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Measuring Sperm DNA Fragmentation and Clinical Outcomes of Medically Assisted Reproduction: A Systematic Review and Meta-Analysis

  • Maartje Cissen,

    Affiliation Department of Obstetrics & Gynaecology, Jeroen Bosch Hospital, ‘s-Hertogenbosch, The Netherlands

  • Madelon van Wely ,

    m.vanwely@amc.uva.nl

    Affiliation Center for Reproductive Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

  • Irma Scholten,

    Affiliation Center for Reproductive Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

  • Steven Mansell,

    Affiliation Department of Molecular and Cell Biology, University of California, Berkeley, California, United States of America

  • Jan Peter de Bruin,

    Affiliation Department of Obstetrics & Gynaecology, Jeroen Bosch Hospital, ‘s-Hertogenbosch, The Netherlands

  • Ben Willem Mol,

    Affiliation The Robinson Institute/School of Paediatrics and Reproductive Health, University of Adelaide, Adelaide, Australia

  • Didi Braat,

    Affiliation Department of Obstetrics and Gynaecology, Radboud University Medical Center, Nijmegen, The Netherlands

  • Sjoerd Repping,

    Affiliation Center for Reproductive Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

  • Geert Hamer

    Affiliation Center for Reproductive Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

Measuring Sperm DNA Fragmentation and Clinical Outcomes of Medically Assisted Reproduction: A Systematic Review and Meta-Analysis

  • Maartje Cissen, 
  • Madelon van Wely, 
  • Irma Scholten, 
  • Steven Mansell, 
  • Jan Peter de Bruin, 
  • Ben Willem Mol, 
  • Didi Braat, 
  • Sjoerd Repping, 
  • Geert Hamer
PLOS
x

Abstract

Sperm DNA fragmentation has been associated with reduced fertilization rates, embryo quality, pregnancy rates and increased miscarriage rates. Various methods exist to test sperm DNA fragmentation such as the sperm chromatin structure assay (SCSA), the sperm chromatin dispersion (SCD) test, the terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labelling (TUNEL) assay and the single cell gel electrophoresis (Comet) assay. We performed a systematic review and meta-analysis to assess the value of measuring sperm DNA fragmentation in predicting chance of ongoing pregnancy with IVF or ICSI. Out of 658 unique studies, 30 had extractable data and were thus included in the meta-analysis. Overall, the sperm DNA fragmentation tests had a reasonable to good sensitivity. A wide variety of other factors may also affect the IVF/ICSI outcome, reflected by limited to very low specificity. The constructed hierarchical summary receiver operating characteristic (HSROC) curve indicated a fair discriminatory capacity of the TUNEL assay (area under the curve (AUC) of 0.71; 95% CI 0.66 to 0.74) and Comet assay (AUC of 0.73; 95% CI 0.19 to 0.97). The SCSA and the SCD test had poor predictive capacity. Importantly, for the TUNEL assay, SCD test and Comet assay, meta-regression showed no differences in predictive value between IVF and ICSI. For the SCSA meta-regression indicated the predictive values for IVF and ICSI were different. The present review suggests that current sperm DNA fragmentation tests have limited capacity to predict the chance of pregnancy in the context of MAR. Furthermore, sperm DNA fragmentation tests have little or no difference in predictive value between IVF and ICSI. At this moment, there is insufficient evidence to recommend the routine use of sperm DNA fragmentation tests in couples undergoing MAR both for the prediction of pregnancy and for the choice of treatment. Given the significant limitations of the evidence and the methodological weakness and design of the included studies, we do urge for further research on the predictive value of sperm DNA fragmentation for the chance of pregnancy after MAR, also in comparison with other predictors of pregnancy after MAR.

Introduction

Traditionally, the diagnosis of male subfertility is based upon the analysis of semen volume and sperm concentration, motility and morphology. Although there is a direct relationship between semen quality and pregnancy rates both in natural conception and after medically assisted reproduction (MAR), there is no definite predictive threshold for success for conventional semen parameters [14]. Conventional semen analysis does not assess all aspects of the function of testis and sperm quality. New tests for predicting the chance of pregnancy would be clinically useful. There have been attempts to propose sperm DNA fragmentation as such a new test for male reproductive capability [5].

The integrity of our genome is continuously challenged by endogenous metabolic by-products and exogenous factors. Depending on variables like cell type, cell cycle stage and the type of DNA damage, a cell has several ways to repair damaged DNA and inaccurate repair can have different consequences [6,7]. While our somatic bodies inevitably die of old age or disease, the germ line has to maintain sufficient DNA integrity to pass on our genome to forthcoming generations. DNA double-strand breaks (DSBs) are endogenously induced during spermatogenesis; first during meiosis, to facilitate the formation of meiotic crossovers, and second during spermiogenesis, when the chromatin of the haploid round spermatids is compacted by the replacement of histones by protamines [8,9]. Furthermore, the sperm may accumulate DNA damage and fragmentation during maturation and storage in the epididymis [1012]. Other causes of sperm DNA fragmentation can be defective apoptosis, excessive reactive oxygen species (ROS) production and decreased seminal antioxidants [1323]. Also toxic effects of drugs, cigarette smoking, pollution, and factors as xenobiotics, high testicular temperature (fever, varicocele) and advanced age have been associated with increased sperm DNA damage [2428].

Recent studies have highlighted the significance of sperm DNA integrity as an important factor that affects functional competence of the sperm. Therefore the detection of sperm DNA fragmentation could be clinically useful as part of fertility workup [29]. For this purpose, several techniques that measure DNA fragmentation are available and have been evaluated in separate studies. In this systematic review we will assess the sperm chromatin structure assay (SCSA), the sperm chromatin dispersion (SCD) test, the terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labelling (TUNEL) and the single cell gel electrophoresis (Comet) assay.

The SCSA bases its results on (1) the DNA fragmentation index (DFI), which is the percentage in the sample that have measurable increased red fluorescence due to acridine orange attaching to a single strand portion of DNA at sites of DNA strand breaks and then collapsing into a crystal that produces a metachromatic shift to red fluorescence under exposure to blue light and (2) the percentage of high DNA stainability (HDS), which is due to excess histones and proteins other than protamines that prevent full condensation of the sperm chromatin [3032].

The SCD test, also known as Halo Sperm assay, estimates the level of DNA fragmentation indirectly by quantification of the amount of nuclear dispersion/halo after sperm lysis and acid denaturation to remove excess nuclear proteins [33,34].

The principle of TUNEL involves labelling of the 3′-ends of single- and double-strand breaks with biotinylated dUTPs. The incorporated labelled nucleotides can be quantified by flow cytometry or (fluorescence) microscopy to determine the number of (apoptotic) sperm cells containing fragmented DNA [31]. However, double strand DNA can have breaks with no exposed 3’OH end and thus not being labelled by the TUNEL assay.

The Comet assay quantifies the shape of the single cell nuclei after gel electrophoresis. Small fragmented DNA has a faster rate of migration towards the anode in an electrophoretic field (tail region) as compared to larger non-fragmented DNA (head region), leading to a typical comet shape [30,31].

Using these tests, the percentage of sperm with fragmented DNA was shown to be comparable in idiopathic subfertile men with normal sperm parameters and in subfertile men with abnormal sperm parameters, and significantly higher in both these groups in comparison to fertile controls [3537]. However, we do not know whether MAR helps to overcome the negative effects of DNA damage on the chance of pregnancy. We therefore performed a systematic review and meta-analysis to assess the value of measuring sperm DNA fragmentation in predicting the ongoing pregnancy chance after in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). 

Materials and Methods

Search and selection strategy

The electronic databases Pubmed, Embase, Cochrane and CINAHL were searched from inception (September 1967) to January 2016 for articles which described sperm DNA fragmentation tests and outcome after MAR. The Medical Subject Headings terms and/or text words that were used in our search can be found in the appendix. We also manually reviewed the bibliographies of retrieved original papers and review articles. We used the preferred reporting items for systematic review and meta-analysis checklist (PRISMA) while conducting this study (S1 Fig).

Titles and abstracts of all identified studies were screened and the full paper of the preselected articles was read by two researchers (S.M. and M.C.). Both researchers extracted the data from the article independently by using standardized data extraction forms. If 2x2 tables could be constructed the study was selected for final inclusion. In the 2x2 tables, the numbers of pregnant and non-pregnant women for different sperm DNA fragmentation cut-off values were recorded. Any disagreement between the two researchers was resolved through discussion or by consultation with a third researcher (I.S.).

Eligibility criteria

All studies investigating the effect of sperm DNA fragmentation detected by the SCSA, the SCD test, the TUNEL assay or the Comet assay on the outcome of IVF and/or ICSI were considered eligible for inclusion. The search was restricted to studies in humans. Studies were excluded if they had no original data available for retrieval and duplicate publications were also excluded. Studies that included cycles with donor oocytes and experiments that asses sperm DNA fragmentation in specific male factor pathologies e.g. azoospermia and co-intervention experiments e.g. antioxidant treatment were excluded from analysis.

