Lipid peroxidation and DNA fragmentation in fresh and cryopreserved spermatozoa of men at different spermatogenesis state

cryopreservation of spermatozoa is widely used in the treatment of infertility by assisted reproductive technologies. however, the cryopreservation causes an oxidative stress which can induce pathological changes in the male gametes. The aim of the research was to evaluate lipid peroxidation (lPO) and DNa fragmentation as well as correlation between these parameters in the fresh and cryopreserved spermatozoa of men with normozoospermia or oligoasthenoteratozoospermia (OaT) and spermatozoa derived from the epididymis of men with azoospermia. The level of malondialdehyde (MDa) in a TBa test, superoxide dismutase (SOD) activity and total antioxidant activity (aOa) were assessed. DNa integrity was estimated by acridine orange staining technique. It was shown that MDA level and SOD activity were significantly higher in the fresh spermatozoa of oligoasthenoteratozoospermic group compared with the fresh spermatozoa of normozoospermic group. after cryopreservation the MDa level increased in all groups and was the highest in OaT group where the greatest aOa decline was detected. DNa fragmentation frequency was 2.6 and 4.1 times higher in the fresh and cryopreserved OaT spermatozoa respectively as compared with the fresh normozoospermic ones (7.2%). DNa fragmentation was found to be the lowest in the fresh (6.2%) and cryopreserved (5.8%) epididymal spermatozoa. after cryopreservation SOD activity in epididymal spermatozoa was lower than in normozoaspermic. In spermatozoa of the studied groups the MDa level positively correlated with DNA fragmentation (0.79 Pearson’s correlation coefficient) both before and after cryopreservation. It is suggested that due to the detected low DNa fragmentation and lPO level in epididymal spermatozoa their use could be an alternative approach for infertility treatment by assisted reproductive technologies.

cryopreservation of spermatozoa is widely used in the treatment of infertility by assisted reproductive technologies. however, the cryopreservation causes an oxidative stress which can induce pathological changes in the male gametes. The aim of the research was to evaluate lipid peroxidation (lPO) and DNa fragmentation as well as correlation between these parameters in the fresh and cryopreserved spermatozoa of men with normozoospermia or oligoasthenoteratozoospermia (OaT) and spermatozoa derived from the epididymis of men with azoospermia. The level of malondialdehyde (MDa) in a TBa test, superoxide dismutase (SOD) activity and total antioxidant activity (aOa) were assessed. DNa integrity was estimated by acridine orange staining technique. It was shown that MDA level and SOD activity were significantly higher in the fresh spermatozoa of oligoasthenoteratozoospermic group compared with the fresh spermatozoa of normozoospermic group. after cryopreservation the MDa level increased in all groups and was the highest in OaT group where the greatest aOa decline was detected. DNa fragmentation frequency was 2.6 and 4.1 times higher in the fresh and cryopreserved OaT spermatozoa respectively as compared with the fresh normozoospermic ones (7.2%). DNa fragmentation was found to be the lowest in the fresh (6.2%) and cryopreserved (5.8%) epididymal spermatozoa. after cryopreservation SOD activity in epididymal spermatozoa was lower than in normozoaspermic. In spermatozoa of the studied groups the MDa level positively correlated with DNA fragmentation (0.79 Pearson's correlation coefficient) both before and after cryopreservation. It is suggested that due to the detected low DNa fragmentation and lPO level in epididymal spermatozoa their use could be an alternative approach for infertility treatment by assisted reproductive technologies. T here is no doubt that reactive oxygen species (ROS) play an important role in the functioning of different cell types, including male gametes [1,2]. Production of ROS is a mandatory step of the natural processes such as posttesticular spermatozoa maturation, apoptosis, in capacitation, hyperactivation, acrosome reaction, and spermatozoa-oocyte fusion. On the other hand, an access ROS level can indicate pathological processes. Thus, ROS influence the seminal parameters such as motili ty, count, volume, activity [3]. ROS implement their effects on spermatozoa functional state by oxidizing the proteins, inducing damage to nucleic acids and peroxidation of lipids (LPO) [4][5][6]. Clinical observations are increasingly showing the relationship between the spermatozoa LPO processes and male infertility as well as play an important role in oocyte fertilization outcomes [7,8]. Since ROS can cause DNA fragmentation and LPO in human spermatozoa, it is important to investigate the relationship of these processes in men with different spermatogene sis state [9]. Additionally, spermatozoa cryopreser-doi: https://doi.org/10.15407/ubj93.03.024 vation can induce injury by ice formation and osmotic stress following reducing membrane fluidity and causing excessive production of endogenous and exogenous ROS [10,11].
