003]) embryo development and implantation/miscarriage. These results suggest that we should avoid the concept of selecting sperm with fewer DNA breaks in a semen sample with abnormal DNA integrity and consider that abnormal DNA integrity results may have an effect on fertility and assisted reproduction (without being a knockout.) These observations support the iceberg effect proposed by Evenson, although iceberg may not be the best term. Finally, when viewing the overall literature on patients who have SCSA, TUNEL, or Comet, it is interesting that these evaluations parallel each other in most patients, whereas experimental models of DNA damage (ie, radiation, etc) may have different results with the various tests for DNA integrity. All this discussion elicited a vigorous defense of the clinical value of SCSA by Donald Evenson. He started by offering a sharp critique of the study by Payne et al: The general conclusion of the article by Payne et al (2005) Redefining the relationship between sperm deoxyribonucleic acid fragmentation as measured by the sperm chromatin structure assay and outcomes of assisted reproductive techniques is that: The poorer the integrity of sperm nuclear DNA, the better the pregnancy outcome. This conclusion stands alone in stark contrast to the couple of hundred manuscripts published on human and animal studies that, overall, conclude that the integrity of the sperm DNA is highly correlated with good embryo development and normal full term pregnancy. Payne et al wish to redefine the relationship between SCSA data and ART outcomes with 100 couples, which is an insufficient number to reach a valid conclusion that the SCSA (over sperm samples measured in our laboratory) must be redefined. Furthermore, most of these couples had female infertility problems. The 100 couples in the Payne et al study included: 45 female infertility unspecified, 17 endometriosis, 17 tubal, 13 ovary-polycystic ovary syndrome, 2 amenorrhea, and 6 male infertility oligospermia. If these female patients were the recipients of sperm in the less than 9% DFI group, this factor alone could explain why pregnancy rates were lower in this group. In the Payne et al (2005) study, a total of 292 embryos were transferred to 97 subjects (average of 3 embryos per transfer). We ask whether any patient had more than 3 embryos transferred, and if so, why, and how does this relate to the conclusions of the study? Pregnancy rates in ICSI obviously depend on the number of embryos obtained and transferred. If DNA fragmentation affects some of the embryos, these embryos will either fail to develop prior to implantation or after implantation leading to abortion. However, the remainder of the embryos may be normal. For example, if the number of embryos obtained in a couple is 10, 8 are of poor quality (some due to sperm DNA fragmentation) and 2 are of high quality, transference of these 2 embryos may result in pregnancy and one may wrongly conclude that DNA fragmentation is not correlated with poor outcome in ART. This would be most obvious in a case where 4 embryos are obtained and all embryos fail. In this case, DNA fragmentation will show a negative correlation with pregnancy rate. An association between sperm DNA fragmentation, poor embryo quality (Tesarik et al 2004), and failed pregnancy in ICSI (Greco et al, 2005; Virro et al, 2004) has already been reported. Dr Evenson then elaborated on the topics brought up by Dr Alvarez regarding the mechanisms of DNA damage with ROS and apoptosis: Concerning the negative predictive value of the SCSA test, I emphasize the views expressed by Dr Juan Alvarez on the relevance of the cause of DNA fragmentation. If this were caused by apoptosis (double strand breaks 5 genetically lethal), the negative predictive value would be much lower than if it were caused by oxidative stress where most of the DNA is affected (primary damage + secondary damage) (primary damage DNA fragmentation) (secondary damage nucleotide damage such as oxo8dg). Secondary damage in cases of apoptosis and DNA nicks is negligible, while in cases of oxidative stress there is extensive secondary damage. Therefore, if the DFI value is 40% but DNA fragmentation was caused by

