Publicaciones Científicas

Agarwal, A. and S. S. Allamaneni (2005). Fertil Steril 84(4): 850-853.

Multiple techniques have been developed to measure the amount of sperm DNA damage in an effort to identify more objective parameters for evaluation of infertile men. We now have evidence to support that integrity of sperm DNA influences a couple's fertility and helps predict the chances of pregnancy and its successful outcome. The available tests of sperm DNA damage require additional large-scale clinical trials before their integration into routine clinical practice.

Benchaib, M., J. Lornage, et al. (2007). Fertil Steril 87(1): 93-100.

OBJECTIVE: To examine sperm DNA fragmentation in semen used for assisted reproduction procedures to establish this factor's prognostic role in fertilization rate, embryo development, pregnancy rate, and outcome. DESIGN: Prospective study. SETTING: Department of Medicine and Biology of Reproduction of the Edouard Herriot Hospital in Lyon, France. PATIENT(S): 322 couples, divided into 88 cycles of in vitro fertilization (IVF) or 234 cycles of intracytoplasmic sperm injection (ICSI). INTERVENTION(S): Sperm DNA fragmentation was detected in sperm obtained 2 to 5 months before the ART procedure. MAIN OUTCOME MEASURE(S): Sperm DNA fragmentation was measured with the terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling (TUNEL) technique. RESULT(S): There was a negative statistical correlation between the rate of fragmentation and the semen characteristics. A statistically significant negative relationship was found for sperm DNA fragmentation and fertilization when ICSI and IVF were compared. With ICSI, a statistically significant negative relationship was found between fertilization rate and percentage of sperm DNA fragmentation (DNA fragmentation index, or DFI). The risk of nontransfer due to blocked embryo development increased when the DFI exceeded 15% (18.2% for ICSI vs 4.2% for IVF) with an odds ratio of 5.05. The miscarriage risk increased fourfold when the DFI exceeded 15% (37.5% for ICSI vs 8.8% for IVF). CONCLUSION(S): Sperm DNA fragmentation measured 2 to 5 months before the assisted reproduction procedure was a prognostic indicator of the fertilization, pregnancy, and miscarriage rates and the pregnancy outcome.

Boe-Hansen, G. B., J. Fedder, et al. (2006). Hum Reprod 21(6): 1576-1582.

BACKGROUND: Sperm DNA integrity has been shown to be necessary for achieving and sustaining embryo development. The objective was to evaluate the sperm chromatin structure assay (SCSA) as a diagnostic tool in clinical practice for intrauterine insemination (IUI), in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) treatments. METHODS: A total of 385 semen samples from 234 couples were frozen for SCSA, and smears were prepared for morphology: 48 IUI, 139 IVF and 47 ICSI. The main SCSA variables were DNA fragmentation index (DFI), standard deviation of DFI (SD-DFI) and high DNA stainability (HDS), and the reproductive outcomes were biochemical pregnancy (BP), clinical pregnancy (CP) and implantation ratio (IR). RESULTS: The results showed no significant difference in the fertility variables BP, CP and IR when <27% DFI was used between the IVF and ICSI groups. A low number of patients received IUI with low success rate, and statistical analysis was therefore not performed. Ongoing pregnancy was achieved for both IVF and ICSI couples with DFI levels >27%, and six couples in ICSI treatment achieved CP full-term. DFI >27% had a high prognostic power for predicting no CP for IVF patients, with a specificity of 97%. Couples diagnosed with male infertility had a significantly higher level of DFI compared to couples with idiopathic fertility. Sperm head morphology showed low but significant correlations with the SCSA variables. CONCLUSION: SCSA is a useful tool in andrological diagnosis and contributes with a prognosis for the fertility outcome of conventional IVF. Although full-term pregnancy can be achieved with assisted reproductive techniques with a DFI >27%, the probability of a successful pregnancy may be reduced.

Bungum, M., P. Humaidan, et al. (2004). Hum Reprod 19(6): 1401-1408.

INTRODUCTION: Sperm chromatin integrity assessment has been suggested as a fertility predictor. The aim of this study was to examine the relationship between the results of sperm chromatin structure assay (SCSA) and the outcome of IVF, ICSI and intrauterine insemination (IUI). METHODS: A total of 306 consecutive couples undergoing assisted reproduction were included. IUI was performed in 131, IVF in 109 and ICSI in 66. SCSA results were expressed as DNA fractionation index (DFI) and highly DNA stainable (HDS) cell fractions. Reproductive outcome parameters were biochemical pregnancy (BP), clinical pregnancy (CP) and delivery (D). RESULTS: For IUI, the chance of pregnancy/delivery was significantly higher in the group with DFI 27% or HDS >10%. The odds ratios (ORs) (95% confidence intervals) were 20 (2.3-117), 16 (1.9-137) and 14 (1.6-110) for BP, CP and D, respectively. No statistical difference between the outcomes of IVF versus ICSI was observed in the group with DFI 27% group, however, the results of ICSI were significantly better than those of IVF. Comparing ICSI with IVF, the OR (95% CI) for BP was 26 (1.9-350). CONCLUSIONS: SCSA is a useful method for prediction of the outcome of assisted reproduction.

Bungum, M., P. Humaidan, et al. (2007). Hum Reprod 22(1): 174-179.

BACKGROUND: The sperm chromatin structure assay (SCSA) has been suggested as a predictor of fertility in vivo as well as in vitro. The available data however, have been based on limited numbers of treatments. We aimed to define the clinical role of SCSA in assisted reproduction. METHODS: A total of 998 cycles [387 intrauterine insemination (IUI), 388 IVF and 223 ICSI] from 637 couples were included. SCSA results were expressed as DNA fragmentation index (DFI) and high DNA stainable (HDS) cell fractions. Outcome parameters were biochemical pregnancy (BP), clinical pregnancy (CP) and delivery (D). RESULTS: For IUI, the odds ratios (ORs) for BP, CP and D were significantly lower for couples with DFI >30% as compared with those with DFI < or =30%. No statistical difference between the outcomes of ICSI versus IVF in the group with DFI < or =30% was seen. In the DFI >30% group, the results of ICSI were significantly better than those of IVF. CONCLUSIONS: DFI can be used as an independent predictor of fertility in couples undergoing IUI. As a result, we propose that all infertile men should be tested with SCSA as a supplement to the standard semen analysis. When DFI exceeds 30%, ICSI should be the method of choice.

Carrell, D. T., L. Liu, et al. (2003). Arch Androl 49(1): 49-55.

Previous studies have indicated that sperm quality may be related to unexplained recurrent pregnancy loss. This study evaluated the degree of sperm DNA fragmentation using the TUNEL assay on sperm from 24 couples with unexplained recurrent pregnancy loss (RPL) compared to sperm from 2 control groups: donors of known fertility and unscreened men from the general population. The percentage of sperm staining positive for DNA fragmentation was increased (p < .001) in the RPL group (38 +/- 4.2) compared to the donor (11.9 +/- 1.0) or general population (22 +/- 2.0) control groups. In the RPL group, no correlation was observed between semen quality parameters and the TUNEL data. These data indicate that some RPL patients have a significant increase of sperm DNA fragmentation, which may be causative of pregnancy loss in some patients.

De Jonge, C. (2002). Hum Fertil (Camb) 5(2): 51-53.

Traditional semen analysis is essential for the diagnosis of male infertility. A number of studies over the past decade have reported that a significant contributing factor to male fertility that is not revealed as part of semen analysis is sperm DNA, specifically its composition and organization. Exogenous and endogenous factors can cause damage to sperm DNA. For example, topoisomerase II activity, which is necessary for sperm DNA packaging, can adversely influence the competence of sperm DNA if the activity of the enzyme is abnormal. Germ cell apoptosis can be induced by oxygen radicals produced from environmental (for example cigarette smoke) or testicular (for example localized ischaemia) sources. Several assays have been developed that are useful for assessing sperm DNA composition and organization. To date, each of these assays has revealed that when sperm DNA has been damaged or packaged improperly there is a concomitant and often significant decline in male fertility.

Evenson, D. P., L. K. Jost, et al. (1999). Hum Reprod 14(4): 1039-1049.

