Correlation between sperm DNA fragmentation index and semen parameters in 418 men seen at a fertility center

Objective: The aim of this study was to determine the correlation between semen parameters and sperm DNA fragmentation index (DFI) in 418 men who attended a fertility center. Methods: This retrospective study includes 418 male patients seen at In Vitro Gestar, Chiclayo - Perú between October 2013 and March 2020. DFI was determined between normozoospermic patients (NORMO) and patients with one altered parameter (ALTERED). The patients were classified as oligozoospermic [Severe/Moderate oligozoospermia (OLIGO SM): <5x106 sperm/mL and Mild oligozoospermia (OLIGO M): 5 to <15x106 sperm/mL], asthenozoospermic [Severe asthenozoospermia (ASTHENO S): <10% PM (progressive motility); Moderate asthenozoospermia (ASTHENO MO): 10 to <20% PM; Mild asthenozoospermia (ASTHENO M): 20 to <32% PM)], teratozoospermic (TERATO) and oligoasthenoteratozoospermic (OAT). DFI was determined between these groups and NORMO. The data was analyzed using the SPSS 22.0 software for Windows. Results: Normozoospermic patients presented lower significant levels of DFI compared with all groups. NORMO (15.64±7.65) vs [ALTERED (18.41±9.43, p=0.003); OLIGO SM (26.38±12.94, p<0.005); ASTHENO S (23.09±11.45, p<0.01); TERATO (17.96±9.23, p<0.05) and OAT (22.05±12.15, p=0.001)]. We determined a significant negative correlation between DFI and progressive motility (r= -0.162 p=0.001), and those with normal morphology (r= -0.100 p=0.040). Likewise the DFI shows a significant positive correlation with age (r= 0.257 p=0.000). Conclusions: Our study establishes that high DFI is accompanied by a significant impairment in all seminal parameters.


INTRODUCTION
Infertility is a disease of the reproductive system, defined by the failure to achieve a clinical pregnancy after 12 months or more of regular unprotected sexual intercourse (De Neubourg et al., 2012), and it affects approximately 8 to 12% of the word population (Kumar & Singh, 2015). Male factor is implicated in almost 50% of cases and out of these; approximately 30 -40% are idiopathic (Kadioglu & Ortac, 2017). These facts determine the importance of semen evaluation to verify fertility potential.
Conventional semen analysis or spermogram (sperm concentration, motility, morphology and vitality) is used for male fertility evaluation (WHO, 2010). However, it is estimated that approximately 10-15% of infertile men present normal semen analysis Agarwal & Allamaneni, 2005;Lewis et al., 2008). Likewise, the spermogram does not measure the fertilizing potential of spermatozoa and the complex changes that occur in the female reproductive tract before fertilization (Wang & Swerdloff, 2014). Therefore, there may be other factors that affect male fertility and are not detected by the spermogram, for example: sperm membrane defects (Rajeev & Reddy, 2004), environmental factors (Oliva et al., 2001), genetic factors (Kim et al., 2017) and sperm DNA fragmentation (Quintero et al., 2015;Ioannou et al., 2016).
Recent studies indicate that sperm DNA fragmentation test is a useful diagnostic tool in male infertility evaluation (Agarwal et al., 2016). Intact sperm DNA is necessary for correct transmission of genetic material to the next generation. High percentage of sperm DNA fragmentation index (DFI) is associated with reduced fertilization rates, early development embryo, embryo quality, pregnancy rates and higher rates of spontaneous miscarriage (Bungum et al., 2007;Lewis et al., 2013;Simon et al., 2014). Sperm DNA fragmentation can be caused by many factors, such as: apoptosis during sperm maturation in the seminiferous tubule epithelium (Gosálvez et al., 2015), defects in chromatin packaging and remodeling during the process of spermiogenesis (Sakkas et al., 2002;Shamsi et al., 2008), increase of reactive oxygen species (ROS) (Aitken & De luliis, 2010;Alahmar, 2019) and decrease of seminal antioxidant (Shamsi et al., 2010). Obesity, stress, alcohol consumption, smoking, use of drugs, caffeine, diet and advanced age are also factors that increase DFI (Durairajanayagam, 2018).
Sperm chromatin structure assay (SCSA), terminal deoxynucleotidyl transferase (dUTP) nick-end labeling (TUNEL) assay, Comet assay, Acridine orange test and sperm chromatin dispersion (SCD) are some methods commonly used to identify sperm DNA fragmentation. SCD is characterized by its easy and fast application, as well as, low-cost.
It is based on the halo test, when sperm are treated with an acid solution prior to lysis buffer. The DNA dispersion halos found in sperms nuclei with fragmented DNA after the removal of nuclear proteins are either minimally present or not produced at all. (Fernandez et al., 2003;Fernandez et al., 2005). Studies have shown that SCD has high sensitivity to detect sperm with DNA fragmentation (Chohan et al., 2006;Zhang et al., 2010;Liffner et al., 2019). Different studies using SCD test have determined the reproductive capacity of the sperm and its correlation with seminal parameters and embryo quality after in vitro fertilization (Velez de La Calle et al., 2008;Feijó & Esteves, 2013;Tandara et al., 2013;Acosta & Dueñas, 2014;Acosta et al., 2015;Borges et al., 2019).
The purpose of our study was to determine the correlation between DFI and semen parameter in patients with normozoospermia, oligozoospermia, asthenozoospermia and teratozoospermia using the sperm chromatin dispersion (SCD) test.