4 Makhlouf and Niederberger N DNA Integrity Tests 319 apoptosis, there is a 60% chance that a healthy spermatozoon would fertilize the oocyte. There seems to be a general consensus that most of the ejaculated sperm DNA damage is caused by ROS activity, although ROS may also induce apoptosis. In that case, the SCSA 30% DFI threshold may reflect the general level of primary and secondary damage. The article published by Greco et al (2005) fully supports that conclusion. The importance is that most of those patients had sperm DNA fragmentation produced post-testicularly, as shown by the fact that of the 18 males, 17 had DNA fragmentation values, as measured by TUNEL, of.15% in ejaculated spermatozoa and,6% in testicular spermatozoa while only 1 male had values.6% in testicular spermatozoa. As suggested by Dr Alvarez, had the levels of oxo8dg been analyzed, they would probably have been very high, and thus our iceberg effect (Evenson and Jost 2000) would have applied in this case. That is likely why the pregnancy rate with ejaculated spermatozoa in the first cycle was 5.6% and 44.4% with testicular spermatozoa. Dr Evenson then argued that the various DNA integrity tests generally correlate: Although the SCSA is targeted for criticism in the Payne et al (2005) article, it was pointed out by Dr Schlegel (2005) that all of the major DNA fragmentation tests are similar in their recognition of a threshold that places a man at an infertility risk. It is not clear at this time what differences exist between TUNEL and SCSA data. However, very significant correlations between human SCSA and TUNEL data have been described, eg, Gorczyca et al (1993), with (r 5.859; P,.01). Correlations have also been made between TUNEL and SCSA data for bulls (r , P,.001), stallions (r 5.65, P,.001), and rams (r 5.84, P,.001) (Sailer et al, 1995). While the TUNEL assay is thought to label mostly double strand (ds) breaks, it may also label less efficiently single strand (ss) DNA breaks (Honda et al, 2004); however, if a ss break in the highly condensed sperm DNA causes partial denaturation, such a ss DNA break may label with TUNEL. He then elaborated on the use of the SCSA to predict outcomes. He clarified that the SCSA does not provide an absolute cutoff for fertility, and that its correlation with pregnancy outcomes depends heavily on the method of conception (intercourse, IUI [intrauterine insemination], IVF, or ICSI). A brief review of the SCSA literature was also presented: Nevertheless, the very important point is: Although we do not know the exact mechanisms of how SCSA and TUNEL targets damaged sperm DNA, the data are very clear that ejaculated semen samples that contain a high proportion of sperm with damaged DNA (,.30% SCSA DFI) have a poorer prognosis for a successful pregnancy. The data are simply statistical probabilities that are a part of most medicine. All SCSA clinical reports have the statement: It is important to note that a DFI value above 30% does not preclude a normal, full term pregnancy. A.30% DFI, if consistent over time, does mean that the male partner is statistically placed into a group of men that demonstrate a longer time period to establish a pregnancy, more IVF/ICSI cycles, increased risk of spontaneous abortions or no pregnancy. SCSA data have clearly been shown to be sufficiently independent from classical semen analysis to provide important and clinically robust information not obtained by any other reasonable method. Under current SCSA conditions, if 30% of the sperm have enough DNA damage to reach the 30% threshold, the other 70% may have DNA damage to lesser and varying degrees, eg, ROS-damaged DNA bases (Evenson et al, 2002; Aitken and Sawyer, 2003; Alvarez, 2005). This is in agreement with miscarriage rates being the highest for ICSI ART procedures in patients whose DFI was.30% (Virro et