The sperm chromatin structure assay (SCSA) was used to measure over 500 human semen samples from two independent studies: Study I, 402 samples from 165 presumably fertile couples wishing to achieve pregnancy over 12 menstrual cycles; Study II, samples from 115 patients seeking fertility counselling. The SCSA measures susceptibility to DNA denaturation in situ in spermatozoa exposed to acid for 30 s, followed by acridine orange staining. SCSA data from the male partners of 73 couples (group 1) achieving pregnancy during months 1-3 of Study I were used as the standard of 'sperm chromatin compatible with high fertility' and were significantly different from those of 40 couples (group 3) achieving pregnancy in months 4-12 (P < 0.01) and those of male partners of 31 couples (group 4) not achieving pregnancy (P < 0.001). Group 2 contained couples who had a miscarriage. SCSA values for Study II were almost twice that of the Study I fertility standards. Within-couple repeatability tended to be less for group 3 than for groups 1, 2 or 4. Based on logistic regression, spermatozoa with denatured DNA (cells outside the main population, COMP alpha t) were the best predictor for whether a couple would not achieve pregnancy. Some 84% of males in group 1 had COMP alpha t < 15%, while no couples achieved pregnancy in group 1 with > or = 30% COMP alpha t, a threshold level considered not compatible with good fertility. Using selected cut-off values for chromatin integrity, the SCSA data predicted seven of 18 miscarriages (39%).

Evenson, D. P., K. L. Larson, et al. (2002). J Androl 23(1): 25-43.

No abstract available

Giwercman, A., L. Lindstedt, et al. (2009). Int J Androl.

Summary Standard sperm parameters have a limited power for prediction of the chance of natural conception. Recent studies have indicated that the sperm chromatin structure assay (SCSA) DNA fragmentation index (DFI), a measure for the fraction of sperms with DNA damage, is associated with fertility in vivo. The aim of this study was to evaluate the value of this parameter for prediction of infertility. One hundred and twenty-seven men from infertile couples with no known female factor and 137 men with proven fertility were included. Semen analysis was performed as recommended by the WHO. DFI was assessed using SCSA. Logistic binary regression was used to compute the odds ratios (OR) for infertility. As compared with men with a DFI <10%, men with a DFI between 10% and 20% had an increased risk for infertility (OR 2.5, 95% CI: 1.0-6.1). This was also true for men with a DFI >20% (OR 8.4; 95% CI: 3.0-23). In men with normal standard semen parameters (sperm concentration, motility and morphology) the OR for infertility was increased with DFI >20% (OR 5.1, 95% CI: 1.2-23), whereas if one of the standard semen parameters was abnormal, the OR for infertility was increased already at DFI above 10% (OR 16, 95% CI: 4.2-60). We conclude that SCSA DFI adds to the value of semen analysis in prediction of the chance of natural conception.

Henkel, R., M. Hajimohammad, et al. (2004). Fertil Steril 81(4): 965-972.
OBJECTIVE: To investigate sperm DNA damage in relation to fertilization and pregnancy. DESIGN: Prospective study. SETTING: The Institute of Reproductive Medicine, Giessen, Germany. PATIENT(S): Semen collected from 249 patients attending the IVF program. MAIN OUTCOME MEASURE(S): The percentage of terminal deoxynucleotidyl transferase-mediated dUDP nick-end labeling- (TUNEL-), Fas-, and annexin-V-positive sperm and the proportion of green-fluorescing sperm in the acridine orange stain was determined and correlated with sperm concentration, motility, fertilization, and pregnancy. RESULT(S): Significant correlations with the concentration of motile sperm were only found for the acridine orange stain (before and after sperm separation) and for the TUNEL assay (after sperm separation). Moreover, patients whose sperm had a high percentage of DNA fragmentations showed significantly lower pregnancy rates (TUNEL assay: 19.05% vs. 34.65%; acridine orange stain: 24.58% vs. 37.93%). The apoptosis parameters (annexin V binding and Fas expression) showed no statistically significant differences. CONCLUSIONS: Our data clearly demonstrate that DNA fragmentation, as determined by the TUNEL assay, is predictive for pregnancy in IVF. This implies that spermatozoa with DNA fragmentation can still fertilize an oocyte but that when paternal genes are 'switched on,' further embryonic development stops, resulting in failed pregnancy. It seems that, at least in the patients we analyzed, apoptosis in the sperm does not play a role for fertilization. This would imply that DNA fragmentation in human spermatozoa is caused by external factors, such as reactive oxygen species, rather than by apoptosis.

Henkel, R., E. Kierspel, et al. (2003). Reprod Biomed Online 7(4): 477-484.
Despite the ever-increasing knowledge of the fertilization process, there is still a need for better understanding of the causes of sperm DNA fragmentation and its impact on fertilization and pregnancy. For this reason, human sperm DNA fragmentation was investigated by means of the terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling (TUNEL) assay and the production of reactive oxygen species (ROS) in the ejaculate and in the spermatozoa themselves. These data were correlated with fertilization and pregnancy data from IVF and intracytoplasmic sperm injection (ICSI) patients. Sperm DNA fragmentation did not correlate with fertilization rate, but there was a significantly reduced pregnancy rate in IVF patients inseminated with TUNEL-positive spermatozoa. ICSI patients exhibited the same tendency. This implies that spermatozoa with damaged DNA are able to fertilize an oocyte, but at the time the paternal genome is switched on, further development stops. The determination of ROS in the ejaculate and the percentage of ROS-producing spermatozoa revealed markedly stronger correlations between sperm functions (i.e. motility) and the percentage of ROS-producing spermatozoa. The influence of seminal leukocytes, known to produce large amounts of oxidants, on sperm DNA fragmentation should not be neglected.

Huang, C. C., D. P. Lin, et al. (2005). Fertil Steril 84(1): 130-140.

OBJECTIVE: To evaluate sperm DNA fragmentation in correlation with sperm parameters and IVF/intracytoplasmic sperm injection (ICSI) outcomes. DESIGN: Retrospective review. SETTING: A tertiary infertility referral clinic. PATIENT(S): We collected 303 semen samples from patients undergoing IVF with or without ICSI. INTERVENTION(S): Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) assay, measurement of fertilization rates, good embryo rates, and pregnancy rates for the IVF/ICSI program. MAIN OUTCOME MEASURE(S): The percentage of sperm with DNA fragmentation, correlated with semen analysis parameters and IVF/ICSI outcomes. RESULT(S): Sperm DNA fragmentation rates were significantly higher in patients with abnormal sperm parameters than in those with normal sperm parameters. When sperm DNA fragmentation was >10%, fertilization rates were affected. Sperm DNA fragmentation rates were negatively correlated with sperm velocity parameters but did not affect pregnancy outcomes. CONCLUSION(S): The results indicated that sperm DNA fragmentation affects fertilization rates and sperm motility but might not affect pregnancy rates.

Larson, K. L., C. J. DeJonge, et al. (2000). Hum Reprod 15(8): 1717-1722.

The predictive value of sperm chromatin integrity for pregnancy outcome following in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) was studied in 24 men attending a university-based assisted reproductive techniques laboratory using the flow cytometric sperm chromatin structure assay (SCSA). The SCSA is a measure of the susceptibility of sperm DNA to low pH-induced denaturation in situ. The mean percentage of spermatozoa in the neat sample demonstrating DNA denaturation was significantly lower in the seven men that initiated a pregnancy (15.4 +/- 4.6, P = 0.01) than in the 14 men who did not initiate a pregnancy (31.1 +/- 3.2). No pregnancies resulted if > or =27% of the spermatozoa in the neat semen sample showed DNA denaturation. These data demonstrate that SCSA parameters are independent of conventional semen parameters. Furthermore, the SCSA may allow physicians to identify male patients for whom IVF and ICSI will be unlikely to result in pregnancy initiation.

Makhlouf, A. A. and C. Niederberger (2006). J Androl 27(3): 316-323.

No abstract available

Muriel, L., N. Garrido, et al. (2006). Fertil Steril 85(2): 371-383.