The primary study outcome was ongoing pregnancy (defined as the presence of a living intrauterine fetus on transvaginal ultrasonography (TVU) at the 12th week of gestation). Other study outcomes were clinical pregnancy (defined as the presence of a gestational sac on TVU or other definitive clinical signs) and live birth (defined as a live-born baby ≥ 24 weeks of gestation). All outcomes were reported per cycle.

Quality assessment

Each selected study was scored for their relevance and methodological quality by using the QUADAS 2 (Quality Assessment of Diagnostic Accuracy Studies) checklist [38]. Furthermore the following characteristics of the studies were taken into consideration: data collection method (prospective of retrospective), study design (cohort or randomized controlled trial (RCT)) and study population.

Statistical analysis

Hierarchical summary receiver operating characteristic.

In order to evaluate the overall accuracy, including the whole range of possible thresholds, we used hierarchical summary receiver operating characteristic (HSROC) plots to display the results of individual studies in a ROC space, each study being plotted as a single sensitivity-specificity point. Reported estimates for sensitivity and specificity from different studies may be based on different positivity thresholds (explicitly due to cut-off values used, or implicitly related to the assessment method or device used). If there are multiple thresholds reported in one study, we chose the threshold that was most comparable to the others. Based on the binomial distributions of the true positives and true negatives we calculated a summary point, with a 95% confidence interval (CI) and predictive interval by using STATA version 14 (Stata-Corp, College Station, Texas, USA). As recommended for meta-analysis of diagnostic accuracy studies [39], we used hierarchical models to obtain summary estimates of sperm DNA fragmentation test in terms of ability to discriminate between men with lower and higher probabilities of pregnancy. Separate HSROC curves for IVF and ICSI were performed when sufficient studies were available.

An area under the curve (AUC) of 1 implies perfect discrimination, whereas an AUC of 0.5 means that the test does not discriminate at all [40]. For this review, a test is considered to have a poor predictive accuracy if the AUC lies between 0.50 and 0.70. An AUC between 0.70 and 0.80 represents a fair predictive accuracy, and an AUC above 0.80 represents a good predictive accuracy.

In cases where insufficient data was available to perform HSROC analyses for clinical pregnancy, (ongoing) pregnancy or live birth independently, different pregnancy outcomes were combined when minimal differences in sensitivity and specificity were found. When minimal differences in sensitivity and specificity were found, studies with different timing of the sperm DNA fragmentation test (pre- and post-wash) were combined.

Pooled sensitivity and specificity.

According to the bivariate method [41], sensitivity and specificity with 95% CI were calculated and displayed in a forest plot. Sensitivity and specificity of original studies were pooled with STATA, using a random effect model. This model was chosen for explicating the heterogeneity between the included studies and to estimate the between-study variance. The amount of heterogeneity was quantified by using the I2 statistic, which represents the percentage of total variability across the studies that is due to heterogeneity instead of chance. Moderate heterogeneity is defined as a value < 50% [42].

Meta-regression.

Meta-regression analysis was performed with type of fertility treatment as covariable to determine if differences in fertility treatment affected the estimated effect of sperm DNA fragmentation. The meta-regression analyses were performed using STATA subroutine MIDAS. If the p-value was < 0.05, results were considered to indicate statistical significance.

Results

Systematic search, selection and data extraction

The electronic search resulted in 859 hits. Following the removal of duplicates and the addition of studies by hand-search and screening of abstracts, 111 studies were identified to be potentially eligible for inclusion (Fig 1).

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Fig 1. Flowdiagram of search and selection strategy in a systematic review and meta-analysis of sperm DNA fragmentation tests and pregnancy rates after MAR.

Legend: not applicable.

https://doi.org/10.1371/journal.pone.0165125.g001

After reading the manuscripts and assessing the inclusion criteria and methodological quality, 67 studies were found that evaluated the association between sperm DNA fragmentation and pregnancy after IVF or ICSI and 30 studies were eligible for final inclusion into the diagnostic meta-analyses. We excluded 81 studies for the reasons shown in Table 1.

Descriptive review

We found 21 studies reported on the SCSA [103,110112,114,117,118,123136], 18 studies on the SCD test [97,98,101,102,106109,113,115,116,120,122,137141], 18 studies reported on the TUNEL assay [9295,96,99,100,104,119,134,142149] and seven studies reported on the alkaline Comet assay [95,105,121,134,150152].

Studies selected for diagnostic meta-analysis

Characteristics of included studies are listed in Table 2. Most studies were of a prospective cohort design and used pregnancy rate as outcome measure. Fig 2 and Table 3 show the scores on overall risk of bias and concerns regarding applicability in this meta-analysis according to QUADAS-2. For about half of the studies the threshold for sperm DNA fragmentation was not pre-specified and hence was judged to be at ‘high risk’ of bias for QUADAS-2 domain ‘index test’. Moreover, different cut-off values for DNA fragmentation were used to assess sperm DNA as fragmented. Studies were at high risk of applicability concerns in domain “index test” when the sperm DNA fragmentation threshold is not comparable to the thresholds of other studies. For QUADAS-2 domain ‘flow and timing’ eight studies judged to be at ‘high risk’ because of an inappropriate interval between the sperm DNA fragmentation test and the fertility treatment. Overall the reference standard was judged to be at ‘low risk’.

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Fig 2. Overall risk of bias in meta-analysis.

This figure illustrates the overall risk of bias in the meta-analysis. The horizontal axis represents the number of studies included. The color of the bars represent the risk of bias. Yellow: high risk, blue: low risk and grey: unclear risk.

https://doi.org/10.1371/journal.pone.0165125.g002

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Table 2. Descriptive data of all studies for the meta-analysis regarding SCSA, SCD test, TUNEL assay and Comet assay as tools for measure sperm DNA fragmentation.

https://doi.org/10.1371/journal.pone.0165125.t002

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Table 3. Study characteristics according to QUADAS II recommendations to report the risk of bias for patient selection and the concerns for applicability of data collected in manuscripts eligible for the meta-analysis.

https://doi.org/10.1371/journal.pone.0165125.t003

SCSA

The predictive accuracy for pregnancy with MAR of the SCSA was poor. The HSROC curve analysis indicated a sensitivity of 0.84 (95% CI 0.77 to 0.88) and specificity of 0.21 (95% CI 0.16 to 0.26) resulting in an AUC of 0.49 (95% CI 0.45 to 0.54) (Figs 3 and 4). Meta-regression indicated a difference in predictive value between IVF and ICSI (p-value: 0.00) (Table 4). For the seven studies on IVF separately the HSROC was 0.53 (95% CI 0.48 to 0.57). For the eight studies on ICSI the HSROC was 0.45 (95% CI 0.40 to 0.49). For IVF the sensitivity of the DNA fragmentation index was higher, however the specificity was lower. The low specificity points to a low proportion of true negatives, indicating low sperm DNA fragmentation does not guarantee more pregnancies. There was significant heterogeneity across studies in sensitivity and specificity (I2 statistic > 50%) (Fig 4, Table 4).

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Fig 3. HSROC curve.

Hierarchical summary receiver operating characteristic (HSROC) plot of sperm DNA fragmentation for prediction of (clinical) pregnancy. Each circle on the plot represents the pair of sensitivity and specificity from a study and the size of the circle is scaled according to the sample size of the study. The solid red block represents the summary sensitivity and specificity, and this summary point is surrounded by a 95% confidence region (yellow dashed line) and 95% prediction region (green dotted line). Sperm DNA fragmentation in the prediction of (clinical) pregnancy for all studies and all cut-off values of the DNA fragmentation index reported: (A) SCSA, (B) SCD test, (C) TUNEL assay and (D) alkaline Comet assay. AUC: Area under the curve; HSROC: Hierarchical summary receiver operating characteristics.

https://doi.org/10.1371/journal.pone.0165125.g003

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Fig 4. Forest plot.

Forest plot of sperm DNA fragmentation according to the DNA fragmentation index for predicting pregnancy. The plot shows study-specific estimates of sensitivity and specificity (with 95% confidence intervals). The studies are ordered according to the type of treatment: (A) SCSA, (B) SCD test, (C) TUNEL assay and (D) alkaline Comet assay. CI: confidence interval.

https://doi.org/10.1371/journal.pone.0165125.g004

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Table 4. Meta-regression analysis with type of fertility treatment as independent variable to determine whether this independent variable could be of influence on the sensitivity and specificity of the sperm DNA fragmentation test.

https://doi.org/10.1371/journal.pone.0165125.t004

SCD test

The predictive accuracy for pregnancy with MAR of the SCD test was poor. The HSROC curve analysis indicated a sensitivity of 0.59 (95% CI 0.43 to 0.73) and specificity of 0.39 (95% CI 0.25 to 0.55) resulting in an AUC of 0.49 (95% CI 0.44 to 0.53) (Figs 3 and 4). There was significant heterogeneity across studies in sensitivity and specificity (I2 statistic > 50%) (Fig 4).