al, 2004; Check et al, 2005). Dr Schlegel (2005) pointed out that men of couples with repeated spontaneous abortions have significantly higher DFI values (Carrell et al 2003). It is also now clear, but was a point of confusion for a number of investigators, that the means of fertilization have a huge impact on the predictive value of the SCSA test results. SCSA results from a study of couples using unprotected sex simply cannot be used to predict the outcomes of ICSI fertilization. In a comprehensive double-blinded study at Georgetown University of male factor infertility (Dr M. Zinaman PI with Dr Evenson s lab performing the SCSA tests) for 200 couples attempting conception by natural intercourse, the odds ratio was 6.56 greater for a successful pregnancy if the DFI was,30% (Evenson et al, 1999; Zinaman et al, 2000). No couple became pregnant when the DFI in that month was.30%. However, some men who had a 3 month average of DFI.30% in months 1 3 fathered a child in months Pregnancies did occur with a previous DFI average of.30% but took a longer time to achieve. Thus, there was a statistical prediction of a lesser success rate, but NOT an absolute cut off at 30%! Furthermore, 39% of the miscarriages were predicted from the SCSA data. Although this paper was published 6 years ago, it is still cited incorrectly! Results from recent meta-analyses performed by our laboratory (Evenson and Wixon [2005]) show the following: a meta-analysis of 4 studies investigating the relationship between sperm DNA fragmentation and pregnancy outcome using in vivo and IUI procedures indicated that the patients were 7.1 (CI 3.37 and 14.91) times more likely to achieve a pregnancy/delivery if the DFI was,30% (n , P,.0001). When routine IVF alone was considered, couples were,2.0 (CI 1.10 and 2.96) times more likely to become pregnant if their DFI was,30% (n 5 375, P,.02). A meta-analysis of 6 studies using ICSI and/or routine IVF showed a nonsignificant trend where patients were 1.7 (CI 0.97 and 3.14) times more likely to achieve a pregnancy/delivery if the %DFI was,30% (n 5 322, P 5.055). Payne et al (2005) state that SCSA marketing states that DFIs of,15% have excellent fertility potential. The term excellent fertility potential was changed some time ago, since sperm DNA integrity is only one factor in the complexity of initiating a pregnancy. The term fertility potential has been changed to: excellent, good, and fair to poor DNA integrity. The SCSA predicts infertility, and NOT fertility.

5 320 Journal of Andrology N May ÙJune 2006 Due to this fact, receiver operating characteristic (ROC) curves generated for SCSA data are useful only for determining the threshold of DFI and high DNA stainability (HDS) populations followed by a determination of the number of patients above that threshold that do, or do not, become pregnant using odds ratio. This is in contrast to the primary utility of the ROC in medicine which makes use of sensitivity and specificity for disease testing where there are absolute outcomes (ie, yes if a disease is present, and no, if it is not). In sharp contrast to this, when considering the SCSA test, the odds for a pregnancy with a DFI.30% are reduced, but there are still chances for pregnancy to occur. Consequently, some data may have a ROC area-under-the-curve approaching 0.5 that could wrongly be interpreted as the test having little predictive power. However, when the ROC is used to derive a threshold for odds ratios, which is the appropriate statistical endpoint for the SCSA analysis, the odds of a pregnancy for a threshold of.30% DFI are up to fivefold greater than when DFI is,30% (De Jonge and Evenson, unpublished data). The value of this same approach was clearly shown in a recent article by Henkel et al (2004) using the TUNEL assay. These authors stated: the performance of receiver operating characteristic (ROC) curve analysis with pregnant yes or no as discriminator and application of calculated cut-off values for further differentiations, significant differences between