OBJECTIVE: To determine the prognostic value of sperm DNA fragmentation levels, as measured by the sperm chromatin dispersion (SCD) test, in predicting IVF and ICSI outcome. DESIGN: Double-blind prospective study. SETTING: University-affiliated private IVF setting. PATIENT(S): A total of 85 couples undergoing infertility treatment with IVF/ICSI. INTERVENTION(S): Analysis of DNA fragmentation by the SCD test in 170 aliquots obtained from the ejaculate and from the processed semen used for assisted reproductive technologies (ART). MAIN OUTCOME MEASURE(S): Percentage of spermatozoa with fragmented DNA was statistically correlated with embryo quality and reproductive success. RESULT(S): Fertilization rate was inversely correlated with DNA fragmentation (r = -0.245 P = .045). Higher DNA fragmentation rate gave an increased proportion of zygotes showing asynchrony between the nucleolar precursor bodies of zygote pronuclei (73.8% vs. 28.8% P < .001). In addition, the slower embryo development and worst morphology on day 6 was correlated with higher sperm DNA fragmentation (47.7% vs. 29.4% P = .044). We also observed a negative correlation between DNA fragmentation and the implantation rate (r = -0.250 P = .042). However, SCD test values were not statistically different in cycles that resulted in a pregnancy compared with those that did not (33.2 vs. 28.2 and 32.4 vs. 34.7). CONCLUSION(S): This is the first report that describes a correlation between sperm DNA integrity, as measured by the SCD test, and fertilization rate, embryo quality, and implantation rate in IVF/ICSI. The degree of DNA fragmentation was inversely correlated with fertilization rate, synchrony of the nucleolar precursor bodies' pattern in pronuclei, embryo ability to achieve blastocyst stage, and embryo morphological quality. Because SCD test values were correlated with embryo quality and blastocyst rate, the lack of correlation between sperm DNA fragmentation and pregnancy outcome in IVF might be due to embryo selection before transfer. The ability of the SCD test to predict the blastocyst rate after IVF/ICSI warrants further study.

Payne, J. F., D. J. Raburn, et al. (2005). Fertil Steril 84(2): 356-364.

OBJECTIVE: To test the hypothesis that couples with sperm chromatin structure assay (SCSA) DNA fragmentation index (DFI) values >27% would not achieve pregnancy with assisted reproductive techniques (ART) and to investigate how DFI and high DNA stainability (HDS), as measured by the SCSA, affect fertilization, cleavage, implantation, and pregnancy rates in IVF cycles. DESIGN: Prospective clinical study. SETTING: Academic human reproduction laboratory. PATIENT(S): One hundred couples undergoing IVF with conventional insemination or intracytoplasmic sperm injection. INTERVENTION(S): Testing with SCSA was performed by SCSA Diagnostics (Brookings, South Dakota) on a semen aliquot taken from ejaculate used for ART. MAIN OUTCOME MEASURE(S): Relating total DFI and HDS to conventional semen parameters and cycle-specific outcomes after ART. RESULT(S): Nine of nineteen couples achieved clinical pregnancy when DFI was > or =27%, and 2 of 22 couples achieved clinical pregnancy when DFI was < or =9%. One of nine couples achieved clinical pregnancy with HDS >17%. The DFI was negatively correlated with sperm density (r = -0.23, P<.03) and motility (r = -0.55, P<.00), and HDS was negatively correlated with sperm density (r = -0.37, P<.00). CONCLUSION(S): Sperm chromatin structure assay failed to identify elevated DFI thresholds for negative pregnancy outcome after ART. Patients with low DFI (< or =9%) were least likely to become pregnant, which is also contradictory to SCSA marketing, which states that DFIs of < or =15% have excellent fertility potential. Patients with HDS > or =17% had low pregnancy rates, indicating decreased fertility potential, which deserves further investigation. Larger studies are necessary to confirm that low DFI is associated with decreased fertility and, if proved, might redefine the use of the SCSA in evaluating infertility.

Rawe, V. Y., H. U. Boudri, et al. (2010). Reprod Biomed Online 20(3): 320-323.

Magnetic activated cell sorting (MACS) with annexin V microbeads recognizes externalized phosphatidylserine (PS) residues on the surface of apoptotic spermatozoa. The successful use of this novel technique applied to a highly apoptotic semen sample before performing intracytoplasmic sperm injection (ICSI) is reported here. The use of annexin V microbeads for selecting non-apoptotic spermatozoa seems to reduce the percentage of altered cells, improving the chance of pregnancy after ICSI.

Rylander, L., B. Wetterstrand, et al. (2009). Asian J Androl 11(6): 723-730.

It is generally thought that a single ejaculate is a bad predictor of semen quality of a subject, because of significant intra-individual variation. Therefore, we investigated the degree to which the results of a first semen analysis differ from that of a second analysis among men from a general population in Norway. In addition, we analysed how the two different semen results mirrored the overall semen quality assessment. A total of 199 volunteers participated in the study and delivered two semen samples with an interval of 6 months. The semen parameters were determined according to the World Health Organization (WHO) 1999 guidelines, which were also used to determine whether semen quality was normal or abnormal. In addition, the DNA fragmentation index (DFI) was determined using the Sperm Chromatin Structure Assay. The two samples from each individual were very similar with regard to standard semen parameters and DFI (r(s:) 0.67-0.72), and there were no significant systematic differences between the two samples. The result of the first sample (normal/abnormal) was highly predictive of the overall conclusion based on the two samples (sperm concentration: in 93% of the cases (95% confidence interval [CI]: 89%-96%); sperm motility: in 85% of the cases (95% CI: 79%-89%); overall semen quality: in 85% of the cases (95% CI: 80%-90%). In epidemiological studies, one ejaculate is a sufficient indicator of semen quality in a group of subjects. In a clinical situation, when the question is whether the semen quality is normal or not, the first ejaculate will, in at least 85% of cases, give a correct overall conclusion.

Sakkas, D. and J. G. Alvarez (2010). Fertil Steril 93(4): 1027-1036.

OBJECTIVE: To review the mechanisms responsible for DNA fragmentation in human sperm, including those occurring during spermatogenesis and transport through the reproductive tract. The mechanisms examined include: apoptosis in the seminiferous tubule epithelium, defects in chromatin remodeling during the process of spermiogenesis, oxygen radical-induced DNA damage during sperm migration from the seminiferous tubules to the epididymis, the activation of sperm caspases and endonucleases, damage induced by chemotherapy and radiotherapy, and the effect of environmental toxicants. The different tests currently used for sperm DNA fragmentation analysis and the factors that determine the predictive value of sperm DNA fragmentation testing and their implications in the diagnosis and treatment of infertility are also discussed. Finally, we also scrutinize how the presence in the embryonic genome of DNA strand breaks or modifications of DNA nucleotides inherited from the paternal genome could impact the embryo and offspring. In particular we discuss how abnormal sperm could be dealt with by the oocyte and how sperm DNA abnormalities, which have not been satisfactorily repaired by the oocyte after fertilization, may interfere with normal embryo and fetal development. CONCLUSION(S): Sperm DNA can be modified through various mechanisms. The integrity of the paternal genome is therefore of paramount importance in the initiation and maintenance of a viable pregnancy both in a natural conception and in assisted reproduction. The need to diagnose sperm at a nuclear level is an area that needs further understanding so that we can improve treatment of the infertile couple.

Saleh RA, A. A., Essam A. Nada, Mohamed H. El-Tonsy, Donald P. and K. L. Evenson (2004). Fertil Steril 79(Suppl 3): 8.

No abstract available

Simon, L., G. Brunborg, et al. (2010). Hum Reprod.

BACKGROUND Sperm DNA damage shows great promise as a biomarker of infertility. The study aim is to determine the usefulness of DNA fragmentation (DF), including modified bases (MB), to predict assisted reproduction treatment (ART) outcomes. METHODS DF in 360 couples (230 IVF and 130 ICSI) was measured by the alkaline Comet assay in semen and in sperm following density gradient centrifugation (DGC) and compared with fertilization rate (FR), embryo cumulative scores (ECS(1)) for the total number of embryos/treatment, embryos transferred (ECS(2)), clinical pregnancy (CP) and spontaneous pregnancy loss. MB were also measured using formamidopyrimidine DNA glycosylase to convert them into strand breaks. RESULTS In IVF, FR and ECS decreased as DF increased in both semen and DGC sperm, and couples who failed to achieve a CP had higher DF than successful couples (+12.2% semen, P = 0.004; +9.9% DGC sperm, P = 0.010). When MB were added to existing strand breaks, total DF was markedly higher (+17.1% semen, P = 0.009 and +13.8% DGC sperm, P = 0.045). DF was not associated with FR, ECS or CP in either semen or DGC sperm following ISCI. In contrast, by including MB, there was significantly more DNA damage (+16.8% semen, P = 0.008 and +15.5% DGC sperm, P = 0.024) in the group who did not achieve CP. CONCLUSIONS DF can predict ART outcome for IVF. Converting MB into further DNA strand breaks increased the test sensitivity, giving negative correlations between DF and CP for ICSI as well as IVF.