TUNEL assay

The predictive accuracy for pregnancy with MAR of the TUNEL assay was fair. The HSROC curve analysis indicated a sensitivity of 0.84 (95% CI 0.75 to 0.90) and specificity of 0.24 (95% CI 0.11 to 0.44) resulting in an AUC of 0.71 (95% CI 0.66 to 0.74) (Figs 3 and 4). For the six studies on IVF the AUC was comparable (0.72; 95% CI 0.68 to 0.76). Meta-regression indicated no difference in predictive value between IVF and ICSI (p-value: 0.59) (Table 4). There was no significant heterogeneity across studies in sensitivity and specificity (I2 statistic = 0%) (Fig 4).

Comet assay

The predictive accuracy for pregnancy with MAR of the alkaline Comet assay was fair. The HSROC curve analysis indicated a sensitivity of 0.79 (95% CI 0.61 to 0.90) and specificity of 0.60 (95% CI 0.48 to 0.71) resulting in an AUC of 0.73 (95% CI 0.19 to 0.97) (Figs 3 and 4). Meta-regression indicated no significant difference in predictive value between IVF and ICSI (p-value: 0.12) (Table 4). There was significant het-erogeneity across studies in sensitivity and specificity (I2 statistic > 50%) (Fig 4).

Discussion

This current review and meta-analysis summarizes the available knowledge concerning the value of sperm DNA fragmentation tests in the prediction of pregnancy after IVF or ICSI. From the HSROC curves (Fig 3) it becomes clear that the SCSA and the SCD test have a poor predictive value, whereas the predictive value of the TUNEL assay and Comet assay was fair. All tests show higher sensitivity and lower specificity for their predictive power, indicating low sperm DNA fragmentation does not guarantee more pregnancies. Overall, there was significant statistical heterogeneity across studies (Fig 4).

We found six meta-analyses investigating the effect of sperm DNA fragmentation on live birth or pregnancy after IVF and/or ICSI. In the meta-analysis of Evenson and Wixon there was a non-significant trend towards the occurrence of pregnancy (odds ratio (OR) 1.6; 95% CI 0.92 to 2.94) when infertile couples were treated with IVF or ICSI and the DFI, determined by the SCSA, was below 30% [153]. The meta-analysis of Li et al. found that the clinical pregnancy rate decreased significantly for IVF patients with a high degree of sperm DNA fragmentation, determined by the TUNEL assay (relative risk (RR) 0.68; 95% CI 0.54 to 0.85) [154]. In ICSI clinical pregnancy rate was unaffected by DFI (RR 0.76; 95% CI 0.55 to 1.04). The meta-analysis of Collins et al. found a significant association between sperm DNA fragmentation, determined by the TUNEL assay or SCSA, and pregnancy after IVF or ICSI (OR 1.44; 95% CI 1.03 to 2.03) [155]. The meta-analysis of Zhao et al. showed that the pregnancy rate decreased significantly for IVF/ICSI patients with a high degree of sperm DNA fragmentation, determined by several sperm DNA fragmentation tests (RR 0.81; 95% CI 0.70 to 0.95) [156]. The meta-analysis of Zhang et al. showed that patients were more likely to get pregnant if DFI was less than 27% (OR 1.44; 95% CI 1.19 to 1.74) [157]. The most recent meta-analysis of Osman et al. found that the live birth rate after IVF and/or ICSI increased significantly in patients with low sperm DNA fragmentation (RR 1.17; 95% CI 1.07 to 1.28) [158]. In all the above meta-analyses the association between the sperm DNA fragmentation test and live birth or pregnancy was determined and expressed as ORs and RRs. An association does however not imply that the test actually has predictive value. Furthermore, we found several studies investigating the significance of sperm DNA fragmentation as a continuous variable in IVF and/or ICSI. These studies were excluded from meta-analysis, because no cut-off values were used so a 2x2 table could not be constructed from the data. Some of these studies found an association between sperm DNA fragmentation and pregnancy [83,92,93,97,104,107], while others did not find an association between sperm DNA fragmentation and conception [47,116]. In conclusion, we think that the best way to judge the value of DNA fragmentation tests is by their predictive capacity for the outcome of interest, ongoing pregnancy. Therefore the outcomes of our review are not comparable with the outcomes of other reviewers that chose to just report on the association between sperm DNA fragmentation and pregnancy. The results of the individual studies that were not included in our and other reviews are conflicting and lack the statistic power of a review.

Many studies investigated the predictive accuracy of sperm DNA fragmentation tests on the outcome of MAR. It is of clinical importance to assess whether these tests can be used as a prognostic tool, to distinguish couples who should be advised to undergo MAR or not. It has been suggested that DNA fragmentation is a useful marker in the prediction of spontaneous pregnancy in couples with unexplained subfertility. The chance of spontaneous conception declines at sperm DNA fragmentation index values above 20% and approaches zero for values over 30–40%. Low sperm DNA fragmentation however does not guarantee normal male fertility [159,160]. To our knowledge no study exists in which the spontaneous pregnancy chance is compared with the chance after MAR for different DNA fragmentation values.

As mentioned before, the methods of assessment of sperm DNA integrity are different for the different assays. The TUNEL assay and Comet assay are direct methods to assess DNA strand breaks, whereas the SCSA and SCD test are indirect methods, which use the higher susceptibility of damaged DNA to denature and/or fragment in an acid solution. Possibly, the fair predictive accuracy of the TUNEL assay and Comet assay, in comparison to the poor accuracy found for other methods in our meta-analysis, is due to its direct method of assessment, which may better reflect genome integrity of the sperm cells.

Besides integrity of sperm DNA, there are other factors that affect the probability to conceive after MAR, primarily the quality and age of the oocyte [161]. Male germ cells are susceptible to the accumulation of DNA lesions in fertilizing sperm because their DNA repair capacity declines during the latter part of spermatogenesis [162]. In contrast, the oocyte is capable of repairing DNA damage throughout oogenesis and provides gene products that are responsible for repairing DNA damage in both parental genomes after fertilization [163,164]. However, the competency for DNA repair depends on the quality of the oocyte which declines with age [108,161].

This review has brought forward some limitations in the available literature on DNA fragmentation tests that need to be addressed. First the study heterogeneity was high. Some studies only included couples suffering with male subfertility, other studies only included couples after previously failed MAR and others included all couples undergoing MAR. Second, the timing of performing the sperm DNA fragmentation test was not uniform. Some studies performed their test a few months before start of the MAR, others performed their test during MAR; before or after semen preparation. In addition, different cut-off values were used to assess sperm DNA as being fragmented and some studies did not even have a pre-specified threshold. However, for this reason, a bivariate model was used for the HSROC curve analysis in the present review, which allows for variations in the assessment of sperm DNA fragmentation and the choice of cut-off values. Another limitation is the reproducibility of sperm DNA fragmentation assessment. Intra-assay variability appears to be different depending on which sperm DNA fragmentation test is being used; previous studies reported this to be either small but significantly different with the TUNEL assay [165] or small and not significantly different using a alkaline Comet assay [166] and no difference using the SCSA test [32]. On the other hand, inter-observer variability was found to be very similar [21,165,167]. Mainly the TUNEL assay has many protocols, which makes comparison between laboratories hard and explains its many clinical thresholds [168]. To take care of these problems, there is need for studies that have been done with exacting protocols in the clinic and in the measuring laboratory on many patients at one center for each kind of sperm DNA fragmentation test. Unfortunately, only few studies/clinics meet these criteria [58,132,134,150152].

It must also be mentioned that there was no correction for confounders possible. Insufficient data were available for potential confounders such as female age, male age, semen parameters and number of oocytes. Further research, for instance an IPD meta-analysis, must reveal the possible interrelation of other factors with pregnancy chance.

Conclusions

Our systematic review and meta-analysis suggests that current sperm DNA fragmentation tests have limited capacity to discriminate between couples who have a low chance to conceive and couples who have a high chance to conceive after MAR. In addition, sperm DNA fragmentation tests have little or no difference in predictive value between IVF and ICSI. At this moment there is insufficient evidence to recommend the routine use of sperm DNA fragmentation tests in couples undergoing MAR both for the prediction of pregnancy and for the choice of treatment. Given the significant limitations of the evidence and the methodological weakness and design of the included studies, we do urge for further research on the predictive value of sperm DNA fragmentation for the chance of a spontaneous pregnancy or a pregnancy after MAR.

Appendix

("dna damage"[MeSH Terms] OR ("dna"[All Fields] AND "damage"[All Fields]) OR "dna damage"[All Fields]) OR ("dna"[All Fields] AND "fragmentation"[All Fields] OR "dna fragmentation"[All Fields]) AND ("humans"[MeSH Terms] OR "humans"[All Fields] OR "human"[All Fields]) AND ("comet assay"[MeSH Terms] OR ("comet"[All Fields] AND "assay"[All Fields]) OR "comet assay"[All Fields] OR "comet"[All Fields]) OR ("Clin Mol Allergy"[Journal] OR "cma"[All Fields]) OR SCSA[All Fields] OR ("chromatin"[MeSH Terms] OR "chromatin"[All Fields]) OR (" acridine orange" [All Fields]) OR ("in situ nick-end labeling"[MeSH Terms] OR ("situ"[All Fields] AND "nick-end"[All Fields] AND "labeling"[All Fields]) OR "in situ nick-end labeling"[All Fields] OR "tunel"[All Fields]) OR ("in situ nick end labelling"[All Fields] OR "in situ nick-end labeling"[MeSH Terms] OR ("situ"[All Fields] AND "nick-end"[All Fields] AND "labeling"[All Fields]) OR "in situ nick-end labeling"[All Fields] OR ("situ"[All Fields] AND "nick"[All Fields] AND "end"[All Fields] AND "labeling"[All Fields]) OR "in situ nick end labeling"[All Fields]) AND ("spermatozoa"[MeSH Terms] OR "spermatozoa"[All Fields] OR "sperm"[All Fields]) AND ("pregnancy"[MeSH Terms] OR "pregnancy"[All Fields]).