TUNEL-negative (,36.5% green-fluorescing sperm) and TUNEL-positive sperm (.36.5% green-fluorescing sperm) were revealed for the pregnancy rate (34.65% vs 19.05%: P ). Thus, the probability for the female partner of becoming pregnant by IVF is almost twice as high, or 50% higher at the same fertility potential, if the percentage of TUNELpositive sperm in the husband s ejaculate is low (,36.5%). DNA Fragmentation in the ART Setting Payne et al (2005), as well as other authors, have frequently highlighted 2 early studies showing no pregnancies when the SCSA DFI.27% while making only minor notes in the discussion that the Virro et al (2004) (ART in Dr Virro clinic, SCSA measures in Evenson lab) clearly showed that pregnancies by IVF/ ICSI were obtained with DFI values.27%. In the case of Virro et al (2004) Payne et al (2005) wrongly stated that no pregnancies occurred. Thus, I feel obligated to comment further on these studies. The first small clinical study of SCSA in ART was performed at the University of Nebraska IVF clinic of Dr C De Jonge on 24 couples (Larson et al, 2000). The SCSA measurements were done blindly in Dr Evenson s laboratory and the data sent to Dr De Jonge. The combined data showed that no pregnancies resulted if.27% of the sperm showed DNA denaturation (DNA strand breaks). Dr De Jonge s current IVF clinic at the University of Minnesota now finds better success for pregnancy with DFI.27% even when our laboratory has measured the SCSA (DeJonge and Evenson, in preparation). Thus, a pertinent question is: Is this sharp contrast simply due to the statistical odds of the couples involved or to the ART techniques/technicians involved in the studies? The same question can also be asked of the Larson-Cook et al (2003) study. Payne et al (2005) state that when the DFI exceeded 30%, they [Virro et al, 2004] observed low rates of blastocyst development and no ongoing pregnancies. This statement is not true! In contrast, Virro et al (2004) reported that the pregnancy rate with a DFI.27% was 40% less than the couples having a DFI,27%. There was a 28% pregnancy rate with DFIs greater than 40%. Payne et al (2005) state: From a biological standpoint, we must remember that the interpretation of the SCSA is based on the assumption that acridine orange (AO) appears red when it binds to fragmented DNA and green when it binds to intact DNA. It is possible that the chemical reaction between this dye and DNA is more complicated than this and that our results are the first hint that red staining with acridine orange does not convey a poor prognosis with patients undergoing IVF. In response, there are hundreds of scientific papers on the interaction of AO and nucleic acids and the correct statement is that AO interaction with single stranded nucleic acids, DNA, or RNA causes a collapse of the stained complex that produces a metachromatic shift from green to red fluorescence. At least 40% of couple infertility is due to a male factor, and sperm DNA fragmentation is definitely one important factor. Patients deserve to know if this is a factor in their specific case. However, they MUST be counseled that a high DFI does not preclude a normal full-term pregnancy and that they should NOT rush to the idea of using donor sperm if their DFI is.30%. However, after working with a number of cases where couples have undergone up to a dozen ICSI cycles with no success, and our finding that the DFI is in the 60% 80% range, this information should direct the doctor/ patient to consider a different direction. A DNA fragmentation test is quick and relatively inexpensive and provides potentially very useful information that will guide the physician toward the most effective course to pregnancy. This thread demonstrated the intense interest surrounding the topic of sperm DNA integrity in the field of male factor infertility. This is also reflected by the number of reviews and letters on this topic published recently (Agarwal and Allamaneni, 2005; Alvarez, 2005; Bungum, 2005; Evenson and Wixon, 