Smit, M., J. C. Romijn, et al. (2009). Fertil Steril.

OBJECTIVE: To establish the diagnostic value of sperm chromatin structure assessment for the evaluation of male factor infertility, in addition to conventional andrological workup. DESIGN: Cross-sectional controlled study. SETTING: A tertiary referral andrology clinic. PATIENT(S): Two hundred seventy-nine male partners of infertile couples. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The DNA fragmentation index (DFI) determined by the sperm chromatin structure assay (SCSA), semen parameters, serum levels of reproductive hormones, and World Health Organization (WHO) classification of male factor subfertility. RESULT(S): In all patient categories, except those including patients with hypogonadotrophic hypogonadism, sperm antibodies, or normospermia, DFI was significantly higher compared with in proven fertile controls. After classification of the quality of spermatogenesis based on mean testicular volume (<10 ml vs. >15 ml), follicle stimulating hormone (FSH; > 10 U/L vs. <5 U/L), and inhibin-B (<100 nmol/L vs. >150 nmol/L), the DFI was significantly higher in patients with poor spermatogenesis (35.9%) than in patients with normal spermatogenesis (25.9%). In a multiple regression analysis, the teratozoospermia index, sperm vitality, and FSH were significant determinants of the DFI level. Male age was associated with DFI, but leukocytospermia, body mass index, and smoking were not confounders of DFI. CONCLUSION(S): Impaired spermatogenesis, irrespective of the WHO classification of male factor subfertility, is generally associated with an increase of sperm DNA damage.

Tarlatzis, B. C. and D. G. Goulis (2010). Gynecol Endocrinol.

No abstract available

Tarozzi, N., D. Bizzaro, et al. (2007). Reprod Biomed Online 14(6): 746-757.

Many studies have shown how a 'paternal effect' can cause repeated assisted reproduction failures. In particular, with increasing experience of intracytoplasmic sperm injection (ICSI), it became evident that spermatozoa from some patients repeatedly fail to form viable embryos, although they can fertilize the oocyte and trigger early preimplantation development. Many authors have shown how this paternal effect can be traced back to anomalies in sperm chromatin organization: the spermatozoa of subfertile men are characterized by numerical abnormalities in spermatozoal chromosome content, Y chromosome microdeletions, alterations in the epigenetic regulation of paternal genome and non-specific DNA strand breaks. In particular, pathologically increased sperm DNA fragmentation is one of the main paternal-derived causes of repeated assisted reproduction failures in the ICSI era. The intention of this review is to describe nuclear sperm DNA damage, with emphasis on its clinical significance and its relationship with male infertility. Assessment of sperm DNA damage appears to be a potential tool for evaluating semen samples prior to their use in assisted reproduction, helping to select spermatozoa with intact DNA or with the least amount of DNA damage for use in assisted conception.

Virro, M. R., K. L. Larson-Cook, et al. (2004). Fertil Steril 81(5): 1289-1295.

OBJECTIVE: To determine the relationship between sperm chromatin structure assay (SCSA) parameters (DNA fragmentation index [DFI] and high DNA stainability [HDS]), and conventional IVF and IVF/intracytoplasmic sperm injection (ICSI) outcomes. DESIGN: Retrospective review and prospective study. SETTING: Private IVF clinic. PATIENT(S): Two hundred forty-nine couples undergoing first IVF and/or ICSI cycle. INTERVENTION(S): IVF, ICSI, blastocyst culture. MAIN OUTCOME MEASURE(S): DFI, HDS, conventional semen parameters, IVF, ICSI. RESULT(S): IVF and ICSI fertilization rates were not statistically different between high- and low-DFI groups. More men with > or =15% HDS had lower (<25% and <50%) IVF fertilization rates. High DNA stainability was not related to ICSI fertilization rates. High DNA stainability did not affect blastocyst rates or pregnancy outcomes. Men with > or =30% DFI were at risk for low blastocyst rates (<30%) and no ongoing pregnancies. Men with > or =30% DFI had more male factors. World Health Organization thresholds were not predictive of ongoing pregnancy. CONCLUSION(S): The relationship between HDS and poor IVF fertilization rates provides preliminary evidence that ICSI may be indicated in men with > or =15% HDS. Men with high levels of DNA fragmentation (> or =30% DFI) were at greater risk for low blastocyst rates and failure to initiate an ongoing pregnancy. The SCSA provides valuable prognostic information to physicians counseling couples before IVF and/or ICSI cycles.

Zini, A. and M. Sigman (2009). J Androl 30(3): 219-229.

The advent of assisted reproductive technologies, particularly intracytoplasmic sperm injection (ICSI), has revolutionized the treatment of male-factor infertility. However, there are many unanswered questions regarding the safety of these techniques. These safety concerns are relevant because 1) these technologies often bypass the barriers to natural selection; 2) infertile men, particularly those with severe male-factor infertility, possess substantially more sperm DNA damage than do fertile men; and 3) experimentally, sperm DNA damage has been shown to adversely affect the developing embryo. This review discusses the etiology of sperm DNA damage, describes the individual tests of sperm DNA damage, and explores the relationship between sperm DNA damage and pregnancy outcomes. Based on a systematic review of the literature, sperm DNA damage is associated with lower natural, intrauterine insemination (IUI), and in vitro fertilization (IVF) pregnancy rates, but not with ICSI pregnancy rates. The literature also suggests that that sperm DNA damage is associated with an increased risk of pregnancy loss in those couples undergoing IVF or ICSI. Nonetheless, the true clinical utility of sperm DNA damage tests remains to be established, because the available studies are small and few in number and the study characteristics are heterogeneous. Although current data suggest that impaired sperm DNA integrity may have the greatest effect on IUI pregnancy rates and pregnancy loss by IVF and ICSI, further prospective studies are needed before testing should become a routine part of patient management.

Zini, A., J. M. Boman, et al. (2008). Hum Reprod 23(12): 2663-2668.

BACKGROUND: Sperm DNA damage is common amongst infertile men and may adversely impact natural reproduction, IUI-assisted reproduction and to a lesser degree IVF pregnancy. The aim of this study was to examine the influence of sperm DNA damage on the risk of spontaneous pregnancy loss after IVF and ICSI. METHODS: We conducted a systematic review and meta-analysis of studies on sperm DNA damage and pregnancy loss after an IVF and/or ICSI pregnancy. RESULTS: Two by two tables were constructed and odds ratios (ORs) were derived from 11 estimates of pregnancy loss (five IVF and six ICSI studies from seven reports). These 11 studies involved 1549 cycles of treatment (808 IVF and 741 ICSI cycles) with 640 pregnancies (345 IVF and 295 ICSI) and 122 pregnancy losses. The combined OR of 2.48 (95% CI 1.52, 4.04, P < 0.0001) indicates that sperm DNA damage is predictive of pregnancy loss after IVF and ICSI. CONCLUSIONS: In conclusion, sperm DNA damage is associated with a significantly increased risk of pregnancy loss after IVF and ICSI. These data provide a clinical indication for the evaluation of sperm DNA damage prior to IVF or ICSI and a rationale for further investigating the association between sperm DNA damage and pregnancy loss.

Mehdi, M., L. Khantouche, et al. (2009). Andrologia 41(6): 383-386.

To determine the prevalence of high levels of sperm DNA damage among infertile men with normal and abnormal semen parameters, 90 patients were subdivided into the following three groups. Group A (n = 30): men with normal semen parameters who acted as the controls. Group B (n = 30): asthenozoospermic men and group C (n = 30): teratozoospermic men, suffering from male infertility. DNA damage was evaluated by the rate of DNA fragmentation index (DFI) as assessed by the terminal desoxynucleotidyl transferase-mediated dUTP nick-end labelling assay. It was found that the difference was not significant between the percentage of DFI in patients with asthenozoospermia and the normospermic men (9.46% +/- 8.68 and 8.19 +/- 6.84 respectively, P-value not significant). The patients with teratozoospermia showed a significantly higher percentage of DNA fragmentation compared with the controls (respectively 21.37 +/- 17.26% and 8.19 +/- 6.84%, P < 0.001). There was a positive correlation between abnormal sperm morphology and the DFI (r = 0.44, P < 0.01) in group C. It is concluded that the impairments of sperm parameters were associated with an increase of DNA fragmentation; this association was strictly related to atypical forms.