Thus, the aim of the study was to investigate the level of lipid peroxidation and DNA fragmentation in fresh and cryopreserved spermatozoa of men with different spermatogenesis state, as well as to establish a correlation between these parameters.

materials and methods
Ejaculated sperm samples obtained from the patients, who underwent infertility treatment from 2017 to 2019, were analyzed. The study included 138 patients (mean age of 32 ± 3.4). The study was approved by the Biomedical Ethical Committee of the Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of Ukraine, in line with the Law of Ukraine "On Personal Data Protection" (2010). Informed consents were obtained from all the participants in line with the WHO regulation [12].
The following study groups were identified: the spermatozoa retrieved from the ejaculates of normozoospermic men (group 1), spermatozoa retrieved from the ejaculates from men with oligoasthenoteratozoospermia (OAT) (group 2), and spermatozoa retrieved by microsurgical or percutaneous epididymal aspiration in men with azoospermia (group 3).
The active motile sperm fraction was isolated in a density gradient Sperm Grade (LifeGlobal, USA) [13]. A two-stage method was used for spermatozoa cryopreservation. Active motile sperm fraction was mixed in 1 : 1 ratio with cryoprotective medium containing 10% glycerol (Sigma Pharmaceutical, Buchs, Switzerland), 20% human serum albumin (LifeGlobal, USA) based on Global total for fertilization medium (LifeGlobal, USA) in 1 ml cryovials. After 10 min of exposure, the cryovials were placed 10 cm above the liquid nitrogen mirror level for 15 min and then plunged into the liquid nitrogen, where they were stored during 1-3 months. Samples were warmed in a water bath at 40°C until complete disappearance of the solid phase. The cell suspension was centrifuged and the supernatant was removed. The culture medium of 0.1 ml was added to the precipitate.
The concentration of MDA was determined by the intensity of the color formed during the reaction of MDA and thiobarbituric acid (TBA), which takes place in an acidic environment at high temperature [14]. The superoxide dismutase (SOD) activity was determined by the reduction of the dye nitrosine tetrazolium and expressed in conventional units: for 1 conventional unit was 50% of inhibited redox reaction [15]. Optical density was measured using a Shimadzu QV-50 spectrophotometer (Japan). Antioxidant activity (AOA) was determined in a model system of suspension of yolk lipoproteins [16]. AOA was expressed as a percentage from inhibition of 50% LPO in the model system compared to the classical antioxidant ionol.
The DNA fragmentation rate of spermatozoa was measured using acridine orange (AO) staining and fluorescent microscopy [17]. AO fluoresces green when it binds to native DNA and it does red when it binds to the fragmented DNA (Fig. 1). In each sample, the number of red and green fluorescence cells were counted among 100 cells and represented in percentage. Statistica 6.0 was used for the results processing . When comparing the samples, the Mann-Whitney Utest was used at a significance level of P ≤ 0.05.

results and discussion
At the first stage, the LPO processes are charac terized by formation of primary (diene, triene, tetraene conjugates) and secondary products. One such compound is MDA, an intermediate product It has been established that there is a quantitative relationship between LPO processes and MDA concentration. When studying the oxidative state and antioxidant systems in spermatozoa of men with different spermatogenesis state, it was found that the level of MDA was significantly higher in group 2 compared with this index in group 1 ( Table 1). The level of MDA in sperm of group 3 was comparable to group 1. The level of SOD in group 2 was significantly higher than in group 1. This fact can be considered as a compensatory response to the excess ROS level in OAT spermatozoa. However, AOA levels in OAT spermatozoa tended to decrease compared with normozoospermic and epididymal spermatozoa.
The study of the DNA fragmentation rate showed that in group 2, this indicator was the highest and amounted to 19.7 ± 7.73%. There were no significant differences in groups 1 and 3.
After cryopreservation, the MDA concentration increased and was the highest in group 2 (Table 2). The accumulation of peroxidation products can lead to appearance of peculiar membrane pores by increasing the hydrophilic hydrocarbon tails content, as well as to increase its stiffness by reducing the content of unsaturated fatty acids and thereby affecting the membrane permeability [18]. Thus, the membranes of OAT spermatozoa undergo significant structural changes, which may affect their cryoresistance.
The activity of SOD in cryopreserved spermatozoa was significantly higher in group 2 compared with group 1. In group 3, there was a decrease in this index by 1.9 times relative to normal spermatozoa. AOA decreased 5.4 times in group 2 cryopreserved spermatozoa compared with group 1. There was also a tendency to decrease in group 3 spermatozoa compared with group 1.
DNA fragmentation rate in group 2 cryopreserved spermatozoa increased 4.1 times compared with group 1 and was the lowest in group 3 (5.8 ± 1.3)% (Fig 2).
The values of MDA concentrations in the studied groups both before and after cryopreservation positively correlated with DNA fragmentation rate (Pearson's correlation coefficient was 0.79 and was significant at 95% confidence interval). There is also a negative correlation between SOD and DNA fragmentation in spermatozoa in the studied groups (Pearson's correlation coefficient was 0.77).
It should be noted that the presence of positive or negative correlation between the indices does not indicate a causal relationship between them. A si- 10.2 ± 0.9 2.3 ± 0.2* 5.8 ± 0.4 multaneous variation is likely due to the influence of another variable and the mechanism or system connections of this phenomenon can be complicated. We assume that the increase in MDA in spermatozoa is associated with a higher level of DNA fragmentation and may indicate apoptosis activation. MDA formation occurs during the cleavage of lipid hydroperoxides induced by oxidative stress [19]. Also, it could cause the destruction of covalent complexes with amino groups of proteins, induces specific nuclease activation leading to DNA fragmentation. The negative impact of cryopreservation increases the ROS effects.
The detected elevated level of LPO products in spermatozoa of OAT men indicates the negative effects of activation of peroxide processes in cells. Normally, the processes of ROS peroxidation, in particular LPO, are maintained at a stationary level due to the antioxidant system. The accumulation of LPO products can be an important factor, leading to disordered integrity of the spermatozoa membrane after freeze-warming [20].
The results of the antioxidant status study indicate a deficiency of antioxidant protection in both fresh and cryopreserved OAT spermatozoa in comparison with normozoospermic and epididymal spermatozoa. As noted above, an increase in SOD level was observed in OAT spermatozoa probably due to the compensatory response to the ROS increasing. However, after cryopreservation, the SOD activity was lower than in fresh OAT spermatozoa despite the increased LPO level and DNA fragmentation rate. Thus, cryopreservation of gametes from OAT men leads to a decrease in SOD activity. And since the SOD addition to spermatozoa can decrease the DNA fragmentation rate, it may be promising for using for OAT spermatozoa cryopreservation in order to increase their fertilization capacity [21]. It is well known that high spermatozoa DNA fragmentation rate is one of the causes of male infertility, and in case of high index of this parameter no patient has reached pregnancy when underwent infertility treatment by assisted reproductive technologies (ART) [22]. An alternative approach may be the use of epididymal spermatozoa for infertility treatment by ART due to the much lower DNA fragmentation rate and LPO level.