Stesura Seveso 125Archivio Italiano di Urologia e Andrologia 2019; 91, 2 ORIGINAL PAPER Relationships between sperm DNA integrity and bulk semen parameters in Bulgarian patients with varicocele Viktor Alargkof 1, Larissa Kersten 2, Romil Stanislavov 3, Zdravko Kamenov 4, Panagiotis Nikolinakos 1 1 Medical University - Sofia, Sofia, BG; 2 University of Essex, UK; 3 Genika, Genetic and Medico-Diagnostic Laboratory, Sofia, BG; 4 Clinic of Endocrinology, Alexandrovska University Hospital, Sofia, BG. Objective: This exploratory retrospective study aimed to compare the level of Sperm DNA Fragmentation (SDF) and investigate its association with bulk semen parameters, for the first time in Bulgarian patients with varicocele, using a distinct methodology. Material and methods: Standard semen analysis was performed according to the 2010 criteria of the European Society of Human Reproduction and Embryology - Nordic Association for Andrology (ESHRE-NAFA-2010) and DNA fragmentation was assessed using the Halosperm® kit. The total sample included 28 males: the control group consisted of men with normal geni- tal examination and unknown fertility (n = 10), group one con- sisted of men with varicocele, normozoospermia and DNA frag- mentation > 15% (n = 9) and group two consisted of men with varicocele, abnormal sperm parameters and DNA fragmenta- tion > 15% (n = 9). Results: DNA fragmentation was found to be higher in patients with abnormal sperm parameters (43.78 ± 30.78) compared to the normozoospermic group (21.22 ± 3.93) (p = 0.008). In nor- mozoospermic patients, no statistically significant correlations were observed between SDF and bulk semen parameters. In patients with abnormal sperm parameters, DNA fragmentation exhibited significant very strong negative association with motility (a+b), vitality and typical morphology (p < 0.001). Conclusions: DNA integrity assays could be used for a better evaluation and management of male infertility, particularly in normozoospermic varicocele patients. KEY WORDS: Male Infertility; Sperm DNA fragmentation; Sperm DNA damage; Varicocele; Semen Analysis. Submitted 26 November 2018; Accepted 15 December 2018 Summary No conflict of interest declared. clinically evident varicocele may present with normal values on primary semen assays. On the other hand, patients may present with altered spermatogenesis, poor bulk seminal parameters and oligoasthenoteratozoosper- mia (OAT syndrome) (4). The mechanisms by which varicoceles lead to spermato- genic failure are not completely elucidated. However, most authors investigating how varicoceles impair the reproductive function consider the primary event to be an increase in intratesticular temperature secondary to interruption in the counter-current heat exchange pro- vided in the plexus pampiniformis with opposing flow vectors in a central arterial system and surrounding veins. The proposed mechanisms by which male fertility is impaired by this effect mainly include DNA fragmen- tation, apoptosis and oxidative stress. Other associated factors include testicular hypoxia secondary to venous stasis, reflux of renal/adrenal toxic metabolites and hypertension in the internal spermatic veins (5). Numerous researchers have recently examined the associ- ation between varicocele and Sperm DNA Fragmentation (SDF). Although a cause-and-effect relationship is not established, multiple reviews and meta-analyses conclude that there is indeed evident association between varicocele and increased DNA fragmentation (6). Although sperm DNA damage represents a reproducible sperm function marker, the American Society for Reproductive Medicine and the American Urological Association (AUA) do not recommend routine clinical use of sperm DNA testing (7). The utility of SDF testing is currently appreciated by the AUA and the European Association of Urology (EAU) for the evaluation of Assisted Reproductive Technology (ART) success (8, 9). Studies have shown that sperm DNA damage is associat- ed with negative influence on embryo development and lower pregnancy rates (lower natural pregnancy rates and lower pregnancy rates after intrauterine insemina- tion and in vitro fertilization) (10, 11). A significant finding is the increased risk for spontaneous abortions with increasing sperm DNA fragmentation. Comprehensive meta-analyses have shown that higher levels of DNA fragmentation are linked with an approxi- mately double risk ratio (RR) of miscarriages (12). The current study included Bulgarian men clinically DOI: 10.4081/aiua.2019.2.125 INTRODUCTION Varicocele, an abnormal dilation and tortuosity of the internal spermatic veins within the plexus pampini- formis, is the most frequently identified lesion in males undergoing infertility evaluation (1). It is a common condition in men with normal spermatogenesis but also in men with abnormal semen parameters. It is found in approximately 15% of the general population and is reported in 35% of men with primary infertility and 75 to 81% of men with secondary infertility (2). Varicocele is a complex entity as its effects on sperm quality are both difficult to define and predict. Not all men with varicocele are infertile (3) and patients with Archivio Italiano di Urologia e Andrologia 2019; 91, 2 V. Alargkof, L. Kersten, R. Stanislavov, Z. Kamenov, P. Nikolinakos 126 diagnosed with varicocele and SDF level higher than 15%, which is defined as the normal threshold for in vivo fertilization using the Halosperm® kit (13). The objective was to compare the levels of DNA frag- mentation between varicocele patients with normo- zoospermia and abnormal sperm parameters and to investigate the correlation of SDF and bulk semen parameters as well as describe the relationships between them, for the first time in the Bulgarian population. At the same time a secondary aim was to collect data using a distinct methodology which is not utilized as frequently in literature. Namely, the normozoospermic patients in the current study are defined according to the ESHRE- NAFA-2010 criteria which are stricter in defining normal parameters and make the additional distinction of border- line parameters in comparison to the WHO manual. Finally, there is great variability in literature in the meth- ods used to determine SDF, which renders making cor- relations and drawing conclusions difficult (14). SDF in the present study was analyzed using the Halsoperm® kit, aiming to add to the pool of data acquired via Sperm Chromatin Dispersion. MATERIALS AND METHODS Study group The study included 28 men assessed by the andrology laboratory department at Genika, Genetic and Medico- Diagnostic Laboratory in Sofia, Bulgaria. A retrospective study was designed involving one control group of men with unknown fertility, normal genital examination and normal bulk semen parameters (n = 10) and two groups of patients with clinically diagnosed varicocele and infer- tility. The varicocele was diagnosed by palpation and Doppler ultrasound examination. Among the patients with varicocele, the first group (n = 9), presented with normozoospermia and levels of DNA fragmentation high- er than 15%. The second group (n = 9) presented with abnormal sperm parameters and levels of DNA fragmen- tation higher than 15%. In all men, medical history was obtained, including occupation, smoking habits, alcohol intake and the use of prescription medication. Exclusion criteria for all groups were: current or previous systemic diseases that would lead to testicular alterations such as cancer and endocrinopathies (and their treatments) and a history of excessive alcohol and drug consumption. According to the laboratory’s protocol, informed consent is acquired from all patients at the time of registration for the anonymous inclusion of their sperm analysis results in potential medical studies and research. Sperm collection and semen analysis Semen samples were collected by masturbation after three to seven days of ejaculatory abstinence and were analyzed within one hour of collection. Additionally, patients were instructed to abstain from alcohol con- sumption for three to seven days and maintain a good general status for three months before the examination as well as present afebrile on the day of the analysis, without having consumed any antibiotics (they were instructed to report any received medications). In all patients, the semen sample was (up to 25 minutes post ejaculation) retained in a 37 °C thermostat for circa 15 minutes. After complete semen liquefaction, pH and volume were meas- ured and a standard seminal analysis was performed according to the criteria of the European Society of Human Reproduction and Embryology - Nordic Association for Andrology 2010 (ESHRE-NAFA-2010). Analysis was per- formed on the computer microscopic fluorescent plat- form Sperm Class Analyzer (SCA - v.5.0) and the parame- ters measured included total sperm count, motility, vital- ity