2005; Gandini, 2005; Schlegel and Paduch, 2005; Seli and Sakkas, 2005; Sergerie et al, 2005; Spano et al, 2005). However, it is clear that integration of DNA tests into routine clinical practice still faces some hurdles (Bungum 2005; Schlegel and Paduch, 2005). The first hurdle is the plethora of different tests that measure different aspects of DNA damage. As outlined by Alvarez and others (Agarwal and Allamaneni, 2005; Alvarez, 2005; Schlegel and Paduch, 2005), DNA integrity tests differ in what they measure. TUNEL (Schlegel and Paduch, 2005), DNA break detection FISH (Muriel et al, 2004), and in situ nick translation (Sakkas et al, 1996) measure breaks in the DNA. The SCSA (Evenson et al, 2002); single-cell electrophoresis (Comet; Schlegel and Paduch, 2005), and SCD or Halosperm (Fernandez et al, 2005) measure susceptibil-

6 Makhlouf and Niederberger N DNA Integrity Tests 321 Studies of the effect of the DNA fragmentation index (DFI) as measured by the sperm chromatin assay on the clinical pregnancy rate for in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) Study n High DFI Predicts Low Pregnancy Rate in IVF/ICSI? DFI Cutoff (%) Low DFI, Pregnant/Total High DFI, Pregnant/Total Larson et al, Yes 27 7/12 0/9 Larson-Cook et al, Yes 27 28/79 0/10 Saleh et al, 2003* 14* Yes 27 N/A* N/A* Bungum et al, No /140 13/35 Gandini et al, No 27 7/24 5/10 Virro et al, Yes 30 84/178 20/71 Chohan et al, Trend 30 25/43 4/9 Gardner et al, 2004*4 128* No 30 35%* 37%* Adams et al, Yes 30 30/41 2/9 Nicopoullos et al, 2005*4 37* Trend 15 50%* 26%* Payne et al, No 27 22/79 9/19 Check et al, No /77 8/29 Zini et al, No 30 25/49 6/11 Total /722 (42%) 67/212 (31%) * Could not obtain actual patient numbers to include in total. 3 A statistically nonsignificant trend toward low pregnancy rates in high-dfi patients was seen in the conventional IVF group, but not ICSI subgroup. Statistically significant difference between DFI groups seen in IUI patients. 4 Conference abstract only. Numbers shown are for implantation rate. 5 Numbers reported are for clinical pregnancy. There was a statistically nonsignificant trend for lower ongoing pregnancy in the high-dfi group. ity to denaturation. In addition to acridine orange used in SCSA, other chromatin-binding dyes such as toluidine blue (Zini et al, 2002) and chromomycin A3 (Sakkas et al, 1996) have also been used to assess sperm chromatin condensation. These assays generally track together (Schlegel and Paduch, 2005) and have in fact been used to validate each other (Erenpreiss et al, 2004; Fernandez et al, 2005). However, it would be naïve to expect that they are equivalent in detecting all conditions of DNA damage. Thus, we should be careful in generalizing results of one assay to another because we do not yet fully understand when and why these tests give different results. Lack of understanding of the pathophysiology of sperm DNA damage is another hurdle. A medical test is classically used to establish a diagnosis (eg, a karyotype to diagnose Klinefelter). Obviously, we cannot use tests like SCSA in a similar fashion because there is yet no characteristic syndrome of poor sperm DNA quality, and cases of isolated DNA damage are rare (Zini et al, 2002). The various clinical studies have been plagued by the inclusion of patients with disparate underlying conditions, ranging from female factors (Payne et al, 2005) to repeated IVF failure (Check et al, 2005), to documented male infertility (Saleh et al, 2003). There is no agreement on the prototype patient in whom these tests would be most useful because we do not know what causes DNA fragmentation. Several putative mechanisms have been proposed, including posttesticular ROS damage (Greco et al, 2005), apoptosis (Rodriguez et al, 1997), abnormal DNA condensation during spermiogenesis (Aoki et al, 2005), and deficiencies in paternal DNA repair mechanisms (McPherson and Longo, 1993; Olsen et al, 2005). Although SCSA and other tests like TUNEL are valuable tools in exploring these possibilities, such use of these