Supporting Information

Author Contributions

  1. Conceptualization: BWM SR.
  2. Data curation: MC MvW.
  3. Formal analysis: MvW MC.
  4. Funding acquisition: SR JPdB.
  5. Investigation: MC IS SM.
  6. Methodology: MvW.
  7. Project administration: SR.
  8. Resources: MC MvW.
  9. Software: MvW.
  10. Supervision: SR DB.
  11. Validation: GH MC.
  12. Visualization: MC.
  13. Writing – original draft: MC MvW GH SR.
  14. Writing – review & editing: MC MvW IS SM JPdB BWM DB SR GH.

References

  1. 1. van der Steeg JW, Steures P, Eijkemans MJ, Habbema JD, Hompes PG, Kremer JA, et al. Role of semen analysis in subfertile couples. Fertil Steril 2011; 95(3): 1013–9. pmid:20338556
  2. 2. Akanji Tijani H, Bhattacharya S. The role of intrauterine insemination in male infertility. Hum Fertil (Camb) 2010; 13(4): 226–32.
  3. 3. van Weert JM, Repping S, Van Voorhis BJ, van der Veen F, Bossuyt P, Mol BW. The performance of the postwash total motile sperm count at the time of intrauterine insemination for the prediction of pregnancy: a meta-analysis. Fertil Steril 2004; 82(3): 612–20. pmid:15374704
  4. 4. van Weert JM, Repping S, van der Steeg JW, Steures P, van der Veen F, Mol BW. A prediction model for ongoing pregnancy after in vitro fertilization in couples with male subfertility. J Reprod Med 2008; 53(4), 250–6. pmid:18472647
  5. 5. Shamsi MB, Imam SN, Dada R. Sperm DNA integrity assays. diagnostic and prognostic challenges and implications in management of infertility. J Assist Reprod Genet 2011; 28(11): 1073–85. pmid:21904910
  6. 6. Vermeij WP, Hoeijmakers JH, Pothof J. Aging: not all DNA damage is equal. Curr Opin Genet Dev 2014; 26: 124–30. pmid:25222498
  7. 7. Hoeijmakers JH. DNA damage, aging, and cancer. N Engl J Med 2009; 361(15): 1475–85. pmid:19812404
  8. 8. Jan SZ, Hamer G, Repping S, de Rooij DG, van Pelt AM, Vormer TL. Molecular control of rodent spermatogenesis. Biochim Biophys Acta 2012; 1822(12): 1838–50. pmid:22366765
  9. 9. Rathke C, Baarends WM, Awe S, Renkawitz-Pohl R. Chromatin dynamics during spermiogenesis. Biochim Biophys Acta 2014; 1839(3): 155–68. pmid:24091090
  10. 10. Ramos L, Kleingeld P, Meuleman E, van Kooy R, Kremer J, Braat D, et al. Assessment of DNA fragmentation of spermatozoa that were surgically retrieved from men with obstructive azoospermia. Fertil Steril 2002; 77(2): 233–7. pmid:11821077
  11. 11. Ramos L, de Boer P, Meuleman EJ, Braat DD, Wetzels AM. Chromatin condensation and DNA damage of human epididymal spermatozoa in obstructive azoospermia. Reprod Biomed Online 2004; 8(4): 392–7. pmid:15149561
  12. 12. Steele EK, McClure N, Maxwell RJ, Lewis SE. A comparison of DNA damage in testicular and proximal epididymal spermatozoa in obstructive azoospermia. Mol Hum Reprod 1999; 5(9): 831–5. pmid:10460221
  13. 13. Aitken RJ, Baker MA, De Iuliis GN, Nixon B. New insights into sperm physiology and pathology. Handb Exp Pharmacol 2010: 198: 99–115.
  14. 14. Boissonneault G. Chromatin remodeling during spermiogenesis: a possible role for the transition proteins in DNA strand break repair. FEBS Lett 2002; 514(2–3): 111–4. pmid:11943135
  15. 15. Carrell DT, Liu L. Altered protamine 2 expression is uncommon in donors of known fertility, but common among men with poor fertilizing capacity, and may reflect other abnormalities of spermiogenesis. J Androl 2011; 22(4): 604–10.
  16. 16. Moustafa MH, Sharma RK, Thornton J, Mascha E, Abdel-Hafez MA, Thomas AJ Jr, et al. Relationship between ROS production, apoptosis and DNA denaturation in spermatozoa from patients examined for infertility. Hum Reprod 2004; 19(1): 129–38. pmid:14688171
  17. 17. McLay DW, Clarke HJ. Remodelling the paternal chromatin at fertilization in mammals. Reproduction 2003; 125(5): 625–33. pmid:12713425
  18. 18. Saalu LC. The incriminating role of reactive oxygen species in idiopathic male infertility: an evidence based evaluation. Pak J Biol Sci 2010; 13(9): 413–22. pmid:20973394
  19. 19. Sakkas D, Moffatt O, Manicardi GC, Mariethoz E, Tarozzi N, Bizzaro D. Nature of DNA damage in ejaculated human spermatozoa and the possible involvement of apoptosis. Biol Reprod 2002; 66(4): 1061–7. pmid:11906926
  20. 20. Shamsi MB, Venkatesh S, Tanwar M, Talwar P, Sharma RK, Dhawan A, et al. DNA integrity and semen quality in men with low seminal antioxidant levels. Mutat Res 2009; 665(1–2): 29–36. pmid:19427508
  21. 21. Shamsi MB, Venkatesh S, Kumar R, Gupta NP, Malhotra N, Singh N, et al. Antioxidant levels in blood and seminal plasma and their impact on sperm parameters in infertile men. Indian J Biochem Biophys 2010; 47(1): 38–43. pmid:21086753
  22. 22. Venkatesh S, Riyaz AM, Shamsi MB, Kumar R, Gupta NP, Mittal S, et al. Clinical significance of reactive oxygen species in semen of infertile Indian men. Andrologia 2009; 41(4): 251–6. pmid:19601938
  23. 23. Venkatesh S, Shamsi MB, Dudeja S, Kumar R, Dada R. Reactive oxygen species measurement in neat and washed semen: comparative analysis and its significance in male infertility assessment. Arch Gynecol Obstet 2011; 283(1): 121–6. pmid:20814688
  24. 24. Colagar AH, Jorsaraee GA, Marzony ET. Cigarette smoking and the risk of male infertility. Pak J Biol Sci 2007; 10(21): 3870–4. pmid:19090244
  25. 25. Enciso M, Muriel L, Fernandez JL, Goyanes V, Segrelles E, Marcos M, et al. Infertile men with varicocele show a high relative proportion of sperm cells with intense nuclear damage level, evidenced by the sperm chromatin dispersion test. J Androl 2006; 27(1): 106–11. pmid:16400086
  26. 26. Rubes J, Selevan SG, Evenson DP, Zudova D, Vozdova M, Zudova Z, et al. Episodic air pollution is associated with increased DNA fragmentation in human sperm without other changes in semen quality. Hum Reprod 2005; 20(10): 2776–83. pmid:15980006
  27. 27. Soares SR, Melo MA. Cigarette smoking and reproductive function. Curr Opin Obstet Gynecol 2008; 20(3): 281–91. pmid:18460944
  28. 28. Weber RF, Dohle GR, Romijn JC. Clinical laboratory evaluation of male subfertility. Adv Clin Chem 2005; 40: 317–64. pmid:16355926
  29. 29. Lewis SE, Agbaje I, Alvarez J. Sperm DNA tests as useful adjuncts to semen analysis. Syst Biol Reprod Med 2008; 54(3): 111–25. pmid:18570047
  30. 30. Palermo GD, Neri QV, Cozzubbo T, Rosenwaks Z. Perspectives on the assessment of human sperm chromatin integrity. Fertil Steril 2014; 102(6): 1508–17. pmid:25456796
  31. 31. Shamsi MB, Kumar R, Dada R. Evaluation of nuclear DNA damage in human spermatozoa in men opting for assisted reproduction. Indian J Med Res 2008; 127(2): 115–23. pmid:18403788
  32. 32. Erenpreiss J, Bungum M, Spano M, Elzanaty S, Orbidans J, Giwercman A. Intra-individual variation in sperm chromatin structure assay parameters in men from infertile couples: clinical implications. Hum Reprod 2006; 21(8): 2061–4. pmid:16684843
  33. 33. Fernandez JL, Muriel L, Goyanes V, Segrelles E, Gosalvez J, Enciso M, et al. Simple determination of human sperm DNA fragmentation with an improved sperm chromatin dispersion test. Fertil Steril 2005; 84(4): 833–42. pmid:16213830
  34. 34. Zhang LH, Qiu Y, Wang KH, Wang Q, Tao G, Wang LG. Measurement of sperm DNA fragmentation using bright-field microscopy: comparison between sperm chromatin dispersion test and terminal uridine nick-end labeling assay. Fertil Steril 2010: 94(3): 1027–32. pmid:19505686
  35. 35. Host E, Lindenberg S, Ernst E, Christensen F. DNA strand breaks in human spermatozoa: a possible factor, to be considered in couples suffering from unexplained infertility. Acta Obstet Gynecol Scand 1999; 78(7): 622–5. pmid:10422909