Moskovtsev, S. I., J. Willis, et al. (2009). Urology 74(4): 789-793.

OBJECTIVES: To analyze the relationship between DNA damage and standard semen parameters (SSP) in patients who present for fertility evaluation. Evaluation of male fertility includes assessment of the SSP and increasingly sperm DNA damage. However, the relationship between DNA damage and SSP remains controversial. METHODS: Following Institutional Research Ethics Board approval, semen samples from 2586 unselected nonazoospermic patients were subjected to computer-assisted semen analysis and flow cytometry-based sperm DNA damage assessment expressed as the DNA fragmentation index. RESULTS: Sperm DNA damage was significantly negatively correlated to sperm SSP (concentration, motility, and normal morphology) and positively correlated to patient's age. DNA damage increased in association with the number of abnormalities in SSP. Patients with oligoasthenoteratozoospermia had significantly higher DNA damage and more frequent DNA damage over 30% compared with normozoospermic patients and patients with abnormalities in 1 or 2 SSP. CONCLUSIONS: Our results indicate that DNA damage is significantly correlated to SSP as well as age. In addition, the degree of DNA damage increases with the number of abnormal parameters in a sample and is most severe in patients with oligoasthenoteratozoospermia. Complex and possibly age-related mechanisms of DNA damage in human spermatozoa may be responsible for the strong relationship between SSP and DNA fragmentation index.

Zini, A., R. Bielecki, et al. (2001). Fertil Steril 75(4): 674-677.

OBJECTIVE: To evaluate two different assays of human sperm DNA integrity, DNA denaturation (DD) and DNA fragmentation (DF), and to correlate these with standard semen parameters. DESIGN: Prospective, observational study. SETTING: University infertility clinic.Patient(s): Forty consecutive semen samples from 33 nonazoospermic men presenting for infertility evaluation and 7 fertile men presenting for vasectomy. Intervention(s): Assessment of sperm concentration, motility, morphology, DD and DF. MAIN OUTCOME MEASURE(S): Sperm DD and DF in fertile and infertile men. RESULT(S): The mean (+/-SE) rates of DD and DF were significantly higher in infertile subjects compared to fertile controls, respectively: 25.4 +/- 3.0 vs. 10.2 +/- 2.3 (P=.028) and 27.6 +/- 2.5 vs. 13.3 +/- 2.5% (P=.016). DF and DD correlated strongly (r = 0.71, P<.0001). Also, DD and DF correlated negatively with standard semen parameters (concentration, motility, and morphology), the strongest correlation being with sperm motility. CONCLUSION(S): The strong correlation between sperm DD and DF, and the higher levels of sperm DNA damage in infertile compared with fertile men, indicate that male infertility is associated with poor sperm DNA integrity. Although infertile men may father children with assisted conception, fertilization with DNA-damaged spermatozoa may increase the risk of genetic disease in the offspring.

Gianaroli, L., M. C. Magli, et al. (2008). Fertil Steril.

OBJECTIVE: To verify clinical outcome after injection of spermatozoa that have undergone the acrosome reaction (reacted spermatozoa) vs. those still having an intact acrosome (nonreacted spermatozoa). DESIGN: Prospective, randomized study. SETTING: Reproductive Medicine Unit, Italian Society for the Study of Reproductive Medicine, Bologna, Italy. PATIENT(S): According to a prospective randomization including 71 couples with severe male factor infertility, intracytoplasmic sperm injection (ICSI) was performed under polarized light that permitted analysis of the pattern of birefringence in the sperm head. Twenty-three patients had their oocytes injected with reacted spermatozoa, 26 patient's oocytes were injected with nonreacted spermatozoa, and in 22 patients both reacted and nonreacted spermatozoa were injected. INTERVENTION(S): Intracytoplasmic sperm injection was performed under polarized light to selectively inject acrosome-reacted and acrosome-nonreacted spermatozoa. MAIN OUTCOME MEASURE(S): Rates of fertilization, cleavage, pregnancy, implantation, and ongoing implantation. RESULT(S): There was no effect on the fertilizing capacity and embryo development of either type of sperm, whereas the implantation rate was higher in oocytes injected with reacted spermatozoa (39.0%) vs. those injected with nonreacted spermatozoa (8.6%). The implantation rate was 24.4% in the group injected with both reacted and nonreacted spermatozoa. The delivery rate per cycle followed the same trend. CONCLUSION(S): Spermatozoa that have undergone the acrosome reaction seem to be more prone to supporting the development of viable ICSI embryos.

Lee, T. H., C. H. Liu, et al. (2010). Hum Reprod.

BACKGROUND Couples with unexplained infertility (UI) tend to have low fertilization rates with current IVF procedures. Here, we attempted to identify spermatozoa with apoptotic markers in couples with UI and unsuccessful intrauterine insemination (IUI) and we investigated the efficiency and benefit of magnetic-activated cell sorting (MACS) for sperm preparation in such patients. METHODS Sixty couples with UI and two IUI failures were recruited. The sperm were prepared by conventional density gradient centrifugation (DGC) and divided into two aliquots. One aliquot was used as a control and the other was further processed by MACS (D + M). Apoptotic markers were identified using fluorescence-labeled dye and flow cytometry, including externalization of phosphatidylserine (EPS), disrupted mitochondrial membrane potential (MMP) and DNA fragmentation. The fertilization potential of prepared spermatozoa was analyzed by basic semen analysis, computer-aided sperm analysis and the induced acrosome reaction test (IART). RESULTS After DGC, spermatozoa showed 18.6% EPS, 28.3% disrupted MMP and 13.5% DNA fragmentation. Numbers of spermatozoa with apoptotic markers were significantly reduced by D + M, versus DGC alone (P < 0.001). Although the motility of spermatozoa was slightly decreased after MACS, most sperm motion characteristics were not impaired. Interestingly, the IART significantly improved after D + M, versus DGC alone, especially for the couples with a normal hemizona assay (P < 0.001). CONCLUSIONS The spermatozoa prepared by D + M showed a reduced level of apoptotic markers. Improvement in the IART suggests a high fertilization potential of the processed spermatozoa. The identification of apoptotic markers and use of MACS may be helpful in directing the management plan for patients with UI and multiple IUI failures.

Nasr-Esfahani, M. H., S. Razavi, et al. (2008). J Assist Reprod Genet 25(5): 197-203.

PURPOSE: To compare the efficiency of routine sperm selection method with HA-selection procedure for fertilization rate, embryo development, implantation and pregnancy rates as well as evaluating the relationship between HA-binding ability with sperm protamine deficiency and DNA fragmentation. METHODS: Semen samples were obtained from the 50 couples undergoing ICSI. The percentage of fertilization rate, cleavage and quality of embryos compared between two procedures (routine sperm selection and HA-binding selection). The semen samples were assessed for DNA fragmentation and protamine deficiency by sperm chromatin dispersion (SCD) test and Chromomycin A3 (CMA3) staining, respectively. RESULTS: A significant inverse correlation was observed between percentage of HA binding with protamine deficiency, DNA fragmentation and abnormal sperm morphology (P < 0.05). Furthermore, in current study, oocytes inseminated by HA sperm selection procedure had significantly higher fertilization rate (P < 0.05). While the pregnancy and implantation rates were insignificantly increased. CONCLUSION: The results suggest that normal sperm have higher chance to bind HA and therefore, HA sperm selection procedure may select sperm with normal protamine content and low DNA fragmentation, but to confirm the effect of HA sperm selection on the ICSI outcome requires further studies.

Parmegiani, L., G. E. Cognigni, et al. (2010). J Assist Reprod Genet 27(1): 13-16.