tests is clearly investigational and not routine. Another hurdle limiting widespread adoption of the SCSA is the technical expertise required. In experienced hands (Evenson and Jost, 2000), SCSA is a reliable test with little repeat variability. However, Boe-Hansen et al (2005) showed that the assay is sensitive to small variations in protocol. This renders comparison of studies from different laboratories more difficult. This interlaboratory variability is partly to blame in the multiplicity of thresholds used with the SCSA in various studies (Sergerie et al, 2005). Despite these limitations, the SCSA has been recommended as an adjunct to predict ART outcomes, particularly costly IVF/ICSI. Unfortunately, this clinical application has yet to be confirmed by large multiinstitutional trials. As outlined by Dr Evenson, the evidence supporting the hypothesis that high DNA fragmentation predicts low fertility is very strong in studies of natural intercourse and IUI (Evenson et al, 1999; Spano et al, 2000; Bungum et al, 2004; Evenson and Wixon, 2005). However, the literature is less clear on how well SCSA predicts IVF success, especially in

7 322 Journal of Andrology N May ÙJune 2006 ICSI cases (Bungum et al, 2004). The meta-analysis mentioned by Dr Evenson in his post (Evenson and Wixon, 2005) combined 6 different studies of SCSA and IVF/ICSI. The analysis showed a trend for improved odds of pregnancy with DFI,30% (odds ratio vs DFI.30%), but the result fell just shy of statistical significance (P 5.06) and did not include some recent studies that found contrary results (Gandini et al, 2004; Payne et al, 2005; Zini et al, 2005). We reviewed all papers and abstracts available to date (December 2005) that have applied SCSA to IVF/ICSI cases (Table). Using clinical pregnancy as an endpoint and DFI cutoffs of 27% or 30%, we found that a majority of studies, but certainly not all, found that high DFI correlated with pregnancy failures. They disagreed on whether SCSA predicted fertilization success, a secondary endpoint. Of note is that many studies have only been presented at conferences, and we look forward to the publication of large series of SCSA, such as that by Bungum et al (2005). The recommendation that a high DFI should guide a couple away from IUI or IVF toward ICSI (Bungum et al, 2004) remains to be confirmed by further studies. The potential value of DNA integrity testing is apparent when one considers that the male gamete is a specialized carrier whose sole function is the delivery of a genetic package to the ooctye. In contrast to the traditional measures of viability, morphology, and motility that examine the carrier, DNA tests assess the content of the package. With ICSI bypassing the carrier role of the spermatozoon, DNA integrity testing might become the more relevant assay. However, we believe that our current state of knowledge regarding DNA fragmentation does net yet allow the routine clinical application of these tests. References Adams C, Anderson L, Wood S. High, but not moderate, levels of sperm DNA fragmentation are predictive of poor outcome in egg donation cycles. Fertil Steril. 2004;82(suppl 2):S44. Agarwal A, Allamaneni SS. Sperm DNA damage assessment: a test whose time has come. Fertil Steril. 2005;84: Aitken RJ, Sawyer D. The human spermatozoon not waving but drowning. Adv Exp Med Biol. 2003;518: Alvarez JG. The predictive value of sperm chromatin structure assay. Hum Reprod. 2005;20: Aoki VW, Moskovtsev SI, Willis J, Liu L, Mullen JB, Carrell DT. Abstract DNA integrity is compromised in protamine-deficient human sperm. J Androl. 2005;26: Benchaib M, Braun V, Lornage J, Hadj S, Salle B, Lejeune H, Guerin JF. Sperm DNA fragmentation decreases the pregnancy rate in an assisted reproductive technique. Hum Reprod. 2003;18: Boe-Hansen GB, Ersboll AK, Christensen P. Variability and laboratory factors affecting the sperm chromatin structure assay in human semen. J Androl. 