Patients
This descriptive retrospective study included 418 patients seen at the Andrology Laboratory of the Assisted Reproduction Center ¨In Vitro Gestar¨, Chiclayo -Perú, between October 2013 and March 2020. We excluded those patients with azoospermia, varicocele, cryptorchidism, antibiotic treatments, diabetes, a history of radiotherapy and chemotherapy, chronic diseases. In Vitro Gestar Committee approved this study.

Sample collection and Semen Analysis
The seminal samples were collected by masturbation into a sterile container after a sexual abstinence period of 3 -7 days. The semen parameters were evaluated according to the WHO guidelines (WHO, 2010). Concentration and sperm motility were determined using a Makler chamber (Sefi-Medical Instruments, Haifa -Israel), and strict Kruger criteria was used for assessing sperm morphology (Papanicolaou staining). To find the normozoospermic patients (NORMO) and patients with an altered parameter (ALTERED), we followed the lower reference limit established by the WHO. The oligozoospermic patients were divided into groups: Severe/Moderate oligozoospermia (OLIGO SM) : <5x10 6 sperm/mL; mild oligozoospermia (OLIGO M): 5 to <15x10 6 sperm/mL. The asthenozoospermic patients were divided Based on their progressive motility (PM): Severe asthenozoospermia (ASTHENO S): <10% PM; Moderate asthenozoospermia (ASTHENO MO): 10 to <20% PM; Mild asthenozoospermia (ASTHENO M): 20 to <32% PM. The teratozoospermic patients (TERATO) are those with <4% normal morphology. The oligoasthenoteratozoospermic patients (OAT) are those with <15x10 6 sperm/m, <32%PM and <4% normal morphology.

Determination of DNA fragmentation Index (DFI)
We measured the DNA fragmentation Index of spermatozoa using the Sperm Chromatin Dispersion test (SCD) (Fernández et al., 2003; with the Halosperm Kit (Halotech DNA) with minor modifications. The normozoospermic patients' seminal samples were diluted in PBS (Phosphate-buffered saline) until reaching a concentration of 5 to 10 x10 6 sperm/mL, and the oligozoospermic patient's seminal samples were diluted in 1:1 (vol/vol) in PBS. The eppendorf with agarose (low melting point) was heated for 5 minutes at 90° to 100°C. Then it was placed in a water bath at 37°C by 5 minutes. Then, 20µL of diluted semen was added and homogenized. 10µL of this homogenate was placed on a pretreated slide with agarose (normal melting point) and covered with a 22x22mm cover slide. The slides were placed under refrigeration at 4°C for 7 minutes. Then the cover slides were gently removed and we added the acid solution (20µL HCl added to 2.5mL of distilled water) for 7 minutes. After the removal of this solution, we added the lysis solution for 15 minutes. Then the lysis solution was removed, and the slide was placed in distilled water and alcohol (70%. 90% and 100%) for two minutes each. We left the slide for drying at room temperature, and then we stained it with Wright dye. We analyzed 500 sperm per slide to determine the DNA fragmentation index (DFI). There were five types of halos: big halo, medium halo, small halo, without halo and degraded. Big and medium halo are considered as spermatozoa with normal DNA fragmentation and small halo, without halo and degraded as fragmented DNA spermatozoa.

Statistical Analysis
The data was analyzed using the SPSS 22.0 software for windows (SPSS, Chicago, IL, USA). The data is presented as mean ± standard deviation (SD). For the statistical comparison between the different groups, we tested the significance of difference using the Mann-Whitney test and the Kruskal-Wallis test. The regression and correlation analyses were performed using the Spearman´s correlation coefficient. The level of significance was set at p<0.05.

RESULTS
The age of the patients ranged from 19 to 59 years with a mean of 38.79±6.91. We analyzed the seminal parameter and DFI of NORMO and ALTERED patients ( Table 1). The values of concentration, progressive motility and normal morphology were significantly lower among the ALTERED patients (p=0.000). The DFI value was significantly high among the ALTERED patients (p=0.003). In addition, the ALTERED patients' ages were significantly higher when compared to the NORMO patients' ages (p=0.004).
In our study, the NORMO patients had significantly higher values (p=0.000) in concentration, progressive motility and normal morphology, and a significantly lower value in DFI (p=0.003), when compared with other groups with oligozoospermia ( Table 2). The same trend occurred in favor of patients with normal concentration, compared with the oligozoospermic group. Although the DFI value is low in normozoospermic patients (NORMO), when compared to patients with normal concentration, there were no significant differences between them. The patients with OLIGO SM had a significantly lower value (p=0.000) in concentration, progressive motility and normal morphology; and significantly higher (p=0.002) percentage of DFI when compared with the NORMO group (Table 2). Table 3 shows the comparison of seminal parameters and DFI between the asthenozoospermic and the normozoospermic patients. The NORMO group presents concentration, progressive motility and normal morphology values significantly higher (p=0.000) and significantly lower ages (p=0.042) and DFI (p=0.000); when compared to the other group. In addition, the patients with normal progressive motility had significantly higher concentration, progressive motility and normal morphology (p=0.000), and a significantly lower DFI (p=0.000), when compared with the group of asthenozoospermic patients. When comparing the NORMO group with patients with normal progressive motility, only the normal morphology group had significant differences (3.76 ± 1.42 vs 4.67 ± 1.65 p = 0.000, respectively). The ASTHENO group patients had significantly lower concentration, progressive motility and normal morphology values, and a significantly low percentage of DFI, when compared with the NORMO patients group.
In the present study, the NORMO group had concentration, progressive motility and normal morphology values significantly higher (p=0.000), and significantly lower ages (p=0.001) and DFI (p=0.005), when compared with the teratozoospermic patients (Table 4). Although the DFI values were lower in NORMO compared with normal morphology patients, there were no significant differences between them (Table 4).