  36. 36. Host E, Lindenberg S, Smidt-Jensen S. DNA strand breaks in human spermatozoa: correlation with fertilization in vitro in oligozoospermic men and in men with unexplained infertility. Acta Obstet Gynecol Scand 2000; 79: 189–93. pmid:10716299
  37. 37. Saleh RA, Agarwal A, Nelson DE, Nada EA, El-Tonsy MH, Alvarez JG, et al. Increased sperm nuclear DNA damage in normozoospermic infertile men: a prospective study. Fertil Steril 2002; 78(2): 313–8. pmid:12137868
  38. 38. Whiting PF, Rutjes AW, Westwood ME, Mallett S, Deeks JJ, Reitsma JB, et al. QUADAS-2: a revised tool for the quality assessment of diagnostic accuracy studies. Ann Intern Med 2011; 155(8): 529–36. pmid:22007046
  39. 39. Macaskill P, Catsonis C, Deeks J, Harbord R, Takwoingi Y. Chapter 10: Analysing and presenting results. In: Deeks J, Bossuyt PM, Gatsonis C (eds Cochrane Handbook for Systematic Reviews of Diagnostic Test Accuracy), version 1.0. The Cochrane Collaboration 2010.
  40. 40. Hanley JA, McNeil BJ. The meaning and use of the area under a receiver operating characteristic (ROC) curve. Radiology 1982; 143(1): 29–36. pmid:7063747
  41. 41. Reitsma JB, Glas AS, Rutjes AW, Scholten RJ, Bossuyt PM, Zwinderman AH. Bivariate analysis of sensitivity and specificity produces informative summary measures in diagnostic reviews. J Clin Epidemiol 2005; 58(10): 982–90. pmid:16168343
  42. 42. Higgins JP, Thompson SG, Deeks JJ, Altman DG. Measuring inconsistency in meta-analyses. BMJ 2003; 327(7414): 557–60. pmid:12958120
  43. 43. Alkhayal A, San Gabriel M, Zeidan K, Alrabeeah K, Noel D, McGraw R, et al. Sperm DNA and chromatin integrity in semen samples used for intrauterine insemination. J Assist Reprod Genet 2013; 30(11): 1519–24. pmid:24068511
  44. 44. Duran EH, Morshedi M, Taylor S, Oehninger S. Sperm DNA quality predicts intrauterine insemination outcome: a prospective cohort study. Hum Reprod 2002; 17(12): 3122–8. pmid:12456611
  45. 45. Muriel L, Meseguer M, Fernandez JL, Alvarez J, Remohi J, Pellicer A, et al. Value of sperm chromatin dispersion test in predicting pregnancy outcome in intrauterine insemination: a blind prospective study. Hum Reprod 2006; 21(3): 738–44. pmid:16311292
  46. 46. Yang XY, Zhang Y, Sun XP, Cui YG, Qian XQ, Mao YD, et al. Sperm chromatin structure assay predicts the outcome of intrauterine insemination. Zhonghua Nan Ke Xue 2011; 17(11): 977–83. pmid:22141266
  47. 47. Dar S, Grover SA, Moskovtsev SI, Swanson S, Baratz A, Librach CL. In vitro fertilization-intracytoplasmic sperm injection outcome in patients with a markedly high DNA fragmentation index (>50%). Fertil Steril 2013; 100(1): 75–80. pmid:23562046
  48. 48. Gosalvez J, Caballero P, Lopez-Fernandez C, Ortega L, Guijarro JA, Fernandez JL, et al. Can DNA fragmentation of neat or swim-up spermatozoa be used to predict pregnancy following ICSI of fertile oocyte donors? Asian J Androl 2013; 15(6): 812–8. pmid:24121979
  49. 49. Greco E, Scarselli F, Lacobelli M, Rienzi L, Ubaldi F, Ferrero S, et al. Efficient treatment of infertility due to sperm DNA damage by ICSI with testicular spermatozoa. Hum Reprod 2005; 20(1): 226–30. pmid:15539441
  50. 50. Morris ID, Ilott S, Dixon L, Brison DR. The spectrum of DNA damage in human sperm assessed by single cell gel electrophoresis (Comet assay) and its relationship to fertilization and embryo development. Hum Reprod 2002; 17(4): 990–8. pmid:11925396
  51. 51. Nunez-Calonge R, Caballero P, Lopez-Fernandez C, Guijarro JA, Fernandez JL, Johnston S, et al. An improved experimental model for understanding the impact of sperm DNA fragmentation on human pregnancy following ICSI. Reprod Sci 2012; 19(11): 1163–8. pmid:23064692
  52. 52. Wang YB, Zhao LW, Xiang ZQ, Zhou XZ, Gui YI, He CH. Correlation analysis of the results of double fluorescence (AO/PI) staining and clinical outcomes. J Reprod Contracep 2012; 23: 111–8.
  53. 53. 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(6): 1401–8. pmid:15117894
  54. 54. Bungum M, Spano M, Humaidan P, Eleuteri P, Rescia M, Giwercman A. Sperm chromatin structure assay parameters measured after density gradient centrifugation are not predictive for the outcome of ART. Hum Reprod 2008; 23(1): 4–10. pmid:17986484
  55. 55. Henkel R, Kierspel E, Hajimohammad M, Stalf T, Hoogendijk C, Mehnert C, et al. DNA fragmentation of spermatozoa and assisted reproduction technology. Reprod Biomed Online 2003; 7(4): 477–84. pmid:14656411
  56. 56. Jiang HH, He XJ, Song B, Cao YX. Sperm chromatin integrity test for predicting the outcomes of IVF and ICSI. Zhonghua Nan Ke Xue 2011; 17(12): 1083–6. pmid:22235675
  57. 57. Larson KL, De Jonge 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(8): 1717–22. pmid:10920092
  58. 58. Simon L, Proutski I, Stevenson M, Jennings D, McManus J, Lutton D, et al. Sperm DNA damage has a negative association with live-birth rates after IVF. Reprod Biomed Online 2013; 26(1): 68–78. pmid:23200202
  59. 59. Cebesoy FB, Aydos K, Unlu C. Effect of sperm chromatin damage on fertilization ratio and embryo quality post-ICSI. Arch Androl 2006; 52(5): 397–402. pmid:16873141
  60. 60. Claassens OE, Menkveld R, Franken DR, Pretorius E, Swart Y, Lombard CJ, et al. The acridine orange test: determining the relationship between sperm morphology and fertilization in vitro. Hum Reprod 1992; 7(2): 242–7. pmid:1577939
  61. 61. Daris B, Goropevsek A, Hojnik N, Vlasisavljevic V. Sperm morphological abnormalities as indicators of DNA fragmentation and fertilization in ICSI. Arch Gynecol Obstet 2010; 281(2): 363–7. pmid:19504115
  62. 62. Lopes S, Sun JG, Jurisicova A, Meriano J, Casper RF. Sperm deoxyribonucleic acid fragmentation is increased in poor-quality semen samples and correlates with failed fertilization in intracytoplasmic sperm injection. Fertil Steril 1998; 69(3): 528–32. pmid:9531891
  63. 63. Marchetti C, Obert G, Deffosez A, Formstecher P, Marchetti P. Study of mitochondrial membrane potential, reactive oxygen species, DNA fragmentation and cell viability by flow cytometry in human sperm. Hum Reprod 2002; 17(5): 1257–65. pmid:11980749
  64. 64. Pregl Breznik B, Kovacic B, Vlaisavljevic V. Are sperm DNA fragmentation, hyperactivation, and hyaluronan-binding ability predictive for fertilization and embryo development in in vitro fertilization and intracytoplasmic sperm injection? Fertil Steril 2013; 99(5): 1233–41. pmid:23290739
  65. 65. Sadeghi MR, Hodjat M, Lakpour N, Arefi S, Amirjannati N, Modarresi T, et al. Effects of sperm chromatin integrity on fertilization rate and embryo quality following intracytoplasmic sperm injection. Avicenna J Med Biotechnol 2009; 1(3): 173–80. pmid:23408716
  66. 66. Sun JG, Jurisicova A, Casper RF. Detection of deoxyribonucleic acid fragmentation in human sperm: correlation with fertilization in vitro. Biol Reprod 1997; 56(3): 602–7. pmid:9047003
  67. 67. Abu-Hassan D, Koester F, Shoepper B, Schultze-Mosgau A, Asimakopoulos B, Diedrich K, et al. Comet assay of cumulus cells and spermatozoa DNA status, and the relationship to oocyte fertilization and embryo quality following ICSI. Reprod Biomed Online 2006; 12(4): 447–52. pmid:16740217
  68. 68. Angelopoulos T, Moshel YA, Lu L, Macanas E, Grifo JA, Krey LC. Simultaneous assessment of sperm chromatin condensation and morphology before and after separation procedures: effect on the clinical outcome after in vitro fertilization. Fertil Steril 1998; 69(4): 740–7. pmid:9548167
  69. 69. Chi HJ, Chung DY, Choi SY, Kim JH, Kim GY, Lee JS, et al. Integrity of human sperm DNA assessed by the neutral comet assay and its relationship to semen parameters and clinical outcomes for the IVF-ET program. Clin Exp Reprod Med 2011; 38(1): 10–7. pmid:22384412
  70. 70. Duran EH, Gurgan T, Gunalp S, Enginsu ME, Yarali H, Ayhan A. A logistic regression model including DNA status and morphology of spermatozoa for prediction of fertilization in vitro. Hum Reprod 1998; 13(5): 1235–9. pmid:9647553
  71. 71. Edwards KW, Chan PJ, Wei AK, Corselli JU, Jacobson JD. Further evidence supporting the Diff-Quik stain for sperm chromatin integrity testing. J Reprod Med 2015; 60(1–2): 6–12. pmid:25745745
  72. 72. Filatov MV, Semenova EV, Vorob’eva OA, Leont’eva OA, Drobchenko EA. Relationship between abnormal sperm chromatin packing and IVF results. Mol Hum Reprod 1999; 5(9): 825–30. pmid:10460220
  73. 73. Hammadeh ME, Hasani SA, Stieber M, Rosenbaum P, Kupker D, Diedrich K, et al. The effect of chromatin condensation (Aniline Blue staining) and morphology (strict criteria) of human spermatozoa on fertilization, cleavage and pregnancy rates in an intracytoplasmic sperm injection programme. Hum Reprod 1996; 11(11): 2468–71. pmid:8981135
  74. 74. Hammadeh ME, Stieber M, Haidl G, Schmidt W. Association between sperm cell chromatin condensation, morphology based on strict criteria, and fertilization, cleavage and pregnancy rates in an IVF program. Andrologia 1998; 30(1): 29–35. pmid:9567167
  75. 75. Hammadeh ME, Al-Hasani S, Gauss C, Rosenbaum P, Georg T, Diedrich K, et al. Predictive value of chromatin decondensation in vitro on fertilization rate after intracytoplasmic sperm injection (ICSI). Int J Androl 2001; 24: 311–316. pmid:11554990
  76. 76. Hammadeh ME, Strehler E, Zeginiadou T, Rosenbaum P, Schmidt W. Chromatin decondensation of human sperm in vitro and its relation to fertilization rate after ICSI. Arch Androl 2001; 47(2): 83–7. pmid:11554688
  77. 77. Hoshi K, Katayose H, Yanagida K, Kimura Y, Sato A. The relationship between acridine orange fluorescence of sperm nuclei and the fertilizing ability of human sperm. Fertil Steril 1996; 66(4): 634–9. pmid:8816630
  78. 78. Jiang H, He RB, Wang CL, Zhu J. The relationship of sperm DNA fragmentation index with the outcomes of in-vitro fertilisation-embryo transfer and intracytoplasmic sperm injection. J Obstet Gynaecol 2011; 31(7): 636–9. pmid:21973140
  79. 79. Karydis S, Asimakopoulos B, Papadopoulos N, Vakalopoulos I, Al-Hasani S, Nikolettos N. ICSI outcome is not associated with the incidence of spermatozoa with abnormal chromatin condensation. In vivo 2005; 19(5): 921–5. pmid:16097447
  80. 80. Katayose H, Yanagida K, Hashimoto S, Yamada H, Sato A. Use of diamide-acridine orange fluorescence staining to detect aberrant protamination of human ejaculated sperm nuclei. Fertil Steril 2003; 79, 670S–6S.
  81. 81. Lazaros LA, Vartholomatos GA, Hatzi EG, Kaponis AI, Makrydimas GV, Kalantaridou SN, et al. Assessment of sperm chromatin condensation and ploidy status using flow cytometry correlates to fertilization, embryo quality and pregnancy following in vitro fertilization. J Assist Reprod Genet 2011; 28(10): 885–91. pmid:21779786
  82. 82. Sakkas D, Urner F, Bianchi PG, Bizzaro D, Wagner I, Jaquenoud N, et al. Sperm chromatin anomalies can influence decondensation after intracytoplasmic sperm injection. Hum Reprod 1996; 11(4): 837–43. pmid:8671337
  83. 83. Tavares RS, Silva AF, Lourenco B, Almeida-Santos T, Sousa AP, Ramalho-Santos J. Evaluation of human sperm chromatin status after selection using a modified Diff-Quik stain indicates embryo quality and pregnancy outcomes following in vitro fertilization. Andrology 2013; 1(6): 830–7. pmid:24124136
  84. 84. Tomlinson MJ, Moffatt O, Manicardi GC, Bizzaro D, Afnan M, Sakkas D. Interrelationships between seminal parameters and sperm nuclear DNA damage before and after density gradient centrifugation: implications for assisted conception. Hum Reprod 2001; 16(10): 2160–5. pmid:11574509
  85. 85. Virant-Klun I, Tomazevic T, Meden-Vrtovec H. Sperm single-stranded DNA, detected by acridine orange staining, reduces fertilization and quality of ICSI-derived embryos. J Assist Reprod Genet 2002; 19(7): 319–28. pmid:12168732
  86. 86. Zhang Y, Wang H, Wang L, Zhou Z, Sha J, Mao Y, et al. The clinical significance of sperm DNA damage detection combined with routine semen testing in assisted reproduction. Mol Med Rep 2008; 1(5): 617–24. pmid:21479459
  87. 87. 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(12): 3476–80. pmid:16123087
  88. 88. Bufang X, Yijuan S, Xiaowei L, Xiaobin Z, Yun F, Aijun Z. Effect of sperm DNA fragment on the outcome of IVF-ET. Chin J Androl 2011; 8: 23–6.
  89. 89. Fang L, Lou LJ, Ye YH, Jin F, Zhou J. A study on correlation between sperm DNA fragmentation index and age of male, various parameters of sperm and in vitro fertilization outcome. Zhonghua Yi Xue Yi Chuan Xue Za Zhi 2011; 28(4): 432–5. pmid:21811987
  90. 90. Xi D, Chen Y, Dai YT. Sperm DNA fragmentation index and the success rate of IVF/ICSI. Zhonghua Nan Ke Xue 2016; 22(1): 77–81. pmid:26931032
  91. 91. Yang XY, Wang LL, Chen P, Zhang Y, Zhang W, Cui YG, et al. Impact of sperm DNA fragmentation index and sperm malformation rate on the clinical outcome of ICSI. Zhonghua Nan Ke Xue 2013; 19(12): 1082–6. pmid:24432618
  92. 92. Avendano C, Franchi A, Duran H, Oehninger S. DNA fragmentation of normal spermatozoa negatively impacts embryo quality and intracytoplasmic sperm injection outcome. Fertil Steril 2010; 94(2): 549–57. pmid:19339003
  93. 93. Bakos HW, Thompson JG, Feil D, Lane M. Sperm DNA damage is associated with assisted reproductive technology pregnancy. Int J Androl 2008; 31(5): 518–26. pmid:17822420
  94. 94. Benchaib M, Braun V, Lornage J, Hadj S, Salle B, Lejeune H, et al. Sperm DNA fragmentation decreases the pregnancy rate in an assisted reproductive technique. Hum Reprod 2003; 18(5): 1023–8. pmid:12721180
  95. 95. Caglar GS, Koster F, Schopper B, Asimakopoulos B, Nehls B, Nikolettos N, et al. Semen DNA fragmentation index, evaluated with both TUNEL and Comet assay, and the ICSI outcome. In vivo 2007; 21(6): 1075–80. pmid:18210759
  96. 96. Garolla A, Cosci I, Bertoldo A, Sartini B, Boudjema E, Foresta C. DNA double strand breaks in human spermatozoa can be predictive for assisted reproductive outcome. Reprod Biomed Online 2015; 31(1): 100–7. pmid:25985994
  97. 97. Gu LJ, Chen ZW, Chen ZJ, Xu JF, Li M. Sperm chromatin anomalies have an adverse effect on the outcome of conventional in vitro fertilization: a study with strictly controlled external factors. Fertil Steril 2009; 92(4): 1344–6. pmid:19368911
  98. 98. Gu LJ, Chen ZW, Lu WH, Xu JF, Li M, Chen ZJ. Effects of abnormal structure of sperm chromatin on the outcome of in vitro fertilization and embryo transfer. Zhonghua Yi Xue Yi Chuan Xue Za Zhi 2011; 28(2): 156–9. pmid:21462126
  99. 99. Hammadeh ME, Radwan M, Al-Hasani S, Micu R, Rosenbaum P, Lorenz M, et al. Comparison of reactive oxygen species concentration in seminal plasma and semen parameters in partners of pregnant and non-pregnant patients after IVF/ICSI. Reprod Biomed Online 2006; 13(5): 696–706. pmid:17169183
  100. 100. Hammadeh ME, Al-Hasani S, Rosenbaum P, Schmidt W, Fischer Hammadeh C. Reactive oxygen species, total antioxidant concentration of seminal plasma and their effect on sperm parameters and outcome of IVF/ICSI patients. Arch Gynecol Obstet 2008; 277(6), 515–26. pmid:18026972
  101. 101. Irez T, Sahmay S, Ocal P, Goymen A, Senol H, Erol N, et al. Investigation of the association between the outcomes of sperm chromatin condensation and decondensation tests, and assisted reproduction techniques. Andrologia 2015; 47(4): 438–47. pmid:24766543
  102. 102. Jin J, Pan C, Fei Q, Ni W, Yang X, Zhang L, et al. Effect of sperm DNA fragmentation on the clinical outcomes for in vitro fertilization and intracytoplasmic sperm injection in women with different ovarian reserves. Fertil Steril 2015; 103(4): 910–6. pmid:25747135
  103. 103. Kennedy C, Ahlering P, Rodriquez H, Levy S, Sutovsky P. Sperm chromatin structure correlates with spontaneous abortion and multiple pregnancy rates in assisted reproduction. Reprod Biomed Online 2011; 22(3): 272–6. pmid:21269883
  104. 104. Khalili MA, Nazari S, Dehghani-Firouzabadi R, Talebi A, Baghazadeh-Naeini S, Sadeghian-Nodoshan F, et al. Comparing the roles of sperm chromatin integrity and apoptosis in intrauterine insemination outcomes of couples with mild male and female factor infertility. J Reprod Infertil 2014; 15(1): 35–40. pmid:24696794
  105. 105. Lewis SE, O’Connell M, Stevenson M, Thompson-Cree L, McClure N. An algorithm to predict pregnancy in assisted reproduction. Hum Reprod 2004; 19(6): 1385–94. pmid:15117906
  106. 106. Li N, Jiang L. Effect of sperm DNA damage on the outcome of in vitro fertilization-embryo transfer. Guangxi Med J 2011; 03.
  107. 107. Lopez G, Lafuente R, Checa MA, Carreras R, Brassesco M. Diagnostic value of sperm DNA fragmentation and sperm high-magnification for predicting outcome of assisted reproduction treatment. Asian J Androl 2013; 15(6): 790–4. pmid:23912311
  108. 108. Meseguer M, Santiso R, Garrido N, Garcia-Herrero S, Remohi J, Fernandez JL. Effect of sperm DNA fragmentation on pregnancy outcome depends on oocyte quality. Fertil Steril 2011; 95(1): 124–8. pmid:20643402
  109. 109. Nasr-Esfahani MH, Razavi S, Vahdati AA, Fathi F, Tavalaee M. Evaluation of sperm selection procedure based on hyaluronic acid binding ability on ICSI outcome. J Assist Reprod Genet 2008; 25(5): 197–203. pmid:18483762
  110. 110. Nicopoullos JD, Gilling-Smith C, Almeida PA, Homa S, Norman-Taylor JQ, Ramsay JW. Sperm DNA fragmentation in subfertile men: the effect on the outcome of intracytoplasmic sperm injection and correlation with sperm variables. BJU Int 2008; 101(12): 1553–60. pmid:18294305
  111. 111. Nijs M, Creemers E, Cox A, Franssen K, Janssen M, Vanheusden E, et al. Chromomycin A3 staining, sperm chromatin structure assay and hyaluronic acid binding assay as predictors for assisted reproductive outcome. Reprod Biomed Online 2009; 19(5): 671–84. pmid:20021715
  112. 112. Nijs M, De Jonge C, Cox A, Janssen M, Bosmans E, Ombelet W. Correlation between male age, WHO sperm parameters, DNA fragmentation, chromatin packaging and outcome in assisted reproduction technology. Andrologia 2011; 43(3): 174–9. pmid:21561463
  113. 113. Rama Raju GA, Jaya Prakash G, Murali Krishna K, Madan K, Siva Narayana T, Ravi Krishna CH. Noninsulin-dependent diabetes mellitus: effects on sperm morphological and functional characteristics, nuclear DNA integrity and outcome of assisted reproductive technique. Andrologia 2012; 44: 490S–8S.
  114. 114. Saleh RA, Agarwal A, Nada EA, El-Tonsy MH, Sharma RK, Meyer A, et al. 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: 1597S–605S.
  115. 115. Sanchez-Martin P, Sanchez-Martin F, Gonzalez-Martinez M, Gosalvez J. Increased pregnancy after reduced male abstinence. Syst Biol Reprod Med 2013; 59(5): 256–60. pmid:23651301
  116. 116. Sharbatoghli M, Valojerdi MR, Amanlou M, Khosravi F, Jafar-abadi MA. Relationship of sperm DNA fragmentation, apoptosis and dysfunction of mitochondrial membrane potential with semen parameters and ART outcome after intracytoplasmic sperm injection. Arch Gynecol Obstet 2012; 286(5): 1315–22. pmid:22760592
  117. 117. Smit M, Wissenburg OG, Romijn JC, Dohle GR. Increased sperm DNA fragmentation in patients with vasectomy reversal has no prognostic value for pregnancy rate. J Urol 2010; 183(2): 662–5. pmid:20018311
  118. 118. Smit M, Romijn JC, Wildhagen MF, Veldhoven JL, Weber RF, Dohle GR. Decreased sperm DNA fragmentation after surgical varicocelectomy is associated with increased pregnancy rate. J Urol 2010; 183(1): 270–4. pmid:19913801
  119. 119. Tarozzi N, Nadalini M, Stronati A, Bizzaro D, Dal Prato L, Coticchio G, et al. Anomalies in sperm chromatin packaging: implications for assisted reproduction techniques. Reprod Biomed Online 2009; 18(4): 486–95. pmid:19400989
  120. 120. Tavalaee M, Razavi S, Nasr-Esfahani MH. Influence of sperm chromatin anomalies on assisted reproductive technology outcome. Fertil Steril 2009; 91(4): 1119–26. pmid:18504041
  121. 121. Tomsu M, Sharma V, Miller D. Embryo quality and IVF treatment outcomes may correlate with different sperm comet assay parameters. Hum Reprod 2002; 17(7): 1856–62. pmid:12093852
  122. 122. Velez de la Calle JR, Muller A, Walschaerts M, Clavere JL, Jimenez C, Wittemer C, et al. Sperm deoxyribonucleic acid fragmentation as assessed by the sperm chromatin dispersion test in assisted reproductive technology programs: results of a large prospective multicenter study. Fertil Steril 2008; 90(5): 1792–9. pmid:18166175
  123. 123. Boe-Hanson GB, Fedder J, Ersboll AK, Christensen P. The sperm chromatin structure assay as a diagnostic tool in the human fertility clinic. Hum Reprod 2006; 21(6): 1576–82. pmid:16543258
  124. 124. Bungum M, Humaidan P, Axmon A, Spano M, Bungum L, Erenpreiss J, et al. Sperm DNA integrity assessment in prediction of assisted reproduction technology outcome. Hum Reprod 2007; 22(1): 174–9. pmid:16921163
  125. 125. 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(2): 121–4. pmid:15804866
  126. 126. Gandini L, Lombardo F, Paoli D, Caruso F, Eleuteri P, Leter G, et al. Full-term pregnancies achieved with ICSI despite high levels of sperm chromatin damage. Hum Reprod 2004; 19(6): 1409–17. pmid:15117904
  127. 127. Guerin P, Matillon C, Bleau G, Levy R, Menezo Y. Impact of sperm DNA fragmentation on ART outcome. Gynecol Obstet Fertil 2005; 33(9): 665–8. pmid:16137906
  128. 128. 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(4): 895–902. pmid:14556809
  129. 129. Lin MH, Lee RKK, Li SH, Lu CH, Sun FJ, Hwu YM. Sperm chromatin structure assay parameters are not related to fertilization rates, embryo quality, and pregnancy rates in in vitro fertilization and intracytoplasmic sperm injection, but might be related to spontaneous abortion rates. Fertil Steril 2008; 90(2): 352–9. pmid:17904130
  130. 130. Micinski P, Pawlicki K, Wielgus E, Bochenek M, Tworkowska I. The sperm chromatin structure assay (SCSA) as prognostic factor in IVF/ICSI program. Reprod Biol 2009; 9(1): 65–70. pmid:19352418
  131. 131. Niu ZH, Shi HJ, Zhang HQ, Zhang AJ, Sun YJ, Feng Y. Sperm chromatin structure assay results after swim-up are related only to embryo quality but not to fertilization and pregnancy rates following IVF. Asian J Androl 2011; 13(6): 862–6. pmid:21841807
  132. 132. Oleszczuk K, Giwercman A, Bungum M. Sperm chromatin structure assay in prediction of in vitro fertilization outcome. Andrology 2016; 4(2): 290–6. pmid:26757265
  133. 133. 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(2): 356–64. pmid:16084876
  134. 134. Simon L, Liu L, Murphy K, Ge S, Hotaling J, Aston KI, et al. Comparative analysis of three sperm DNA damage assays and sperm nuclear protein content in couples undergoing assisted reproduction treatment. Hum Reprod 2014; 29(5): 904–17. pmid:24619433
  135. 135. Speyer BE, Pizzey AR, Ranieri M, Joshi R, Delhanty JD, Serhal P. Fall in implantation rates following ICSI with sperm with high DNA fragmentation. Hum Reprod 2010; 25(7): 1609–18. pmid:20495207
  136. 136. 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(5): 1289–95. pmid:15136092
  137. 137. Anifandis G, Bounartzi T, Messini CI, Dafopoulos K, Markandona R, Sotiriou S, et al. Sperm DNA fragmentation measured by Halosperm does not impact on embryo quality and ongoing pregnancy rates in IVF/ICSI treatments. Andrologia 2015; 47(3): 295–302. pmid:24621442
  138. 138. Muriel L, Garrido N, Fernandez JL, Remohi J, Pellicer A, de los Santos MJ, et al. Value of the sperm deoxyribonucleic acid fragmentation level, as measured by the sperm chromatin dispersion test, in the outcome of in vitro fertilization and intracytoplasmic sperm injection. Fertil Steril 2006; 85(2): 371–83. pmid:16595214
  139. 139. Ni W, Xiao S, Qiu X, Jin J, Pan C, Li Y, et al. Effect of sperm DNA fragmentation on clinical outcome of frozen-thawed embryo transfer and on blastocyst formation. PLoS One 2014; 9(4): e94956. pmid:24733108
  140. 140. Wang M, Sun J, Wang L, Gao X, Lu X, Wu Z, et al. Assessment of density gradient centrifugation (DGC) and sperm chromatin dispersion (SCD) measurements in couples with male factor infertility undergoing ICSI. J Assist Reprod Genet 2014; 31(12): 1655–63. pmid:25227693
  141. 141. Yilmaz S, Zergeroglu AD, Yilmaz E, Sofuoglu K, Delikara N, Kutlu P. Effects of sperm DNA fragmentation on semen parameters and ICSI outcome determined by an improved SCD test, Halosperm. Int J Fertil Steril 2010; 4(2): 73–8.
  142. 142. Benchaib M, Lornage J, Mazoyer C, Lejeune H, Salle B, Guerin JF. Sperm deoxyribonucleic acid fragmentation as a prognostic indicator of assisted reproductive technology outcome. Fertil Steril 2007; 87(1): 93–100. pmid:17074327
  143. 143. Borini A, Tarozzi N, Bizzaro D, Bonu MA, Fava L, Flamigni C, et al. Sperm DNA fragmentation: paternal effect on early post-implantation embryo development in ART. Hum Reprod 2006; 21(11): 2876–81. pmid:16793992
  144. 144. Esbert M, Pacheo A, Vidal F, Florensa M, Riqueros M, Ballesteros A, et al. Impact of sperm DNA fragmentation on the outcome of IVF with own or donated oocytes. Reprod Biomed Online 2011; 23(6): 704–10. pmid:22019617
  145. 145. Frydman N, Prisant N, Hesters L, Frydman R, Tachdjian G, Cohen-Bacrie P, et al. Adequate ovarian follicular status does not prevent the decrease in pregnancy rates associated with high sperm DNA fragmentation. Fertil Steril 2008; 89(1): 92–7. pmid:17482180
  146. 146. Henkel R, Hajimohammad M, Stalf T, Hoogendijk C, Mehnert C, Menkveld R, et al. Influence of deoxyribonucleic acid damage on fertilization and pregnancy. Fertil Steril 2004; 81(4): 965–72. pmid:15066449
  147. 147. Huang CC, Lin DPC, Tsao HM, Cheng TC, Liu CH, Lee MS. Sperm DNA fragmentation negatively correlates with velocity and fertilization rates but might not affect pregnancy rates. Fertil Steril 2005; 84(1): 130–40. pmid:16009168
  148. 148. Ozmen B, Caglar GS, Koster F, Schopper B, Diedrich K, Al-Hasani S. Relationship between sperm DNA damage, induced acrosome reaction and viability in ICSI patients. Reprod Biomed Online 2007; 15(2): 208–14. pmid:17697499
  149. 149. Seli E, Gardner DK, Schoolcraft WB, Moffatt O, Sakkas D. Extent of nuclear DNA damage in ejaculated spermatozoa impacts on blastocyst development after in vitro fertilization. Fertil Steril 2004; 82(2): 378–83. pmid:15302287
  150. 150. Simon L, Brunborg G, Stevenson M, Lutton D, McManus J, Lewis SE. Clinical significance of sperm DNA damage in assisted reproduction outcome. Hum Reprod 2010; 25(7): 1594–608. pmid:20447937
  151. 151. Simon L, Lutton D, McManus J, Lewis SE. Sperm DNA damage measured by the alkaline Comet assay as an independent predictor of male infertility and in vitro fertilization success. Fertil Steril 2011; 95(2): 652–7. pmid:20864101
  152. 152. Simon L, Castillo J, Oliva R, Lewis SE. Relationships between human sperm protamines, DNA damage and assisted reproduction outcomes. Reprod Biomed Online 2011; 23(6): 724–34. pmid:22036908
  153. 153. Evenson D, Wixon R. Meta-analysis of sperm DNA fragmentation using the sperm chromatin structure assay. Reprod Biomed Online 2006; 12(4): 466–72. pmid:16740220
  154. 154. Li Z, Wang L, Cai J, Huang H. Correlation of sperm DNA damage with IVF and ICSI outcomes: a systematic review and meta-analysis. J Assist Reprod Genet 2006; 23(9–10): 367–76. pmid:17019633
  155. 155. Collins JA, Barnhart KT, Schlegel PN. Do sperm DNA integrity tests predict pregnancy with in vitro fertilization? Fertil Steril 2008; 89(4): 823–31. pmid:17644094
  156. 156. Zhao J, Zhang Q, Wang Y, Li Y. Whether sperm deoxyribonucleic acid fragmentation has an effect on pregnancy and miscarriage after in vitro fertilization/intracytoplasmic sperm injection: a systematic review and meta-analysis. Fertil Steril 2014; 102(4): 998–1005. pmid:25190048
  157. 157. Zhang Z, Zhu L, Jiang H, Chen H, Chen Y, Dai Y. Sperm DNA fragmentation index and pregnancy outcome after IVF or ICSI: a meta-analysis. J Assist Reprod Genet 2015; 32(1): 17–26. pmid:25392073
  158. 158. Osman A, Alsomait H, Seshadri S, El-Toukhy T, Khalaf Y. The effect of sperm DNA fragmentation on live birth rate after IVF or ICSI: a systematic review and meta-analysis. Reprod Biomed Online 2015; 30(2): 120–7. pmid:25530036
  159. 159. Evenson DP, Jost LK, Marshall D, Zinaman MJ, Clegg E, Purvis K, et al. Utility of the sperm chromatin structure assay as a diagnostic and prognostic tool in the human fertility clinic. Hum Reprod 1999; 14(4): 1039–49. pmid:10221239
  160. 160. Spano M, Bonde J, Hjollund HI, Kolstad HA, Cordelli E, Leter G. Sperm chromatin damage impairs human fertility. Fertil Steril 2000; 73(1): 43–50. pmid:10632410
  161. 161. de Ziegler D, Frydman R. Different implantation rates after transfers of cryo-preserved embryos originating from donated oocytes or from regular in vitro fertilization. Fertil Steril 1990; 54(4): 682–8. pmid:2209890
  162. 162. Olsen AK, Lindeman B, Wiger R, Duale N, Brunborg G. How do male germ cells handle DNA damage? Toxicol Appl Pharmacol 2005; 207: 521S–31S.
  163. 163. Ashwood-Smith MJ, Edwards R. DNA repair by oocytes. Mol Hum Reprod 1996; 2(1): 46–51. pmid:9238657
  164. 164. Brandriff B, Pedersen RA. Repair of the ultraviolet-irradiated male genome in fertilized mouse eggs. Science 1981; 211(4489): 1431–3. pmid:7466400
  165. 165. Sharma RK, Sabanegh E, Mahfouz R, Gupta S, Thiyagarajan A, Agarwal A. TUNEL as a test for sperm DNA damage in the evaluation of male infertility. Urology 2010; 76(6): 1380–6. pmid:20573380
  166. 166. Hughes CM, Lewis SE, McKelvey-Martin VJ, Thompson W. Reproducibility of human sperm DNA measurements using the alkaline single cell gel electrophoresis assay. Mutat Res 1997; 374(2): 261–8. pmid:9100849
  167. 167. McEvoy A, Roberts P, Yap K, Matson P. Development of a simplified method of human semen storage for the testing of sperm DNA fragmentation using the Halosperm G2 test kit. Fertil Steril 2014; 102(4): 981–8. pmid:25212839
  168. 168. Lewis SE. The place of sperm DNA fragmentation testing in current day fertility management. Middle East Fertil Society J 2013; 18(2): 78–82.