2005;26: Bungum M. Reply to: sperm chromatin structural assay versus hypoosmotic swelling test in predicting the need for in vitro fertilization with intracytoplasmic sperm injection. Hum Reprod. 2005;20: Bungum M, Humaidan P, Axmon X, Spano M, Bungum L, Erenpreiss J, Giwercman A. Sperm chromatin structure assay (SCSA) predicts the outcome of ART. Abstracts of the 21st annual meeting of the European Society for Human Reproduction and Embryology; June 19 22, 2005; Copenhagen, Denmark. i199. Bungum M, Humaidan P, Spano M, Jepson K, Bungum L, Giwercman A. The predictive value of sperm chromatin structure assay (SCSA) parameters for the outcome of intrauterine insemination, IVF and ICSI. Hum Reprod. 2004;19: Carrell DT, Liu L, Peterson CM, Jones KP, Hatasaka HH, Erickson L, Campbell B. Sperm DNA fragmentation is increased in couples with unexplained recurrent pregnancy loss. Arch Androl. 2003;49: Check JH, Graziano V, Cohen R, Krotec J, Check ML. Effect of an abnormal sperm chromatin structural assay (SCSA) on pregnancy outcome following (IVF) with ICSI in previous IVF failures. Arch Androl. 2005;51: Chohan KR, Griffin JT, Lafromboise M, De Jonge CJ, Carrell DT. Sperm DNA damage relationship with embryo quality and pregnancy outcome in IVF patients. Fertil Steril. 2004;82(suppl 2):S55. Cui J, Holmes EH, Greene TG, Liu PK. Oxidative DNA damage precedes DNA fragmentation after experimental stroke in rat brain. FASEB J. 2000;14: Erenpreiss J, Jepson K, Giwercman A, Tsarev I, Erenpreisa J, Spano M. Toluidine blue cytometry test for sperm DNA conformation: comparison with the flow cytometric sperm chromatin structure and TUNEL assays. Hum Reprod. 2004;19: Evenson D, Jost L. Sperm chromatin structure assay is useful for fertility assessment. Methods Cell Sci. 2000;22: Evenson DP, Jost LK, Marshall D, Zinaman MJ, Clegg E, Purvis K, de Angelis P, Claussen OP. Utility of the sperm chromatin structure assay as a diagnostic and prognostic tool in the human fertility clinic. Hum Reprod. 1999;14: Evenson DP, Larson KL, Jost LK. Sperm chromatin structure assay: its clinical use for detecting sperm DNA fragmentation in male infertility and comparisons with other techniques. J Androl. 2002;23: Evenson DP, Wixon R. Clinical aspects of sperm DNA fragmentation detection and male infertility. Theriogenology. 2006;65: , [Epub ahead of print]. Fernandez JL, Muriel L, Goyanes V, Segrelles E, Gosalvez J, Enciso M, LaFromboise M, De Jonge C. Simple determination of human sperm DNA fragmentation with an improved sperm chromatin dispersion test. Fertil Steril. 2005;84: Gandini L. Reply to: sperm chromatin structural assay versus hypoosmotic swelling test in predicting the need for in vitro fertilization with intracytoplasmic sperm injection. Hum Reprod. 2005;20: Gandini L, Lombardo F, Paoli D, Caruso F, Eleuteri P, Leter G, Ciriminna R, Culasso F, Dondero F, Lenzi A, Spano M. Full-term pregnancies achieved with ICSI despite high levels of sperm chromatin damage. Hum Reprod. 2004;19: Gardner DK, Schoolcraft WB, Surrey ES, Stilley K, Stevens J, Evenson D. The sperm chromatin structure assay (SCSA) and its relationship to IVF outcome: a prospective trial. Fertil Steril. 2004;82(suppl 2):S129. Gorczyca W, Traganos F, Jesionowska H, Darzynkiewicz Z. Presence of DNA strand breaks and increased sensitivity of DNA in situ to denaturation in abnormal human sperm cells: analogy to apoptosis of somatic cells. Exp Cell Res. 1993;207:

8 Makhlouf and Niederberger N DNA Integrity Tests 323 Greco E, Scarselli F, Iacobelli M, Rienzi L, Ubaldi F, Ferrero S, Franco G, Anniballo N, Mendoza C, Tesarik J. Efficient treatment of infertility due to sperm DNA damage by ICSI with testicular spermatozoa. Hum Reprod. 2005;20: Henkel R, Hajimohammad M, Stalf T, Hoogendijk C, Mehnert C, Menkveld R, Gips H, Schill WB, Kruger TF. Influence of deoxyribonucleic acid damage on fertilization and pregnancy. Fertil Steril. 