and morphology. Sperm morphology was assessed using Kruger’s strict criteria. Sperm vitality was assessed using Eosin Y to stain spermatozoa and Negrosin to stain the background. In comparison to WHO-5, 2010, the cri- teria of ESHRE-NAFA-2010 interpret results in three cat- egories: normal, borderline and pathologic. The border- line results represent a buffer zone of clinical importance. Patients in this zone are regarded as having the potential to shift to either normal or pathological spectra depend- ing on the persistence of offending factors. Determination of DNA integrity Sperm nuclear DNA integrity was evaluated by use of the Halosperm® kit (Halotech® DNA SL, Madrid, Spain). This test is a modified and improved version of the sperm chromatin dispersion (SCD) test (15). The clinical threshold for percentage of spermatozoa with DNA fragmentation has been established as (1) < 15% for in vivo fertilization, (2) < 30% for in vitro fertilization. The kit included: 10 Super Coated Slides, 10 Eppendorf agarose tubes, one tube with denaturizing solution (1 mL), and two bottles of Lysis solution (60 mL ×2). The first step included insert- ing the semen sample in agarose microgel. The lysis solu- tion was first left in room temperature. The semen sample was diluted with an extender or PBS until a concentration of 5-10 ×106/ml. The Eppendorf tube with the agarose was placed in a float for five minutes at 90 to 100 °C until the agarose melted. The tube was then moved along with the float into a water bath with a temperature of 37 °C and was left there for five minutes. 60 mL of the semen sam- ple were transferred to the Eppendorf tube and were gen- tly mixed. The Super-Coated Slide to be used was placed on a cold surface area (glass plate) at 4 °C. Once the slide had been cooled, cell suspension was transferred from the Eppendorf tube to the slide, towards the side that was treated and marked by a dark point. A coverslip was placed on top very carefully, to avoid creating bubbles. A drop of 14, 20 or 50 μL for the slides is used for the respective magnifications of 18×18mm, 22×22 mm or 24×60 mm. The slides were maintained in horizontal ori- entation throughout the whole procedure. The cooled glass plate with the slide was then placed in the fridge and the sample was left to jellify for five minutes. Processing the sample followed. The denaturizing agent was pre- pared by adding 80 μL from the acid denaturizing solu- tion to 10 μL distilled water and was placed in an incu- bator tray. The cover slip was slid off the slide and the slide was immediately immersed in the denaturizing solu- tion in horizontal orientation, leaving it to incubate for seven minutes at room temperature (22°C). After putting on gloves, the slide was picked up with the help of pin- cers. While remaining in horizontal position it was placed in another incubation tray containing 10 μL of lysis solu- tion. It was left to incubate for 25 minutes. The slide was picked up and placed in a Petri dish filled with distilled water in order to wash off the lysis solution. It was left to incubate for five minutes. The slide was picked up and was placed horizontally in a Petri dish with 70% ethanol for two minutes, 90% ethanol for two minutes and 100% ethanol for two minutes. The slide was left to dry and once completely dried the prepared slide was stained for observation under light microscopy. Wright’s staining solution was mixed with Phosphate-buffered saline (PBS) in 1:1 ratio and one layer of the staining solution was placed horizontally in order to cover the wet slide. The slide was left to stain for five to 10 minutes. The slide was careful- ly rinsed with running water and was left to dry. Subsequently direct microscopic visual analysis was per- formed. The nucleoid that corresponds to deproteinized nuclei of spermatozoa is made up of two parts: a core located centrally and a peripheral halo of chromatin/DNA dispersion. The spermatozoa tails are also visible. A min- imum of 500 spermatozoa are studied per sample and scored according to the following criteria. Sperm classification The categorization of the different halo sizes is performed using the minor diameter of the core from the own nucleoid as a reference to which the halo width is com- pared. The SCD patterns established are the following (15): Sperm cells without DNA fragmentation a) Sperm cells with large halo: those whose halo width is similar or higher than the minor