DISCUSSION
The semen conventional analysis (sperm concentration, progressive motility and morphology) is used to assess fertility in men. However, the lack of correlation with the fertilizing potential make necessary to introduce new routine tests, such as sperm DNA fragmentation. High DFI values are correlated with reduced fertilization rates, early development embryo, bad embryo quality, low pregnancy rates and higher rates of spontaneous miscarriage (Bungum et al., 2007;Lewis et al., 2013;Simon et al., 2014). For these reasons, various authors have recommended the introduction of sperm DNA fragmentation analysis as a routine and complementary test in semen analysis (Lewis & Aitken, 2005;Fortunato et al., 2013). In this study, we uses the SCD test due its high sensitivity to detect sperm with DNA fragmentation (Chohan et al., 2006;Zhang et al., 2010;Liffner et al., 2019).
This study found that sperm concentration, progressive motility and normal morphology were significantly lower in ALTERED patients, when compared with NORMO patients, while semen volume did not differ (Table 1). Several studies have confirmed the correlation between altered semen parameters and high ROS concentrations (Agarwal et al., 2014;Majzoub et al., 2018;; and up to 80% of infertile men have elevated seminal ROS (Agarwal & Sekhon, 2014). Likewise, the current evidence recognizes oxidative stress as an important cause of sperm DNA fragmentation (Aitken & De luliis, 2010;Iommiello et al., 2015). Thus, we believe that high DFI values among the ALTERED patients is due to high concentrations of ROS produced by decreased semen quality in these patients. These results are consistent with other studies from Peru, such as those of Acosta & Dueñas (2014), which found a mean DFI value among ALTERED patients to be significantly higher than in the NORMO patients (22.95 ± 12.25 vs 14.39 ± 9.06); and Acosta et al. (2016) found significant differences in the mean value of DFI between ALTERED and NORMO patients (21.51 ± 14.18 vs 14.08 ± 7.08, respectively). Similar results were reported by other studies evaluating fertile and infertile patients using the SCD test (Fernández et al., 2005;Chohan et al., 2006;Evgeni et al., 2015;Wiweko & Utami, 2017;Majzoub et al., 2019) and other tests (Sergerie et al., 2005;Chohan et al., 2006;Sheikh et al., 2008;Mayorga-Torres et al., 2013;Di Santo et al., 2016). On the other hand, the study by Khalili et al. (2006) did not find significant differences in the value of DFI between fertile and infertile patients using the acridine orange staining test. We believe that the lack of correlation with DFI in this study is due to the assay used. The acridine orange staining assay is questionable, and is not recommended as a screening test for sperm quality and functional capacity due its very low clinical significance for infertility testing, and lack of relationship with other test, such as SCSA, TUNEL and SCD (Chohan et al., 2006).
We found that age has a high significance in ALTERED patients when compared with NORMO patients (39.50 ± 6.87 vs 37.26 ± 6.76, respectively) ( Table 1). Male aging is associated with infertility, semen altered parameters and sperm DNA fragmentation. Aging causes accumulation of ROS, leading to increased oxidative stress that induces lipid peroxidation and further ROS generation in the mitochondria (Gunes et al., 2016). An excessive amount of ROS and decreased antioxidant capacity in the course of aging may induce apoptosis or oxidative damage to DNA.
Our results are consistent with the study from Majzoub et al. (2019), and discordant with the studies from Acosta & Dueñas (2014) and Acosta et al. (2016).
Our study shows significant differences in the DFI between normozoospermic and oligozoospermic patients (Table 2). Our data is congruent to several studies (Varshini et al., 2011;Aydos et al., 2015;Choucair et al., 2016). A possible explanation for this relationship is the increased apoptosis of mature spermatozoa. Apoptosis controls the overproduction of male gametes (Sakkas et al., 1999). Increased levels of ROS are correlated with increased apoptosis of mature spermatozoa. (Agarwal & Sekhon, 2011). Oxidative stress-induced DNA damage may accelerate the process of germ cell apoptosis, leading to a decline in sperm count (Agarwal et al., 2003).
Progressive motility is one of the important parameters involved in sperm fertilizing capacity. This study shows DFI significant differs between normozoospermic patients and the asthenozoospermic group (Table 3). Our results are consistent with several studies. Belloc et al. (2014) determined that sperm motility is the seminal parameter that is most intimately related to sperm DNA fragmentation. In their study, they found a significant high level of DFI in males with asthenozoospermia alone when compared to oligozoospermia, or isolated teratozoospermia. Elbashir et al. (2018) found a significant negative correlation between DFI and progressive motility between infertile asthenozoospermic men and men with proven fertility. On the other hand, Varshini et al. (2011) reported a statically non-significant difference in DFI using TUNEL between asthenozoospermic patients and normozoospermic patients, despite having high median distribution. A possible explanation of the association between DFI and asthenozoospermia can be found in the development of the flagellum in the spermatogenesis. Cho et al. (2001) showed that the DNA compaction (using protamine or transition protein insufficiency models) is associated with development of an abnormal flagellum and defective motility. Another reason that explains this correlation, may be due to the increased oxidative stress causing sperm DNA damage, and in turn induce the lipid peroxidation of the sperm membrane, which results in oxidation of polyunsaturated fatty acids in the plasma membrane and the formation of malondialdehyde (MDA), thereby leading to structural and functional damage to the spermatozoa (Agarwal & Said, 2005). High levels of MDA is correlated with high DFI, both having a negative correlation with the progressive motility (Dorostghoal et al., 2016). Thus, high concentration of ROS can cause decreased sperm motility due to the damage to the axonemal structure or the reduction in intracellular adenosine triphosphate (Tsunoda et al., 2012). The decreased sperm motility has also been explained by apoptosis. Oxidative stress causes the generation of spermatozoa with poorly remodelled chromatin. These defective cells have a tendency to enter in an apoptotic pathway associated with motility loss (Aitken & Koppers, 2011).
Teratozoospermia presents a high phenotypic variability and limited information is available about its pathophysiological mechanism. Morphological anomalies of the spermatozoa play a very important role in determining the male fertility potential. DNA damage is correlated with different abnormal shape of the head and flagellum of sperms, being the anomaly of the head that presents a highly significant DFI value (Eskandari et al., 2017). In this study, we found a significant DFI correlation between teratozoospermic and normozoospermic patients (Table 4). Previous studies have demonstrated a positive correlation between teratozoospermia and DFI (Varshini et al., 2011;Garcia-Ferreyra et al., 2014;Aydos et al., 2015); and other studies have not reported a significant relationship between these parameters (Avendaño et al., 2009;Choucair et al., 2016). One of the processes that explains this relationship is the incomplete replacement of histone by protamine, which induces abnormal chromatin condensation, producing deformations of the nucleus and overall head shape in the sperm (Ma et al., 2019). Another explanation may be the abnormal apoptosis causing oxidative stress. The abnormal apoptosis may cause the persistence of abnormal spermatozoa that are marked for elimination, thereby increasing teratozoospermia (Sakkas et al., 1999). Aydos et al. (2015) determined that the positive correlation between sperm DNA damage and impaired sperm morphology might be associated with the fact that sperm DNA damage leads to impairment in the sperm chromatin structure.
Our results indicate that DFI is significantly higher in men with OAT when compared with normozoospermic men (Table 5). Several studies have investigated the association between DFI and conventional seminal parameters. Most of these studies are consistent with our results (Varshini et al., 2011, Acosta et al., 2015Aydos et al., 2015;Choucair et al., 2016), confirming that male infertility is associated with poor sperm DNA integrity.
The present data demonstrated a statically significant inverse correlation between the DFI percentage, sperm progressive motility and sperm normal morphology ( Figure  1, 2). We did not find a significant correlation with sperm concentration. Many studies have reported a significant negative correlation between DFI and semen parameters, mainly concentration (Velez de la Calle et al., 2008;Acosta & Dueñas, 2014;Aydos et al., 2015;Acosta et al., 2015;Choucair et al;, progressive motility (Sheikh et al., 2008;Velez de la Calle et al., 2008;Acosta & Dueñas, 2014;Aydos et al., 2015;Evgeni et al., 2015;Choucair et al;Elbashir et al., 2018) and normal morphology (Velez de la Calle et al., 2008;Fortunato et al., 2013;Acosta & Dueñas, 2014;García-Ferreyra et al., 2014;Aydos et al., 2015), alone or in combination. On the other hand, the study by Khalili et al. (2006) did not find correlations between DFI and sperm concentration, progressive motility and normal morphology using the acridine orange staining test. Likewise, we found a significant positive correlation between DFI and age, but this is considered a weak correlation (r = 0.257) (  (Winkle et al., 2009;Brahem et al., 2011). Petersen et al. (2018) concluded that unfortunately not all studies follow these statistical analyzes, which makes it difficult to interpret this data.
In conclusion, our results suggest that an abnormal spermogram not only reflects altered spermatogenesis but also a negative effect on sperm DNA, and high DFI is accompanied by significant impairment to all seminal parameters.

CONFLICTS OF INTEREST
The authors have no conflict of interest to declare.