2004;81: , Honda S, Sugita I, Miki K, Saito I. The semi-quantitative comparison of oxidative stress mediated DNA single and double strand breaks using terminal deoxynucleotidyl transferase mediated end labeling combined with a slot blot technique. Free Radic Res. 2004;38: Larson KL, DeJonge CJ, Barnes AM, Jost LK, Evenson DP. Sperm chromatin structure assay parameters as predictors of failed pregnancy following assisted reproductive techniques. Hum Reprod. 2000;15: Larson-Cook KL, Brannian JD, Hansen KA, Kasperson KM, Aamold ET, Evenson DP. Relationship between the outcomes of assisted reproductive techniques and sperm DNA fragmentation as measured by the sperm chromatin structure assay. Fertil Steril. 2003;80: McPherson S, Longo FJ. Chromatin structure-function alterations during mammalian spermatogenesis: DNA nicking and repair in elongating spermatids. Eur J Histochem. 1993;37: Muriel L, Segrelles E, Goyanes V, Gosalvez J, Fernandez JL. Structure of human sperm DNA background damage, analysed by in situ enzymatic treatment and digital image analysis. Mol Hum Reprod. 2004;10: Nicopoullos JDM, Ramsay JWA, Almeida P, Gilling-Smith C. Can the outcome of intracytoplasmic sperm injection be predicted by genetic screening of spermatozoa? Abstracts of the 21st annual meeting of the European Society for Human Reproduction and Embryology; June 19 22, 2005; Copenhagen. Denmark. i198. Olsen AK, Lindeman B, Wiger R, Duale N, Brunborg G. How do male germ cells handle DNA damage? Toxicol Appl Pharmacol. 2005;207(2 suppl): Payne JF, Raburn DJ, Couchman GM, Price TM, Jamison MG, Walmer DK. Redefining the relationship between sperm deoxyribonucleic acid fragmentation as measured by the sperm chromatin structure assay and outcomes of assisted reproductive techniques. Fertil Steril. 2005;84: Rodriguez I, Ody C, Araki K, Garcia I, Vassalli P. An early and massive wave of germinal cell apoptosis is required for the development of functional spermatogenesis. EMBO J. 1997;16: Sailer BL, Jost LK, Evenson DP. Mammalian sperm DNA susceptibility to in situ denaturation associated with the presence of DNA strand breaks as measured by the terminal deoxynucleotidyl transferase assay. J Androl. 1995;16: Sakkas D, Urner F, Bianchi PG, Bizzaro D, Wagner I, Jaquenoud N, Manicardi G, Campana A. Sperm chromatin anomalies can influence decondensation after intracytoplasmic sperm injection. Hum Reprod. 1996;11: Saleh RA, Agarwal A, Nada EA, El-Tonsy MH, Sharma RK, Meyer A, Nelson DR, Thomas AJ. Negative effects of increased sperm DNA damage in relation to seminal oxidative stress in men with idiopathic and male factor infertility. Fertil Steril. 2003;79(suppl 3): Schlegel PN, Paduch DA. Yet another test of sperm chromatin structure. Fertil Steril. 2005;84: Seli E, Sakkas D. Spermatozoal nuclear determinants of reproductive outcome: implications for ART. Hum Reprod Update. 2005;11: Sergerie M, Bleau G, Teule R, Daudin M, Bujan L. Sperm DNA integrity as diagnosis and prognosis element of male fertility. Gynecol Obstet Fertil. 2005;33: Spano M, Bonde JP, Hjollund HI, Kolstad HA, Cordelli E, Leter G. Sperm chromatin damage impairs human fertility. The Danish First Pregnancy Planner Study Team. Fertil Steril. 2000;73: Spano M, Seli E, Bizzaro D, Manicardi GC, Sakkas D. The significance of sperm nuclear DNA strand breaks on reproductive outcome. Curr Opin Obstet Gynecol. 2005;17: Tesarik J, Greco E, Mendoza C. Late, but not early, paternal effect on human embryo development is related to sperm DNA fragmentation. Hum Reprod. 2004;19: Virro MR, Larson-Cook KL, Evenson DP. Sperm chromatin structure assay (SCSA) parameters are related to fertilization, blastocyst development, and ongoing pregnancy in in vitro fertilization and intracytoplasmic sperm injection cycles. Fertil Steril. 2004;81: Zinaman MJ, Brown CC, Selevan SG, Clegg ED. Semen quality and human fertility: a prospective study with healthy couples. J Androl. 2000;21: Zini A, Fischer MA, Sharir S, Shayegan B, Phang D, Jarvi K. Prevalence of abnormal sperm DNA denaturation in fertile and infertile men. Urology. 2002;60: Zini A, Meriano J, Kader K, Jarvi K, Laskin CA, Cadesky K. Potential adverse effect of sperm DNA damage on embryo quality after ICSI. Hum Reprod. 2005;20: