Sperm DNA integrity testing: big halo is a good predictor of embryo quality and pregnancy after conventional IVF

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1 ISSN: ORIGINAL ARTICLE Correspondence: Marijan Tandara, Department of Human Reproduction, Clinic for Women s Diseases and Obstetrics of University Hospital Center Split, Split, Croatia. marijan.tandara@inet.hr Keywords: big halo, conventional IVF, DFI, embryo quality, halosperm test, pregnancy Received: 25-Feb-2014 Revised: 19-Apr-2014 Accepted: 12-May-2014 doi: /j x Sperm DNA integrity testing: big halo is a good predictor of embryo quality and pregnancy after conventional IVF 1 M. Tandara, 1 A. Bajić, 2 L. Tandara, 3 L. Bilić-Zulle, 1 M. Šunj, 4 V. Kozina, 5 T. Goluza and 1 M. Jukic 1 Department of Human Reproduction, Clinic for Women s Diseases and Obstetrics of University Hospital Center Split, 2 Department of Medical Laboratory Diagnosis, University Hospital Center Split, Split, 3 Clinical Department of Laboratory Diagnosis, Clinical Hospital Center Rijeka and Department of Medical Informatics, Rijeka University School of Medicine, Rijeka, 4 Department of Histology and Embryology, and 5 Department of Gynecology and Obstetrics, School of Medicine, University of Zagreb, Zagreb, Croatia SUMMARY Sperm DNA integrity is a sperm functional parameter of male fertility evaluation. Two parameters of sperm DNA integrity were observed: DNA damage expressed as DNA fragmentation index (DFI) and percentage of the DNA undamaged spermatozoa expressed as big halo. Halosperm test was used for sperm DNA integrity determination. The aim of this study was to evaluate which DNA integrity parameter is better as an embryo quality and pregnancy prognostic parameter after the conventional IVF. We evaluated two embryo groups (positive and negative group) according to the 3rd day cumulative embryo score. Big halo and DFI, as we expected, showed good correlation (r = 0.69; p < 0.001). Receiver operating characteristic (ROC) analyses show that DFI and big halo are significant (p < 0.001) as prognostic parameters of embryo quality. ROC curves comparison of DFI and big halo revealed the AUC value for big halo to be significantly higher (DFI AUC = 0.71 vs. big halo AUC = 0.83; p = 0.025) than for DFI. Big halo was found to be the only independent predictor of embryo quality. Sperm DNA integrity both parameters are good prognostic parameters of embryo quality after the conventional IVF where big halo seems to be better. ROC analyses show DFI and big halo as significant prognostic parameters for achieved pregnancy (AUC SE for DFI was and for big halo). To our knowledge, this is the first study demonstrating the correlation between sperm DNA undamaged rate expressed as big halo parameter and semen characteristics as well as the influence on fertilization rate, embryo quality and pregnancy in conventional IVF. INTRODUCTION The use of assisted reproductive techniques (ART) has enhanced recently in treatment of those couples who cannot conceive naturally thus making infertility generally become not only medical but social issue as well (Simon et al., 2010). Over the years new infertility treatment methods have been developed and modified in hope of finding an ideal treatment for each infertile couple (Said & Land, 2011). Still, live birth rate after ART is relatively low and could be further improved (Said & Land, 2011). In a vast majority of fertility clinics male fertility evaluation is based on the determination of conventional semen parameters: concentration, motility and morphology (Barratt et al., 2010). However, conventional semen parameter analysis is insufficiently precise, poorly standardized and subjective, making diagnostic sensitivity of such evaluation low (Lewis, 2007). Sometimes it is hard to establish whether the male factor causes infertility or only contributes to it so the additional sperm function testing is required (Natali & Turek, 2010). The crucial thing is spermatozoa s ability to fertilize an oocyte regardless of the sperm number and motility, as ~15% men with fertility problem are normozoospermic patients (Agarwal & Allamaneni, 2004). Spermatozoa are a highly specialized cell undergoing various biological changes necessary to achieve fertilization (Aitken & Henkel, 2011). Although, there is no direct measurement of sperm fertilization capability, different assays were developed for better understanding of male fertility pathophysiology and aetiology mostly in 1980s. Unfortunately, the majority of these tests did not manage to become part of the ART laboratory practice. After intracytoplasmic sperm injection (ICSI) was introduced in routine treatment, andrology investigations became secondary whereas sperm role in fertilization and embryo development has been reduced only to father s genetic material transfer (Palermo et al., 1992; Said & Land, 2011). Studies have shown 678 Andrology, 2014, 2, American Society of Andrology and European Academy of Andrology