PURPOSE: Hyaluronic Acid (HA) has a role as 'physiologic selector' for spermatozoa prior to intracytoplasmic sperm injection (ICSI). The objective of this study is to analyze the results achievable by the introduction of a routine HA-ICSI programme. METHODS: We retrospectively observed 293 couples treated with HA-ICSI versus 86 couples treated with conventional PVP-ICSI (historical control group). ICSI was performed on a limited number of oocytes per patient (1-3) according to Italian IVF law at the time of the study. Main outcome measures observed were: fertilization, embryo quality, implantation and pregnancy. RESULTS: This study showed that Injection of HA-bound spermatozoa (HA-ICSI) significantly improves embryo quality and implantation. CONCLUSIONS: If wider multi-center randomized studies will confirm these beneficial effects on ICSI outcome, HA could be considered as a routine choice for 'physiologic' sperm selection prior to ICSI.

Parmegiani, L., G. E. Cognigni, et al. (2010). Reprod Biomed Online 20(3): 437-438.

No abstract available

Polak de Fried, E. and F. Denaday (2010). Fertil Steril.

OBJECTIVE: To treat couples with intracytoplasmic sperm injection (ICSI) after annexin V sperm sorting. DESIGN: Two case reports. SETTING: Department of Reproductive Medicine at a private medical institute. PATIENT(S): Couples on infertility treatment, donor oocytes. INTERVENTION(S): Sperm sorted with annexin V magnetic microbeads before ICSI, day 3 embryo transfer; case 1: ovum donation; case 2: patient oocytes. MAIN OUTCOME MEASURE(S): 1) Sperm DNA fragmentation (terminal deoxynucleotide transferase-mediated dUTP nick-end labeling [TUNEL]) and active caspase-3 (immunocytochemistry); 2) fertilization rate, embryonic quality, blastocyst development of nontransferred embryos, and pregnancy outcome after ICSI of sorted sperm. RESULT(S): Case 1: Premature ovarian failure patient with previous fertilization failures: asthenoteratozoospermia, abnormal DNA fragmentation (TUNEL 30%; normal <20%). ICSI with annexin V-treated sperm done on six donated metaphase II (MII) oocytes; four fertilized, and a 5-cell/grade-2 and a 6-cell/grade-2-3 embryo were transfered. A day 5 blastocyst was cryopreserved. The patient was in the last trimester of gestation. Case 2: Couple with >4 years of primary infertility and recent ICSI failure. Semen with teratozoospermia (5% normal forms [Kruger]) and abnormal active caspase-3 (16%; normal <11%). ICSI with annexin V-treated sperm done on 9 MII oocytes. All fertilized; a 7-cell/grade-1 and an 8-cell/grade-1-2 embryo were transferred. A day 5 expanded blastocyst was cryopreserved. The patient was in the second trimester of a twin normal pregnancy. CONCLUSION(S): Sperm sorting with annexin V columns was effective in the treatment of two cases of ICSI failure, resulting in a single and a twin pregnancy after transfer of two embryos in each case.

Rawe, V. Y., H. U. Boudri, et al. (2010). Reprod Biomed Online 20(3): 320-323.

Magnetic activated cell sorting (MACS) with annexin V microbeads recognizes externalized phosphatidylserine (PS) residues on the surface of apoptotic spermatozoa. The successful use of this novel technique applied to a highly apoptotic semen sample before performing intracytoplasmic sperm injection (ICSI) is reported here. The use of annexin V microbeads for selecting non-apoptotic spermatozoa seems to reduce the percentage of altered cells, improving the chance of pregnancy after ICSI.

Tarozzi, N., M. Nadalini, et al. (2009). Reprod Biomed Online 19 Suppl 3: 35-43.

Hyaluronan has recently been employed in the development of a commercial diagnostic kit for assessing sperm maturity, the so-called sperm-hyaluronan-binding assay (HBA). The aim of this study was to evaluate the usefulness of this test, in addition to routine semen analysis, to identify patients with poor reproductive prognosis in conventional IVF. Furthermore, the ability of hyaluronan to select spermatozoa with low DNA fragmentation was investigated. A total of 60 IVF patients were analysed with regard to reproductive outcome, sperm parameters, HBA score and sperm DNA fragmentation. The DNA fragmentation analysis was performed using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling assay on the total sperm population and on the hyaluronan-bound spermatozoa obtained from the same samples. No relationship between hyaluronan binding and fertilization, cleavage, good-quality embryos, implantation, clinical pregnancy, miscarriages and biochemical pregnancy rates was found. Otherwise, correlations between spermatozoa hyaluronan binding and morphology (P < 0.01) and a significant difference between DNA fragmentation of the total sperm population and DNA fragmentation of the hyaluronan-bound spermatozoa (P = 0.029) were found. The results underline the ability of hyaluronan to select spermatozoa with higher DNA integrity and morphology. Nevertheless, the clinical value of the HBA in the management of male infertility seems to be limited.

Yagci, A., W. Murk, et al. (2010). J Androl.

During human spermiogenesis, the elongated spermatids undergo a plasma membrane remodeling step which facilitates formation of the zona pellucida and hyaluronic acid (HA)-binding sites. Various biochemical sperm markers indicated that human sperm bound to HA exhibit attributes similar to that of zona pellucida-bound sperm, including minimal DNA fragmentation, normal shape, and low frequency of chromosomal aneuploidies. In this work, we tested the hypothesis that HA-bound sperm, would be enhanced in sperm of high DNA chain integrity and green acridin orange fluorescence (AOF) as compared to the original sperm in semen. Sperm DNA integrity in semen and in their respective HA-bound sperm fractions was studied in 50 men tested for fertility workup. In the semen samples the proportions of sperm with green AOF (high DNA integrity) and red AOF (DNA breaks) were 54.9 +/- 2.0% and 45.0 +/- 1.9%, whereas in the HA-bound sperm fraction, the respective proportions were 99% and 1.0%. The data indeed demonstrated that HA show high degree of selectivity for sperm with high DNA integrity. These findings are important from the points of view of human sperm DNA integrity, sperm function, and the potential efficacy of hyaluronic acid mediated sperm selection for intracytoplasmic sperm injection.

Akmal, M., J. Q. Qadri, et al. (2006). J Med Food 9(3): 440-442.

This study was carried out to monitor the effect of oral supplementation of vitamin C on various semen parameters in oligospermic, infertile, otherwise healthy individuals. Various semen parameters, including sperm motility, sperm count, and sperm morphology, were studied before and after the vitamin C treatment. A total of 13 infertile patients were included. Their ages ranged between 25 and 35 years. They had no genital infection or varicocele. Physical examination and other routine laboratory investigations were normal. General semen analysis revealed oligozoospermia (mean sperm count was 14.3 +/- 7.38 x 10(6) sperms/mL, mean sperm with normal morphology was 43 +/- 7.87%, and mean sperm motility was 31.2 +/- 9.61%). Testicular biopsy was not done. These patients received in an open trial of 1,000 mg of vitamin C twice daily for a maximum of 2 months. Results showed that the mean sperm count was increased to 32.8 +/- 10.3 x 10(6) sperms/mL (P < .001) after 2 months of vitamin C intake. The mean sperm motility was increased significantly to 60.1 +/- 8.47% (P < .001), and mean sperms with normal morphology increased significantly to 66.7 +/- 4.77% (P < .001). This study showed that vitamin C supplementation in infertile men might improve sperm count, sperm motility, and sperm morphology and might have a place as an additional supplement to improve the semen quality towards conception.

Greco, E., M. Iacobelli, et al. (2005). J Androl 26(3): 349-353.

Sperm DNA fragmentation is known to compromise male fertility. Previous findings have suggested the implication of oxidative stress in the etiology of this pathological condition. The present study was conducted to find out if the pathologically increased incidence of DNA fragmentation in ejaculated spermatozoa can be reduced by oral treatment with two antioxidants, vitamins C and E. Sixty-four men with unexplained infertility and an elevated (> or = 15%) percentage of DNA-fragmented spermatozoa in the ejaculate were randomized between an antioxidant treatment (1 g vitamin C and 1 g vitamin E daily for 2 months) group and a placebo group. Sperm DNA fragmentation was evaluated by terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling assay before and after treatment. No differences in basic sperm parameters were found between the antioxidant treatment and the placebo group before or after treatment. However, the percentage of DNA-fragmented spermatozoa was markedly reduced (P < .001) in the antioxidant treatment group after the treatment (9.1 +/- 7.2) as compared with the pretreatment values (22.1 +/- 7.7). No difference in the pretreatment and posttreatment incidence of sperm DNA fragmentation was observed in the placebo group. These data show that sperm DNA damage can be efficiently treated with oral antioxidants administered during a relatively short time period.