diameter of the core. b) Sperm cells with medium-size halo: the halo size is between: maximum one third of the minor diameter of the core and minimum the length of the minor diameter of the core. Sperm cells with DNA fragmentation c) Sperm cells with small halo: the halo width is similar or smaller than one third of the minor diameter of the core. The core may have irregular form or barely dis- tinguishable (< 150 μm2). d) Sperm cells without halo e) Sperm cells without halo-degraded: there is no halo and the core presents granule-like fragmentation and is weakly stained. ’’Others’’ Nucleoids that do not correspond to spermatozoa. One of the morphological characteristics that distinguish them is the absence of tail. These cells are not included in the estimation of frequency of sperm with fragmented DNA. Statistical analysis Data analysis was performed using the R software envi- ronment for statistical computing and graphics. For each variable the mean, the standard deviation and interquar- tile range (IR) are presented to indicate the relevant dis- tribution. The Shapiro-Wilk test was applied to test whether variables are normally distributed. For normal- ly distributed data a Student’s test was conducted to test whether means differ across groups. In the case of not normally distributed variables a Mann- Whitney-Wilcoxon test was applied to test whether the distribution across two groups differs. For both tests, the Null-Hypothesis (difference in means/populations) was rejected if the p-value was smaller than 0.05. Correlation coefficients were calculated according to the Pearson method. Statistically significant correlation coefficients were interpreted as: weak from 0.20 to 0.39 (or -0.20 to -0.39); moderate from 0.40 to 0.59 (or -0.40 to -0.59); strong from 0.60 to 0.79 (or -0.60 to -0.79); very strong from 0.80 to 1.0 (or -0.80 to -1.0). If the p-value was smaller than 0.05 then the correlation coefficient was interpreted as being statistically significant. RESULTS All 18 patients entering the study presented varicocele in the left testis, which was detected by physical examina- tion and confirmed by Doppler ultrasound. The median age (years) was 31 ± 6.182 (26.25-34.5) for controls, 32 ± 6.648 (30-37) for patients with normal semen param- eters (G1) and 36 ± 6.464 (35-42) for patients with abnormal semen parameters (G2). No statistically signifi- cant difference was found between the groups with respect to age apart from between the controls age and the G2 age, p = 0.01905. Standard semen analysis parameters The main sperm parameters of controls and varicocele patients are presented in Table 1. G1 (n = 9) had normal 127Archivio Italiano di Urologia e Andrologia 2019; 91, 2 SDF and bulk semen parameters in varicocele Table 1. Comparison of standard sperm parameters between control subjects, varicocele patients with normal sperm parameters (G1) and varicocele patients with abnormal sperm parameters (G2). Numbers represent mean ± standard deviation and range is shown in parentheses. Control (n = 10) Normal Sperm Parameters Abnormal Sperm Parameters A: p value B: p value (G1) n = 9 (G2) n = 9 between controls between controls and G1 and G2 pH 7.54 ± 0.07 (7.5-7.575) 7.611 ± 0.078 (7.6-7.7) 7.733 ± 0.132 (7.6-7.8) 0.052 0.002 Volume (mL) 3.87 ± 2.465 (2.525-4.4) 3.944 ± 1.474 (2.7-5.1) 3.889 ± 2.070 (2.6-5.5) 0.595 0.744 Number (x 106/mL) 448.4 ± 317.518 (251.2-651.7) 293.9 ± 109.582 (214.6-362.3) 117.3 ± 115.782 (41.1-169.3) 0.488 0.008 Motility (% a+b) 57.8 ± 3.225 (56-60.5) 57 ± 3.640 (55-58) 24.89 ± 13.897 (26-32) 0.594 <0.001 Vitality (%) 78.2 ± 3.676 (78-81) 76.67 ± 3.464 (73-79) 48.89 ± 19.127 (46-62) 0.337 < 0.001 Morphology: Typical (%) 17.7 ± 1.947 (16-18) 16.56 ± 1.667 (16-17) 4.111 ± 2.028 (4-5) 0.256 < 0.001 Archivio Italiano di Urologia e Andrologia 2019; 91, 2 V. Alargkof, L. Kersten, R. Stanislavov, Z. Kamenov, P. Nikolinakos 128 sperm parameters. G2 (n = 9) showed an abnormality in one or more of the bulk semen parameters. Particularly, five patients had oligoasthenoteratozoospermia and four had asthenoteratospermia. Furthermore, six patients presented with more than 20% decapitated forms. Sperm DNA fragmentation measured by the Halosperm® kit In the control group the mean SDF percentage was cal- culated as 11.4 ± 1.35 (10.25-12). This was lower in comparison to G1 (p < 0.001) and G2 (p < 0.001). SDF was found to be higher in patients with abnormal sperm parameters 43.78 ± 30.78 (26-38) compared to the nor- mozoospermic group 21.22 ± 3.93 (18-23) (p = 0.008). SDF by group is presented in Figure 1. Relationships between DNA fragmentation and bulk semen parameters In patients with varicocele and normal sperm parame- ters, DNA fragmentation was negatively correlated with sperm pH (r = -0.54, p = 0.135) and motility (a+b) (r = -0.31, p = 0.410), and positively correlated with sperm volume (r = 0.10, p = 0.799), number (r = 0.14, p = 0.713), vitality (r = 0.05, p = 0.894) and typical mor- phology (r = 0.38, p = 0.314). In all cases the strength of association ranges from very weak to moderate only, of which none is significant (i.e., p > 0.05). On the other hand, in the group of patients with abnormal sperm parameters, DNA fragmentation was positively cor- related with pH (r = 0.08, p = 0.840) and volume (r = 0.15, p = 0.706); and negatively correlated with number (r = -0.47, p = 0.204), motility (a+b) (r = -0.94; p < 0.001), vitality (r = -0.93; p < 0.001) and typical morphology (r = -0.89; p < 0.001). The latter three indicate a very strong association. DISCUSSION This exploratory study aimed to answer the following question: do varicocele patients with abnormal sperm parameters have higher levels of SDF than varicocele patients with normozoospermia? According to the literature research performed by the authors, it was impossible to identify other studies using the same design: directly comparing the DNA fragmentation levels, by use of the Halosperm® kit, between varicocele patients with normozoospermia and varicocele patients with abnormal sperm parameters, while performing semen analysis according to the ESHRE- NAFA-2010 criteria. The results of our study indicate that there is clearly a higher degree of SDF in patients with varicocele. Furthermore, in our study varicocele patients with abnormal sperm parameters were found to have higher levels of SDF than varicocele patients with normozoospermia. This finding is consistent with what other authors have also demonstrated regarding infertile men (16). The fact that the levels of fragmentation in the group of patients with abnormal sperm parameters are higher, could reflect the more advanced effects of varicocele on sper- matogenesis. Moreover, in men with abnormal sperm parameters, we established statistically significant, very strong correla- tions between DNA fragmentation and three semen parameters: progressive motility, vitality and typical morphology. These results are comparable to what other authors have observed (17). Weaknesses of this study are the limited number of par- ticipating patients as well as the absence of data regard- ing the grade of varicocele which do not allow the draw- ing of any major conclusions. This being an exploratory study, the authors urge other researchers both in Bulgaria and abroad to collect more data using the methodology described, to obtain a more thorough understanding of the correlations between SDF and bulk semen parameters in patients with varicocele. Criticisms regarding the utility of SDF tests to evaluate male infertility include: that the minimum number of spermatozoa without fragmentation required for in vivo conception is unknown, that a simple sperm vitality assessment could suffice given its correlation to SDF (18), and that more studies investigating the cost effec- tiveness of SDF testing are required to determine its role alongside the standard sperm analysis. While the Halosperm® kit is only available in a few laboratories in Figure 1. DNA fragmentation by group. Abnormal Sperm Control Normal Sperm Parameters Parameters Group Pe rc en ta ge 20 40 60 80 10 0 our country, it can be argued that it is cost-and-time effective, since the results are produced on the same day and the average price of 100 EUR for the combined stan- dard semen and SDF analysis is affordable. 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Int Urol Nephrol. 2018; 50:583. 129Archivio Italiano di Urologia e Andrologia 2019; 91, 2 SDF and bulk semen parameters in varicocele Correspondence Viktor Alargkof, MD valargoff@yahoo.gr 24 Rue du Bugnon, Lausanne, 1005, CH Larissa Kersten, MD larissa.kersten@gmail.com 4 Theodor-Heuss STR, 45731 Waltrop, DE Romil Stanislavov, MD rstanik@abv.bg 84 Ami Bue STR. 1000, Sofia, BG, Zdravko Kamenov, MD zkamenov@hotmail.com 1 Sveti Georgi Sofiyski STR, 1431, Sofia, BG Panagiotis Nikolinakos, MD pnikolinakos@yahoo.gr 139 Pentelis AVE, 15127, Athens, GR