2 BIG HALO AS A EMBRYO QUALITY AND PREGNANCY PREDICTOR that ICSI has better success in achieving fertilization than conventional IVF especially in patients with poor semen samples (Xi et al., 2012). Successful oocytes fertilization by the ICSI has made this method used in almost 70% of all ART cycles according to the European IVF-monitoring program s report for 2010 (De Mouzon et al., 2010). However, ICSI represents an invasive method in which natural steps in sperm selection are bypassed and based on embryologist s subjective evaluation of fertilizing sperm morphology and motility. Thus, spermatozoa that perhaps would never have been part of the fertilizing pool in a natural sperm selection might fertilize an oocyte (Jakab et al., 2005). Therefore, using ICSI for oocytes fertilization without clear indication can lead to even lower fertilization and implantation rates than conventional IVF (Poehl et al., 2001). On the other hand, conventional IVF represents in vitro simulation of natural fertilization process where oocyte-sperm self-selection is present, with the assumption that only the best spermatozoa, morphologically normal and motile with undamaged DNA will fertilize an oocyte (Borini et al., 2006). Over the past decade, DNA integrity was recognized as a significant sperm functional parameter with an important role in human ART (Lewis et al., 2013). During spermatogenesis, sperm DNA is subjected to various changes to achieve highly organized, compact and condensed structure (Agarwal & Said, 2003). That unique chromatin structure protects father s genome from external influences enabling its correct transfer towards oocyte (Simon et al., 2010). The DNA integrity is the sperm functional parameter that together with conventional semen parameters gives more reliable and precise diagnosis of male reproductive potential (Agarwal & Allamaneni, 2004). Consequently, DNA integrity testing should be incorporated into standard semen analysis (Barratt et al., 2010). There have been a great deal of studies evaluating DNA fragmentation rate in human semen and its influence on fertilization, embryo development and quality, implantation and pregnancy outcome (Virro et al., 2004; Zini et al., 2008; Sakkas & Alvarez, 2010; Simon et al., 2010). A big variations in conclusions off all those studies indicates that our knowledge of sperm DNA damage mechanisms and their effects on reproductive outcomes is still controversial (Schulte et al., 2010). Understanding the complex sperm chromatin structure is necessary for finding the best possible method of assessing DNA integrity considering its influence on the outcome in ART (Agarwal & Said, 2003; Ward, 2010). So far, there is no unique test to determine sperm DNA integrity, only numerous test measuring DNA fragmentation of which some have become part of laboratory practice: TUNEL (Terminal dutp Nick-End Labeling) test, Comet test (Single Cell Gel Electrophoresis), SCSA (Sperm Chromatin Structure Assay) test and SCD (Sperm Chromatin Dispersion) test (Singh & Stephens, 1998; Evenson et al., 2002; Erenpreisa et al., 2003; Fernandez et al., 2003). Halosperm is the SCD test based on the ability of intact DNA with deproteinized nuclei to create loops around nuclear matrix. Deproteinized nuclei create the halos of dispersed DNA that correspond to relaxed DNA loops attached to the residual nuclear structure (core) (Fernandez et al., 2005). Nuclei with fragmented DNA produce either small halos (sh) or no halos (wh) of dispersed DNA. DNA fragmentation is expressed as DNA fragmentation index (DFI). In contrast, the sperm nuclei without DNA fragmentation release DNA creating big halos (bh) or those slightly fragmented creating medium halos (mh). Halosperm test, actually measures undamaged spermatozoa rather than spermatozoa with DNA fragmentation (Lewis et al., 2013). The aim of this study was to examine whether the percentage of spermatozoa with undamaged DNA showing big halo was better prognostic parameter of embryo quality and achieved pregnancy in conventional IVF outcome than DFI. MATERIALS AND METHODS Study design This was prospective observational study conducted at Department of Human Reproduction, Clinic for Women s Diseases and Obstetrics of University Hospital Center Split from September 2012 to May The study group represents consecutive cohort of male patients included in conventional IVF infertility couple treatment. Informed consent was signed by all participants. Study was approved by the ethical committee of the University Hospital Center Split. The study involved 88 male participants, average age of 37 years, ranging from 26 to 55 years. The following data were collected: sperm concentration, sperm morphology, sperm motility, sperm vitality, DFI, big halo, fertilization and embryo quality evaluation. Semen quality assessment Semen samples were collected by masturbation, on aspiration day, after 2 4 days of sexual abstinence. The samples were processed within an hour from ejaculation. Semen quality evaluation was made according to the WHO manual (World Health Organization, 2010). AutoSperm (Version 5, MedCalc Software, Ostend, Belgium) system for computer semen analysis was used for sperm concentration (10 6 spermatozoa/ml) and motility determination. Motility was determined as% of progressive motility (PR) or total motility (PR + NP, non-progressive motility). Reference value was > /ml for concentration and for motility was as follows: PR > 32% or PR + NP > 40% (World Health Organization, 2010). Smears (aliquot = 20 ll) were made for sperm morphology (% normal forms) determination and stained with Giemsa dye (Merck KGaA, Darmstadt, Germany) after dehydration in 96% alcohol. Two hundred spermatozoa were scored