Tremellen, K., G. Miari, et al. (2007). Aust N Z J Obstet Gynaecol 47(3): 216-221.

BACKGROUND: Evidence has accumulated supporting the role of reactive oxygen species (ROS) in the pathogenesis of sperm dysfunction among men with infertility. Damage to sperm DNA by ROS can lead to failure of conception, miscarriage or potentially even childhood cancer. The objective of this study was to examine the effect of male antioxidant treatment on embryo quality and pregnancy outcome during in vitro fertilisation-intracytoplasmic sperm injection (IVF-ICSI) treatment. METHODS: Sixty couples with severe male factor infertility were enrolled in a prospective randomised double-blind placebo-controlled trial. Male participants were randomly assigned to take either one capsule per day of the Menevit antioxidant or an identical in appearance placebo for three months prior to their partner's IVF cycle. The primary outcome was cleavage stage embryo quality and the secondary outcomes were oocyte fertilisation rate, pregnancy rates and treatment side-effects. Approval by the local Human Research Ethics Committee was obtained prior to the commencement of this study. RESULTS: The antioxidant group recorded a statistically significant improvement in viable pregnancy rate (38.5% of transferred embryos resulting in a viable fetus at 13 weeks gestation) compared to the control group (16% viable pregnancy). No significant changes in oocyte fertilisation rate or embryo quality were detected between the antioxidant and the placebo groups. Side-effects on the Menevit antioxidant were rare (8%) and mild in nature. CONCLUSIONS: The Menevit antioxidant appears to be a useful ancillary treatment that significantly improves pregnancy rates in couples undergoing IVF-ICSI treatment for severe male factor infertility.

Zini, A., M. San Gabriel, et al. (2009). J Assist Reprod Genet.

PURPOSE: Infertile men possess substantially more sperm DNA damage than do fertile men, damage that may impact negatively on reproductive outcomes. In this era of assisted reproductive technologies there is mounting concern regarding the safety of utilizing DNA-damaged spermatozoa in this setting. Therefore, it is important to identify strategies that may reduce sperm DNA damage. The purpose of this review is to discuss the rationale for antioxidant therapy in men with sperm DNA damage and to evaluate the data on the efficacy of dietary and in vitro antioxidant preparations on sperm DNA damage. METHODS: We reviewed the literature on antioxidants and sperm DNA damage. RESULTS: To date, the data suggest that dietary antioxidants may be beneficial in reducing sperm DNA damage, particularly, in men with high levels of DNA fragmentation. However, the mechanism of action of dietary antioxidants has not been established and most of the clinical studies are small. A beneficial effect of in vitro antioxidant supplements in protecting sperm DNA from exogenous oxidants has been demonstrated, however, the effect of these antioxidants in protecting sperm from endogenous ROS, gentle sperm processing and cryopreservation has not been established.

Dalzell, L. H., C. M. McVicar, et al. (2004). Fertil Steril 82(5): 1443-1445.

DNA fragmentation in testicular sperm from men with obstructive azoospermia is increased by 4-hour and 24-hour incubations and after cryopreservation with the effect is intensified by post-thaw incubation. Testicular sperm for use in intracytoplasmic sperm injection (ICSI) should be injected without delay.

Dalzell, L. H., M. E. Thompson-Cree, et al. (2003). Fertil Steril 79 Suppl 3: 1670-1672.

No abstract available

Greco, E., F. Scarselli, et al. (2005). Hum Reprod 20(1): 226-230.

BACKGROUND: Sperm DNA damage (fragmentation) is a recently discovered cause of male infertility for which no efficient treatment has yet been found. Previous findings have suggested that clinically relevant sperm DNA damage may occur at the post-testicular level. This study was undertaken to assess the clinical usefulness of ICSI with testicular spermatozoa in this indication. METHODS: The percentage of spermatozoa with fragmented DNA, assessed by terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labelling assay, and ICSI outcomes were compared in two sequential attempts performed, respectively, with ejaculated and testicular spermatozoa in 18 men with increased sperm DNA fragmentation. RESULTS: The incidence of DNA fragmentation was markedly lower in testicular spermatozoa as compared with ejaculated spermatozoa. No differences in fertilization and cleavage rates and in embryo morphological grade were found between the ICSI attempts performed with ejaculated and with testicular spermatozoa. However, eight ongoing clinical pregnancies (four singleton and four twin) were achieved by ICSI with testicular spermatozoa (44.4% pregnancy rate; 20.7% implantation rate), whereas ICSI with ejaculated spermatozoa led to only one pregnancy which was spontaneously aborted. CONCLUSIONS: These data show that ICSI with testicular spermatozoa provides the first efficient assisted reproduction treatment option for men with high levels of sperm DNA damage.

Meseguer, M., R. Santiso, et al. (2009). Fertil Steril 92(5): 1638-1645.

OBJECTIVE: To analyze sperm DNA fragmentation (SDF) in testicular sperm samples from patients with azoospermia either from spermatogenic failure or from duct obstruction. Several technologies can be applied in the evaluation of SDF, but given the ease and low costs, the sperm chromatin dispersion test (SCD) has emerged as a promising standard. DESIGN: Prospective blind observational cohort study. SETTING: University-affiliated private IVF setting. PATIENT(S): Azoospermic patients from couples undergoing intracytoplasmic sperm injection cycles. INTERVENTION(S): Testicular sperm extraction (TESE). MAIN OUTCOME MEASUREMENT(S): We determined testicular SDF, and a basic comparison between nonobstructive (n = 22) and obstructive azoospermia (n = 40) was performed. We also correlated SDF with embryo quality and pregnancy outcome. RESULT(S): SDF in the testicular sperm of patients with nonobstructive azoospermia was significantly higher, 46.92% (SEM = 4.47), than that of patients with obstructive azoospermia, 35.96% (SEM = 2.63). A moderate relationship between embryo morphology and testicular SDF was detected. Logistic regression analysis of the effect of testicular SDF on pregnancy outcome revealed no significant effect (odds ratio = 1.015). CONCLUSION(S): Ours is the first report of SDF analysis in testicular sperm by using SCD in azoospermia. This result suggests that spermatogenesis failure may result in a severe affectation of sperm DNA integrity. The degree of DNA fragmentation using the SCD test is not reflected in pregnancy chances, and the explanation could be that embryos have been selected.

Bertolla, R. P., A. P. Cedenho, et al. (2006). Fertil Steril 85(3): 625-628.

OBJECTIVE: To verify if sperm from adolescents with varicocele have an increased rate of DNA fragmentation when compared with adolescents without varicocele. DESIGN: Controlled prospective study. SETTING: Patients in an academic research environment. PATIENT(S): Adolescent patients with a clinical diagnosed bilateral varicocele, grades II and III, and adolescent patients without a varicocele. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Rate of sperm DNA fragmentation as assessed by the Comet assay, graded as class I (no DNA fragmentation), II (little DNA fragmentation), III (meaningful DNA fragmentation), or IV (high DNA fragmentation). RESULT(S): A higher percentage of cells with no DNA fragmentation (class I) was found in the nonvaricocele group (47.62 +/- 7.69) when compared with the varicocele group (27.52 +/- 10.73). A higher percentage of sperm with class III and class IV DNA fragmentation was found in the varicocele group (20.43 +/- 8.97, and 19.57 +/- 10.68) when compared with the nonvaricocele group (11.38 +/- 5.55, and 5.71 +/- 2.35). CONCLUSION(S): Although standard semen analysis showed no difference between the groups, adolescents with varicocele have an increase in sperm nuclear DNA fragmentation. Thus, DNA fragmentation evaluation could be important in deciding treatment options for adolescent patients.

Blumer, C. G., R. M. Fariello, et al. (2008). Fertil Steril 90(5): 1716-1722.

OBJECTIVE: To verify the impact of varicocele on semen quality and sperm function (DNA integrity and mitochondrial activity). DESIGN: Prospective study. SETTING: Patients in an academic research environment. PATIENT(S): Seventeen patients with a clinical diagnosed varicocele of grade II or III and 20 men without a varicocele. MAIN OUTCOME MEASURE(S): Rate of sperm DNA fragmentation as assessed by the Comet assay and categorized as classes I (no DNA fragmentation), II (little DNA fragmentation), III (meaningful DNA fragmentation), and IV (high DNA fragmentation). Rate of mitochondrial activity as assessed by the diaminobenzidine (DAB) assay and categorized as grades I (all mitochondria active), II (most mitochondria active), III (most mitochondria inactive), and IV (all mitochondria inactive). RESULT(S): No statistically significant differences were found between the study and control groups with respect to age, ejaculatory abstinence, and round cell count. Men with varicocele had significantly higher ejaculate volume, concentration of immotile sperm, and neutrophil count and lower mean percentage of sperm concentration, progressive motility, and morphology than men in the control group. The study group presented a lower percentage of sperm with little DNA fragmentation (class II) and a higher percentage of sperm with DNA fragmentation (class IV). In addition, the study group presented a greater percentage of sperm with inactive mitochondria (class III). CONCLUSION(S): Compared with men without varicocele, men with varicocele had a higher percentage of cells with DNA fragmentation and sperm with inactive mitochondria. Indeed, varicocele causes a decrease in motility, concentration, and morphology and an increase in volume and concentration of immotile sperm and neutrophils. The sperm functional evaluation (DNA fragmentation and mitochondrial activity) could be important factors in deciding treatment options for men with varicocele.

Enciso, M., L. Muriel, et al. (2006). J Androl 27(1): 106-111.

The frequency of sperm cells with fragmented DNA was studied in a group of 18 infertile patients with varicocele and compared with those obtained in a group of 51 normozoospermic patients, 103 patients with abnormal standard semen parameters, and 22 fertile men. The spermatozoa were processed to discriminate different levels of DNA fragmentation using the Halosperm kit, an improved Sperm Chromatin Dispersion (SCD) test. In this technique, after an acid incubation and subsequent lysis, those sperm cells without DNA fragmentation show big or medium-sized halos of dispersion of DNA loops from the central nuclear core. Otherwise, those spermatozoa containing fragmented DNA either show a small halo, exhibit no halo with solid staining of the core, or show no halo and irregular or faint stain of the remaining core. The latter, that is, degraded type, corresponds to a much higher level of DNA-nuclear damage. The varicocele patients showed 32.4% +/- 22.3% of spermatozoa with fragmented DNA, significantly different from the group of fertile subjects (12.6% +/- 5.0%). Nevertheless, this was not different from that of normozoospermic patients (31.3% +/- 16.6%) (P = .83) and with abnormal semen parameters (36.6% +/- 15.5%) (P = .31). No significant differences were found between the normozoospermic patients and the patients with abnormal semen parameters. Strikingly, the proportion of the degraded cells in the total of sperm cells with fragmented DNA was 1 out of 4.2 (23.9% +/- 12.9%) in the case of varicocele patients, whereas it was 1 out of 8.2 to 9.7 in the normozoospermic patients (11.1% +/- 9.9%) in the patients with abnormal sperm parameters (12.2% +/- 8.3%) and in the fertile group (10.3% +/- 7.2%). Thus, whereas no differences in the percentage of sperm cells with fragmented DNA were evident with respect to other infertile patients, individuals with varicocele exhibit a higher yield of sperm cells with the greatest nuclear DNA damage level in the population with fragmented DNA. This finding illustrates the value of assessing different patterns of DNA-nuclear damage within each sperm cell and the particular ability of the Halosperm kit to reveal them.

Gallegos, G., B. Ramos, et al. (2008). Fertil Steril 90(2): 328-334.

OBJECTIVE: To determine the frequency of sperm cells with fragmented DNA in semen samples from men with genitourinary infection by Chlamydia trachomatis and Mycoplasma and the influence of antibiotic therapy, using the sperm chromatin dispersion test with the Halosperm kit. DESIGN: Prospective study. SETTING: University-affiliated reproductive medicine center, medical genetics laboratory, and academic biology center. PATIENT(S): One hundred forty-three male member of couples attending the andrology infertility center and a group of 50 fertile subjects. The effect of antibiotic treatment was evaluated in 95 male patients. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Classical semen analysis (concentration, motility, morphology, and vitality), sperm DNA fragmentation, and clinical outcome. RESULT(S): The mean percentage of sperm cells with fragmented DNA was 35.2% +/- 13.5%, 3.2 times higher than in the control fertile group (10.8% +/- 5.6%). Concentration, morphology, and motility were also significantly affected but to a much lower degree. Sperm vitality was not significantly affected. After 3.8 +/- 2.2 months of antibiotic treatment, the mean frequency of spermatozoa with fragmented DNA decreased from 37.7% +/- 13.6% to 24.2% +/- 11.2%. Sperm concentration and motility were not significantly improved. In a group of 16 couples who attempted pregnancy during antibiotic treatment course, only 12.5% achieved pregnancy. However, in a group of 14 couples who attempted pregnancy after finishing the antibiotic treatment, 85.7% achieved it. The only significant differences found between groups was the rate of sperm DNA fragmentation and morphology. CONCLUSION(S): Patients with genitourinary infection by Chlamydia trachomatis and Mycoplasma have increased sperm DNA fragmentation in comparison with fertile controls. This increase is proportionally greater than the influence on classical semen parameters and could result in a decreased fertility potential. Antibiotic therapy appears to be important in providing a remedy for infection-induced high DNA fragmentation levels.

sperm concentration after varicocelectomy was applied, 31 of 49 patients (63%) responded to varicocelectomy. After varicocelectomy 37% of the couples conceived spontaneously and 24% achieved pregnancy with assisted reproductive technique. The mean postoperative DNA fragmentation index was significantly higher in couples who did not conceive spontaneously or with assisted reproductive technique (p = 0.033). CONCLUSIONS: After varicocelectomy sperm parameters significantly improved and sperm DNA fragmentation was significantly decreased. Low DNA fragmentation index values are associated with a higher pregnancy rate (spontaneous and with assisted reproductive technique). We suggest that varicocelectomy should be considered in infertile men with palpable varicocele, abnormal semen analysis and no major female factors.

Werthman, P., R. Wixon, et al. (2008). Fertil Steril 90(5): 1800-1804.

OBJECTIVE: To measure sperm DNA integrity values before and after varicocelectomy in patients with elevated preoperative levels of sperm DNA fragmentation. DESIGN: Retrospective. SETTING: Private urology clinic. PATIENT(S): Eleven patients with grade 1, 2, or 3 varicocele. INTERVENTION(S): Varicocelectomy. MAIN OUTCOME MEASURE(S): Sperm DNA fragmentation values were assessed before and after varicocelectomy. RESULTS(S): Ninety percent of the patients showed a significant decrease in sperm DNA fragmentation levels. CONCLUSIONS(S): Although this study was small, 10 of the 11 patients with varicocele showed a significant decrease in sperm DNA fragmentation after varicocele repair. Elevated sperm DNA fragmentation has been shown to have a significant negative effect on pregnancy outcome with use of in vivo, IUI, routine IVF, and to a lesser extent intracytoplasmic sperm injection fertilization; therefore pregnancy outcome may improve after varicocelectomy

Zini, A., R. Azhar, et al. (2010). Int J Androl.

Summary There is evidence from retrospective studies that varicocelectomy can improve sperm DNA damage in infertile men with a clinical varicocele. The objective of this prospective study was to examine further the effect of varicocelectomy on sperm chromatin and DNA integrity. We evaluated a consecutive series of infertile men (n = 25) who underwent microsurgical varicocelectomy for treatment of clinical varicocele. We examined conventional sperm parameters and sperm chromatin structure assay parameters (percentage DFI - DNA fragmentation index and percentage HDS - high DNA stainability, an index of chromatin compaction) before and 4 and 6 months after microsurgical varicocelectomy. Sperm DNA integrity improved significantly after surgery (percentage DFI decreased from 18 +/- 11% before surgery to 10 +/- 5%, and 7 +/- 3%, at 4 and 6 months after surgery respectively). Sperm chromatin compaction also improved significantly after surgery (percentage HDS decreased from 11 +/- 7% before surgery to 8 +/- 6%, and 7 +/- 5%, at 4 and 6 months after surgery, respectively). Sperm concentration and progressive motility improved after surgery, although the differences were not statistically significant when compared with that before surgery. The data show that varicocelectomy is associated with an improvement in sperm DNA integrity and chromatin compaction. These findings support the concept that correction of a varicocele can improve spermatogenesis, particularly spermiogenesis (the stage in spermatogenesis where compaction and stability of the sperm DNA and chromatin occur).