at 9400 magnification and a percentage of morphologically normal spermatozoa was determined with a lower reference value of 4% (World Health Organization, 2010). Hypoosmotic test was used for sperm vitality estimation. Slides were made and examined with a phase-contrast microscope at 9400 magnification, and 200 spermatozoa were counted. Vital spermatozoa with intact membranes create different flagellar shapes in hypoosmotic medium, while non-vital spermatozoa have damaged membrane and tails that do not change shape (Jeyendran et al., 1984). The reference limit was set at 58% according to the WHO manual (World Health Organization, 2010). Determination of big halo and DFI Halosperm test (Halotech DNA, Madrid, Spain) is a modified and improved SCD fragmentation test version (Fernandez et al., 2005). Semen samples were diluted with Sperm washing medium (Sage, Trumbull, CT, USA) to the concentration of / ml. Agarose gel from kit was incubated for 5 min at C and 5 min at 37 C, this was followed by adding and mixing of 2014 American Society of Andrology and European Academy of Andrology Andrology, 2014, 2,

3 M. Tandara et al. 25 ll of semen in marked eppendorf test tube. Twenty microlitres of mixture were placed on previously supercoated slide from kit and covered with 22 mm 9 22 mm coverslip. Slides were kept for 5 min at 4 C in refrigerator to create a microgel with an implanted spermatozoa. Coverslips were then carefully removed, a procedure followed by immersion of slides into the previously prepared acid solution (80 ll HCl in 10 ml of distilled water) for 7 min. Slides were then transferred to the tray with a lysing solution from the kit and incubated for 25 min. Rinsing with distilled water followed by dehydration for 2 min in increasing concentrations of ethanol (70, 90 and 100%). After drying, slides were stained with Giemsa dye (Merck KGaA), rinsed under tap water and allowed to dry at room temperature. Each slide was examined under the light microscope at 9100 magnification and 200 spermatozoa were scored. Non-fragmented spermatozoa immersed in agarose matrix and exposed to lysing solution to deproteinize nuclei create the halos of dispersed DNA. The halos correspond to relaxed DNA loops attached to the residual nuclear structure (Fernandez et al., 2003). The spermatozoa without DNA fragmentation show halos of dispersed DNA which can be large (big halo, bh) or medium (medium halo, mh), whereas those sperm nuclei with fragmented DNA produce either small halos (small halo, sh) or no halos at all (wh). Fragmented spermatozoa can also be degraded (d). Big halo spermatozoa produce halos with thickness equal to or greater than the length of the minor diameter of the core, medium halo spermatozoa produce halos with thickness smaller than the length of the minor diameter of the core and greater than 1/3 of the minor diameter of the core, whereas small halo spermatozoa produce halos with thickness equal to or smaller than 1/3 diameter of the minor diameter of the core (Fernandez et al., 2005). The spermatozoa were scored on each slide and the big halo sperm rate was defined. Sperm preparation Swim-up technique was used for sperm preparation (Younglai et al., 2001; World Health Organization, 2010). The semen samples were diluted with Sperm Washing medium double volume (Sage) and centrifuged two times for 15 min at 1400 g. After removing the supernatant, 0.5 ml of Sperm Washing medium (Sage) was layered and the tube was put into an incubator (6.0% CO 2,37 C). During min incubation, motile spermatozoa migrate to the above layered medium. After incubation, the supernatant was aspirated with pipette, centrifuged and the sediment was used for insemination in the conventional IVF. Oocyte collection, insemination and fertilization Collection and fertilization of oocytes was carried out in accordance with the Law on Medically Assisted Reproduction (NN 86/ 12) of the Republic of Croatia. With the size of dominant follicle of 17 mm, the injection of hcg (6500 IUI) was received. After 36 h the puncture and aspiration of follicles was performed transvaginally, tracked by ultra sound. Conventional IVF was used for oocyte insemination. Only mature oocytes were used for fertilization whereas evaluation was performed based on cumulus-corona complex morphology (Veeck & Zaninovic, 2003). Oocytes were inseminated after 4 6 h of incubation with ~ /ml per oocyte (Bungum et al., 2004). Fertilization was determined after 18 h with the appearance of zygote with two pronuclei. Evaluation of human zygotes is based on the pronuclei size and position, as well as on the nucleoli size, distribution and number (Tesarik & Greco, 1999). Embryo development and quality evaluation Gametes, zygotes and embryos were cultivated in Fertilization and Cleavage medium. (Cook, Bloomington, IL, USA). The 3rd day embryo scoring system was used to estimate the development and quality of embryos. Embryos were observed for 68 h (1 h) after insemination for morphological evaluation. Puissant et al. (1987) have described embryo development and quality as embryo score (ES) value based on the blastomeric number and symmetry and cytoplasm fragmentation. The assessment of embryos used in this study was based on ES value expression. Given the blastomeric number and equality and cytoplasm fragmentation, dynamics of embryo development and quality was evaluated. Embryos were given grades from 1 to 5: grade 5 embryos with 8 equal blastomeres without cytoplasm fragmentation, grade 4 embryos with equal 7 8 blastomeres and with small cytoplasm fragmentation, grade 3 embryos with 6 7 unequal blastomeres, small or medium fragmentation, grade 2 significantly fragmented 4 6 cell embryos of equal or unequal blastomeres, grade 1 embryos with a few blastomeres (<4) of any size with a strong or complete cytoplasm fragmentation (Tandara et al., 2013). Cumulative embryo score (CES) was calculated as an average value of all embryos developed from each IVF couple. Embryos of grade 1, 2 and 3 were considered as low quality embryos and those of grade 4 and 5 were considered as high quality embryos. Pregnancy and IVF outcome Serum bhcg levels were measured 12 days after embryo transfer. Clinical pregnancy was confirmed by transvaginal ultrasound 5 weeks after embryo transfer. Statistical analyses Collected data were presented as mean standard deviation or median and interquartile range (IR) with 95% CI of mean or median depending on normal distribution of data. Kolmogorv Smirnov test was used to investigate normal distribution. Correlations between semen characteristic variables were investigated by Pearson correlation. Statistically significant correlation coefficients were interpreted as no relation for correlations from 0 to 0.25 (or 0.25), those from 0.25 to 0.50 (or 0.25 to 0.50) as fair degree of relation, those from 0.50 to 0.75 as moderate to good relations and those greater than 0.75 (or 0.75) as very good to excellent relation. Receiver operating characteristic (ROC) curve analysis were performed for all collected variables according to the two criterions: embryo quality criterion (positive group contained embryos of quality 4 and 5 and negative group those embryos of quality number 1, 2 and 3) and achieved pregnancy criterion (positive group was established pregnancy and negative group was without pregnancy). For significant variables according to ROC analyses optimal cut-off value, specificity, sensitivity, positive and negative likelihood ratios and positive and negative predictive values with 95% CI were calculated. ROC curves for variables that were significant according to ROC analyses were compared. To identify independent predictors among all observed variables stepwise method of logistic multiple regression was performed and odds ratio values with 95% CI were presented for variables included in the model. All p values were 680 Andrology, 2014, 2, American Society of Andrology and European Academy of Andrology

4 BIG HALO AS A EMBRYO QUALITY AND PREGNANCY PREDICTOR considered significant if p < The power of study was calculated considering primary question, the prognostic value of big halo for achieving pregnancy. Type I error (alpha) and type II error (beta) were set to 0.05 (power 95%). Desirable AUC was set to 0.75 and the ratio of sample sizes in negative and positive groups was set to 2 (expected pregnancies in one-third of cases). Such input gave sample size of 72 participants (24 positive and 48 negative). With this consideration, all 88 (27 positive and 61 negative) consecutive couples undergone conventional IVF infertility treatment during study period were included. All computations were made using MedCalc statistical software ver (MedCalc Software, Ostend, Belgium, licensed to Rijeka University School of Medicine Department of Medical Informatics). RESULTS Characteristics of semen (concentration, motility, morphology, vitality, DFI, big halo) and fertilization are presented in Table 1. Of 88 patients 37 (46%) obtained embryos of higher quality (CES 4 or 5) and 43 (54%) obtained embryos of lower quality (CES 1, 2 or 3). Correlations between all parameters are shown in Table 2. Age showed fair correlation with all observed semen characteristics. Fertilization also showed fair correlation with other parameters. All other parameters correlate between themselves in moderate to good degree. DFI correlated negatively with concentration (r = 0.35) and with motility (r = 0.53). Good negative correlation with statistical significance was found between DFI and big halo (r = 0.69; p < 0.001). Also, moderate to good correlations were found between DFI and morphology (r = 0.48; p < 0.001), big halo and morphology (r = 0.43; p < 0.001) and between big halo and vitality (r = 0.49; p < 0.001), all with statistical significance. According to ROC analyses presented in Table 3 only DFI and big halo were significant as prognostic parameters of embryo quality criterion (AUC SE for DFI was and for big halo ). Values with the best ratio of sensitivity and specificity were taken as cut-off criterion value and that is 26% for DFI and 28% for big halo. There is good sensitivity and specificity of DFI for prediction of embryo of higher quality (60 and 72) and even better for big halo (84 and 74). Comparison of ROC curves of DFI and big halo for embryo quality criterion (Fig. 1) revealed that value of the AUC for big halo is significantly higher than for the DFI (difference between areas is , p = 0.025) indicating big halo as better prognostic parameter of Table 1 Characteristics of semen samples collected and fertilization Variable N Mean SD/Median (IR range) 95% CI of mean/median Concentration (910 6 /ml) ( ) Motility (% a + b) Morphology (% normal forms) (27 43) Vitality (%) DNA fragmentation index (%) (24 34) Big halo (%) Fertilization (%) (80 100) Data presented as mean standard deviation and 95% CI of average or median and interquartile range (IR) where appropriate (depending on normal distribution of data and motility, vitality and big halo were normally distributed). embryo quality than the DFI. Logistic multiple regression (stepwise method) revealed that the only independent predictor of embryo quality is big halo with odds ratio = 1.12 and 95% CI = with 73% of cases correctly classified. Others parameters were removed from model owing to the lack of significance. ROC analyses presented in Table 5 show vitality, DFI and big halo as significant prognostic parameters for reached pregnancy criterion (AUC SE for vitality was , for DFI and for big halo). Values with the best ratio of sensitivity and specificity were taken as cut-off criterion value: >60% for vitality, 28% for DFI and >38% for big halo (Table 6). Sensitivity and specificity of vitality (81 and 43), DFI (70 and 61) and big halo (56 and 89) were presented in Table 6. Comparison of ROC curves of vitality, DFI and big halo for achieved pregnancy criterion showed there is no significant difference in AUC for any pair of three parameters (Fig. 2). DISCUSSION Halosperm test is DNA integrity test which measures the absence of sperm DNA fragmentation rather than DNA fragmentation itself (Lewis et al., 2013). According to Halosperm test sperm classification has been defined with five groups (Fig. 3), where non-fragmented spermatozoa creates big halo or medium halo (Fernandez et al., 2005). Unlike many other studies which are focused on DNA fragmentation, this study was interested only in spermatozoa showing big halo because we had assumed that such spermatozoa has undamaged DNA or slightly damaged which is undetectable by Halosperm test. We determined subpopulation of so called big halo spermatozoa of each sample regardless to its DFI value because we had observed that samples with DNA fragmentation 30% could still have notable share of spermatozoa with undamaged DNA and successfully fertilize oocytes. We wanted to see if DNA integrity parameter, expressed as big halo was better prognostic parameter of embryo quality and achieved pregnancy after conventional IVF than DFI. For embryo quality, two embryo groups (positive and negative group) were evaluated on the third day of development when according to some authors, embryonic genome expression is present (Borini et al., 2006). Such classification was made because it was assumed that embryos from the positive group have a better implantation and chance of pregnancy success than negative group embryos (Alpha Scientists 2011). We observed ROC analysis data showing embryo quality expressed as CES groups and semen parameters (concentration, motility, morphology, vitality, DFI and big halo) and fertilization (Table 3). According to the ROC data results only the DFI and big halo were significant as predictors of embryo quality (Table 3). Furthermore, results have shown that sensitivity and specificity for big halo were higher than for the DFI indicating big halo as a better embryo quality prognostic parameter than the DFI (Table 4; Fig. 1). Nevertheless, from further logistic multiple regression analysis we found big halo as the only independent predictor of embryo quality. We consider this as a valuable result of DNA integrity impact on embryo quality, and believe that a strong focus should be put on this parameter in future researches. For pregnancy, positive and negative group was set considering pregnancy establishment. We observed ROC analysis data showing pregnancy and semen parameters (concentration, 2014 American Society of Andrology and European Academy of Andrology Andrology, 2014, 2,

5 M. Tandara et al. Variable Concentration Motility Morphology Vitality DFI b Big halo Fertilization Age (years) r a p Concentration (910 6 /ml) r p <0.001 <0.001 < Motility (% a + b) r p < < Morphology (% normal forms) r p <0.001 <0.001 < Vitality (%) r p <0.001 < DFI b (%) r p < Big halo (%) r 0.01 p Table 2 Correlations of semen characteristic parameters, age and fertilization in observed patients. Italic figures indicate a fair degree of correlations (for correlation coefficients from 0.25 to 0.50 or 0.25 to 0.50) and bold figures indicate moderate to good correlations (for correlation coefficients from 0.50 to 0.75 or 0.50 to 0.75) a Pearson correlation coefficient. b DNA fragmentation index. Table 3 ROC analyses data of observed semen characteristics, age and fertilization. Criterion parameter was embryo quality that was assessed for N = 80 patients. Positive group was embryos of quality 4 and 5 (N = 37, 46%) and negative group was with embryos of quality 1, 2 and 3 (N = 43, 54%). Bold figures indicate significant prognostic parameters of embryo quality Parameter AUC a SE 95% CI Significance b p Age (years) Concentration (910 6 /ml) Motility (% a + b) Morphology (% normal forms) Vitality (%) Fertilization (%) DFI c (%) <0.001 Big halo (%) <0.001 a Area under ROC curve. b Significance level p vs. AUC = 0.5. c DNA fragmentation index. Figure 1 Comparison of ROC curves for DNA fragmentation index (DFI) and big halo. Criterion variable is embryo quality. There is significant difference in AUC for two parameters (for DFI AUC = 0.71 vs. big halo AUC = 0.83; p = 0.025; difference between areas = 0.12 with standard error = 0.06 and 95% CI for difference = ). Sensitivity DFI Big halo Specificity motility, morphology, vitality, DFI and big halo) and fertilization (Table 5). According to the ROC data results vitality, DFI and big halo were significant as pregnancy predictors with cut-off value for big halo >38% and for DFI 28% (Tables 5 and 6), but there was no significant difference for any pair of the three parameters, nor between the big halo and DFI (Fig. 2). However, our results show that both, big halo and DFI, are good pregnancy prognostic parameters in conventional IVF (Table 6). It has been reported by some authors that sperm DNA integrity is statistically significantly associated with pregnancy (Collins et al., 2008). Our results show DFI value of 28% in Halosperm test to be a good pregnancy prognostic parameter which is close to DFI value of 30% in SCSA test reported by Evenson et al. (1999). Sperm DNA is the most compact eucariotic DNA undergoing numerous modifications in chromatin structure during spermatogenesis (Ward & Coffey, 1991). All this reorganization stages insure sperm chromatin protection during transmission through male and female reproductive tract (Sakkas et al., 1999). Given that sperm DNA contributes with 50% to zygote s genome, DNA integrity of a fertilizing spermatozoa is essential for a normal embryonic development and a healthy baby birth (Agarwal & Allamaneni, 2004). Sperm DNA integrity is, therefore, an important parameter of male reproductive potential and semen quality (Agarwal & Allamaneni, 2004). Origins of DNA damage of ejaculated spermatozoa are chromatin remodelling by topoisomerase, oxidative stress and abortive apoptosis (Aitken & De Iuliis, 2010). Sperm DNA fragmentation can appear as a result of one or combination of different mechanisms (Aitken & De Iuliis, 2010). It has been proven that damaged DNA has an influence on fertility in both natural and assisted cycles (Larson et al., 2000; Spano et al., 2000). Studies have found that fertilization achieved with spermatozoa that has fragmented DNA impacts negatively on the embryo development and quality, implantation rate and impacts positively on foetus malformations and pregnancy loss (Borini et al., 2006; Muriel et al., 2006; Zini et al., 2008; Aitken et al., 2009). Importance of DNA integrity in fertility evaluation and its influence on early embryogenesis, implantation and pregnancy outcome has been recognized (Barratt et al., 2010; 682 Andrology, 2014, 2, American Society of Andrology and European Academy of Andrology

6 BIG HALO AS A EMBRYO QUALITY AND PREGNANCY PREDICTOR Table 4 Sensitivity and specificity, positive and negative likelihood ratio and positive and negative predictive values with 95% confidence interval for DNA fragmentation index and percentage of big halo in semen specimens for cut-off value determined by ROC analyses according to criterion of embryos quality. Positive group was embryos of quality 4 and 5 (N = 37, 46%) and negative group was with embryos of quality 1, 2 and 3 (N = 43, 54%) Variable Cut-off value Sensitivity (95% CI) Specificity (95% CI) PLR a (95% CI) NLR b (95% CI) PPV c (95% CI) NPV d (95% CI) DFI e (%) (42 75) 72 (56 84) 2.1 ( ) 0.6 ( ) 64 (47 80) 67 (52 81) Big halo (%) >28 84 (68 94) 74 (59 87) 3.3 ( ) 0.22 ( ) 74 (58 86) 84 (69 94) a Positive likelihood ratio. b Negative likelihood ratio. c Positive predictive value. d Negative predictive value. Table 5 ROC analyses data of observed semen characteristics, age and fertilization. Criterion parameter was achieved pregnancy that was assessed for N = 88 patients. Positive group was established pregnancy (N = 27, 31%) and negative group was without pregnancy (N = 61, 69%). Bold figures indicate significant prognostic parameters of pregnancy Figure 3 Spermatozoa assessed by Halosperm test (image taken from the microscope); Spermatozoa without DNA fragmentation: big halo spermatozoa (1) and medium halo spermatozoa (2). Spermatozoa with DNA fragmentation: small halo spermatozoa (3), spermatozoa without halo (4) and degraded spermatozoa (5). Parameter AUC a SE 95% CI Significance b p Age (years) Concentration (910 6 /ml) Motility (%) Morphology (%) Vitality (%) Fertilization (%) DFI c (%) Big halo (%) <0.001 a Area under ROC curve. b Significance level p vs. AUC = 0.5. c DNA fragmentation index. Figure 2 Comparison of ROC curves for sperm vitality, DNA fragmentation index (DFI) and big halo. Criterion variable is achieved pregnancy. There is no significant difference in AUC for any pair of three parameters (for vitality (AUC = 0.66) vs. DFI (AUC = 0.67) p = 0.847; DFI vs. big halo (AUC = 0.75) p = and vitality vs. big halo p = 177). 100 Sensitivity Vitality DFI Big halo Specificity Lewis et al., 2013). Therefore, DNA integrity testing needs to be incorporated into the standard semen analysis. In our study, sperm DNA fragmentation is measured with Halosperm test and expressed with DFI value (Fernandez et al., 2005). Today, there are numerous studies considering correlation of DNA fragmentation with semen and ART parameters, where DFI has been measured with various tests which are probably expressing different aspects of DNA damage. However, the results off those studies show diverse conclusions with no universal opinion about the DNA fragmentation role in male fertility as well as pregnancy achievement and outcome. Studies have shown that 18% of men with normal conventional semen parameters have high DNA fragmentation rate with a risk for poor blastocyst development and failure to initiate an ongoing pregnancy (Virro et al., 2004). Contrary to those studies, Huang et al. (2005) reported that sperm DNA fragmentation might have no effect on pregnancy rate. Also, some authors have found that fertilization, embryo development and subsequent pregnancy are possible despite a high level of DNA fragmentation in the sperm population (Twigg et al., 1998; Bungum et al., 2004). All these studies indicate the importance of DNA integrity testing, but the imparity of results led us to evaluate another parameter of DNA integrity beside the DNA fragmentation. So, in this study, we have evaluated both DNA integrity parameters, DFI and big halo but the focus was set on those spermatozoa without DNA fragmentation so called big halo spermatozoa. Gandini et al. (2004) reported full-term pregnancies despite high levels of DNA fragmentation. Their results might be explained with the fact that despite the high levels of DNA damaged spermatozoa, fertilization, embryo cleavage and pregnancy can still be achieved if an ejaculate contains high percentage of DNA undamaged spermatozoa. Our results show that if cut-off value of big halo is >38%, pregnancy may be achieved even if DNA fragmentation rate is high. Considering DNA fragmentation, in the previous study we obtained statistically significant difference in embryo quality (good/medium and low) between the two DFI groups 2014 American Society of Andrology and European Academy of Andrology Andrology, 2014, 2,

7 M. Tandara et al. Table 6 Sensitivity and specificity, positive and negative likelihood ratio and positive and negative predictive values with 95% confidence interval for sperm vitality (%), DNA fragmentation index and percentage of big halo in semen specimens for cut-off value determined by ROC analyses according to achieved pregnancy criterion. Positive group was established pregnancy (N = 27, 31%) and negative group was without pregnancy (N = 61, 69%) Variable Cut-off value Sensitivity (95% CI) Specificity (95% CI) PLR a (95% CI) NLR b (95% CI) PPV c (95% CI) NPV d (95% CI) Vitality (%) >60 81 (62 94) 43 (30 56) 1.4 ( ) 0.43 ( ) 39 (26 52) 84 (66 95) DFI e (%) (50 86) 61 (47 73) 1.8 ( ) 0.49 ( ) 44 (29 60) 82 (68 92) Big halo (%) >38 56 (35 74) 89 (78 95) 4.8 ( ) 0.50 ( ) 68 (45 86) 82 (70 90) a Positive likelihood ratio. b Negative likelihood ratio. c Positive predictive value. d Negative predictive value. e DNA fragmentation index. (DFI < 30% and DFI 30%). Reference value for DFI was set at 30% as suggested by Halosperm. We concluded that higher sperm DNA damage has an impact on embryo quality (Tandara et al., 2013). Negative correlation between DNA fragmentation and embryo quality after IVF was shown in some other studies (Muriel et al., 2006; Velez De La Calle et al., 2008; Simon et al., 2010). In this study, we found that DFI was significant predictor of embryo quality with a cut-off value 26% (Table 4). The DFI cutoff values proposed by authors differ in DNA fragmentation tests, but also within the same test, so predictive value of sperm DNA damage assessment may vary depending on the DNA fragmentation test and cut-off value that is used (Zini et al., 2008). For example, some suggested Halosperm DFI cut-off values are as follows: <18 and 30% (Velez De La Calle et al., 2008; Venkatesh et al., 2011). Because of this variations in the DFI cut-off value, sperm DNA undamaged rate expressed as big halo parameter may be a useful parameter in predicting ART success. Regarding the big halo parameter, the cut-off value for predicting embryo quality was set at >28% (Table 4). We found that fertilization correlates in fair degree with DFI and big halo (Table 2) which is in concordance for DFI with some other studies that demonstrated similar results using TUNEL fragmentation tests (Henkel et al., 2004; Li et al., 2006). Our results could be explained with a paternal effect. It is assumed that the first steps of development are subjected to maternal control, whereas the paternal gene expression starts at the 4 8 cell stage in humans (Braude et al., 1988; Borini et al., 2006). Consequently, at this stage sperm DNA fragmentation may have a negative effect on the embryo development. Furthermore, conventional IVF represents in vitro simulation of the natural conditions with a natural sperm selection, so morphologically abnormal spermatozoa with low motility and DNA damage have low potential in oocytes fertilization (Borini et al., 2006). Cut-off value of big halo parameter for embryo quality and pregnancy achievement obtained in this study is rather different (Tables 4 and 6) which could be explained with the fact that embrionic genome expression starts at the 4 8 cell stage (Borini et al., 2006). During that time, father s genome may have significant impact on embryo quality and development. We performed embryo transfer on the 3rd day. In that stage of in vitro development, the embryo could be of good quality but that does not mean development to the blastocyst stage will occur, and even if it occurs, the implantation could fail. That could be explained with the fact that every stage of embryo development, after embryonic genome expression starts, could be associated with the DNA integrity, whereas each step of normal development could be interrupted with sperm DNA fragmentation presence, leading to increasing criteria as the embryo goes further on its path to pregnancy achievement. We believe that with higher big halo ratio probability for pregnancy achievement increases. Studies have demonstrated that if an oocyte is fertilized by a spermatozoa with fragmented DNA, further development will depend on fragmentation rate and ability of the oocyte to repair that damage (Derijck et al., 2008). The ability of the oocyte to repair DNA damage in fertilizing spermatozoa is still poorly understood and limited to gene expression studies showing the presence of mechanisms in oocytes that possibly cope with paternal DNA anomalies (Sakkas & Alvarez, 2010). It was shown that DNA repair initiation by oocyte depends on the cytoplasmic and genomic quality of each oocyte. Also, the oocyte repair ability is impacted with the increase in woman s age (Sakkas & Alvarez, 2010). We still cannot know neither the extent of oocyte ability to repair DNA damage of fertilizing spermatozoa nor determine whether that particular oocyte even has repair capability. Some studies have pointed to the present DNA fragmentation tests inability to give information about the repairability of DNA damage and postulated that if unrepairable sperm DNA damage is not compatible with normal embryo development a pregnancy should not be achieved (Sakkas & Alvarez, 2010). Conversely, there are studies that demonstrated there was correlation between DNA damage and pregnancy rate (Bungum et al., 2004; Zini et al., 2005). That might be explained with the share of spermatozoa with undamaged DNA in those patients where high DNA fragmentation rate has been established. So, it is possible that the patients with high DFI can still have significant amount of big halo spermatozoa and be fertile. If we assume that natural sperm selection exists in conventional IVF and morphologically normal and motile spermatozoa without DNA damage will fertilize an oocyte then the amount of undamaged spermatozoa in semen is a significant parameter regardless to DNA fragmentation rate. Studies have demonstrated that DNA integrity represents an individual parameter that complements conventional semen parameters to give more precise male fertility evaluation (Agarwal & Allamaneni, 2004). In this study we have put two parameters of DNA integrity in correlation, big halo and DFI, mutually and separately with semen parameters (Table 2). Big halo and DFI show good negative correlation with statistical significance (Table 2) which was expected. Results indicate the importance of measuring both DNA integrity parameters. Correlation between sperm DNA fragmentation and conventional semen parameters has been shown in several studies with various DNA fragmentation tests (Khalili et al., 2006; Sheikh et al., 2008; Tandara et al., 2013). In this study, we also have found negative correlation between DFI and all conventional semen parameters and vitality with statistical significance for motility, morphology and vitality (Table 2). As for big halo parameter results have shown positive correlation with conventional semen parameters 684 Andrology, 2014, 2, American Society of Andrology and European Academy of Andrology

8 BIG HALO AS A EMBRYO QUALITY AND PREGNANCY PREDICTOR and vitality with statistical significance for morphology and vitality (Table 2). Our results demonstrate that DNA integrity evaluation, expressed as DFI and big halo parameters, represents a significant role in male fertility analysis. Chohan et al. (2006) demonstrated that a fertile group has a significantly higher percentage of spermatozoa showing big halo (large halo) than an infertile group. So, the evaluation of DNA undamaged spermatozoa expressed as big halo parameter can provide valuable information about patient s fertility potential and maybe facilitate embryologist s decision in finding proper ART method for infertile couple. CONCLUSION From analysis of Halosperm results from our patients included in conventional IVF program, we have noticed how some patients with relatively high DFI have significant big halo ratio and finally good embryo quality. That finding has led us to focus on big halo parameter and investigate that particular parameter further. We found that big halo also correlates with conventional semen parameters in good degree. Also, our results showed that big halo was better prognostic parameter of embryo quality and pregnancy after conventional IVF than DFI. The aim of this study was based on our assumption that with higher ratio of big halo spermatozoa in ejaculate, the probability of oocyte fertilization by big halo spermatozoa increases. To our knowledge, this is the first study demonstrating the correlation between DNA undamaged rate expressed as big halo parameter and semen characteristics and also the influence on fertilization rate, embryo quality and pregnancy in conventional IVF. We believe that big halo represents very interesting parameter which potential may be worth investigating in the future. ACKNOWLEDGEMENTS This work has been supported by the University of Split School of Medicine. 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